CN110294720A - For detecting fluorescence probe and its application of butyrylcholine esterase - Google Patents
For detecting fluorescence probe and its application of butyrylcholine esterase Download PDFInfo
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- CN110294720A CN110294720A CN201810247083.2A CN201810247083A CN110294720A CN 110294720 A CN110294720 A CN 110294720A CN 201810247083 A CN201810247083 A CN 201810247083A CN 110294720 A CN110294720 A CN 110294720A
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- fluorescence probe
- butyrylcholine esterase
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- 101710083761 Cholinesterase Proteins 0.000 title claims description 106
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 86
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 82
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 22
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- 229940123923 Butyrylcholinesterase inhibitor Drugs 0.000 claims description 10
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- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000012512 characterization method Methods 0.000 claims description 4
- 125000003236 benzoyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C(*)=O 0.000 claims description 2
- 239000000243 solution Substances 0.000 description 18
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 13
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- 239000012086 standard solution Substances 0.000 description 9
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- 102000005367 Carboxypeptidases Human genes 0.000 description 6
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 3
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- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
- C07D277/60—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings condensed with carbocyclic rings or ring systems
- C07D277/62—Benzothiazoles
- C07D277/64—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2
- C07D277/66—Benzothiazoles with only hydrocarbon or substituted hydrocarbon radicals attached in position 2 with aromatic rings or ring systems directly attached in position 2
-
- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1003—Carbocyclic compounds
- C09K2211/1007—Non-condensed systems
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- C—CHEMISTRY; METALLURGY
- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
- C09K2211/10—Non-macromolecular compounds
- C09K2211/1018—Heterocyclic compounds
- C09K2211/1025—Heterocyclic compounds characterised by ligands
- C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
- C09K2211/1037—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with sulfur
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Materials Engineering (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The present invention provides a kind of for detecting the fluorescence probe of the activity of BuChE, and the fluorescence probe is non-peptides fluorescence probe, and the structure of the non-peptides fluorescence probe is as shown in following formula 1:
Description
Technical field
The invention belongs to field of biotechnology more particularly to a kind of fluorescence probe for detecting butyrylcholine esterase and its
Using.
Background technique
Butyrylcholine esterase (BChE) is to be present in a kind of intracorporal acetylcholine isodynamic enzyme of biology, it is synthesized by liver
A kind of nonspecific esterase, be immediately released into blood plasma after being synthesized in liver due to the enzyme, in serum BChE activity
It is the sensitive indexes for measuring Hepatocyte matter synthesis function.With the continuous improvement of detection technique and clinic diagnosis level, fourth
Acetylcholinesterase is in addition to extending also to malignant tumour, nervous system disease, cardiovascular disease for assessing liver reserve function
In the detection and diagnosis field of numerous diseases such as disease, metabolic syndrome.The method master of existing measurement the activity of BuChE
Will there are two types of.The first is the analysis detection of toxic agent and protein adduct, mainly detects mind by mass spectral analysis means
Addition product through property toxic agent (GB, GD and VX) formation in conjunction with the butyrylcholine esterase in blood plasma reaches testing goal.The
Two kinds of methods are then fluorine ion Reactivation methods, and mainly nerve toxicant is made by butyrylcholine esterase phosphorylated, then with fluorine ion
With rear, the phosphate part of nerve toxicant can be released from choline vinegar enzyme, and regenerated Organic fluoride phosphate can be with
It is detected with means such as GC, GC-MS, while can use large volume sample injection technology also to measure the regeneration Organic fluoride of low concentration
Phosphate.Not only operating process is complex for both the above method, and being required to expensive large-scale instrument and testing cost could be real
It is existing, and it is only used for the detection of polypeptide fluorescence probe.Since that there are stability is poor, storage and transportation condition is severe for polypeptide fluorescence probe
It carves, the disadvantages of atom utilization is low, fluorescence intensity is weak, therefore, designs and develops the fluorescence probe based on non-peptides and its detection side
Method, it appears very urgent and important.
Summary of the invention
The fluorescence probe and its application that the purpose of the present invention is to provide a kind of for detecting butyrylcholine esterase, it is intended to solve
Certainly it is an object of the invention to overcome the detection method of butyrylcholine esterase in the prior art to need to rely on expensive large-scale instrument
The problem of equipment and detection process complexity, and that there are stability is poor, storage and transportation condition is harsh, atom utilizes for polypeptide fluorescence probe
The problem that rate is low, fluorescence intensity is weak.
For achieving the above object, The technical solution adopted by the invention is as follows:
One aspect of the present invention provides a kind of for detecting the fluorescence probe of the activity of BuChE, and the fluorescence probe is
Non- peptides fluorescence probe, and the structure of the non-peptides fluorescence probe is as shown in following formula 1:
Another aspect of the present invention provides a kind of kit for detecting the activity of BuChE, including as above-mentioned non-peptide class is glimmering
The standard items of light probe and butyrylcholine esterase.
Further aspect of the present invention provides a kind of application method of kit for detecting the activity of BuChE, including following
Step:
Butyrylcholine esterase standard items, butyrylcholine esterase sample to be tested and non-peptides fluorescence probe are provided；
By the butyrylcholine esterase standard items and the non-peptides fluorescence probe hybrid reaction, obtains fluorogen reaction and produce
Object；The fluorescence intensity change value of reaction product under detection excitation light stimulus changes with time value, acquisition butyrylcholine esterase-
Fluorescence intensity change value standard curve obtains butyrylcholine esterase-fluorescence intensity change value linear equation；
Under testing conditions identical with the butyrylcholine esterase standard items, by the butyrylcholine esterase sample to be tested
With the non-peptides fluorescence probe hybrid reaction, fluorogen reaction product is obtained；Detection excites the reaction product under light stimulus
Fluorescence intensity change value substitutes into the butyrylcholine esterase-fluorescence intensity change value linear equation, calculates and obtains the butyryl
The concentration of cholinesterase sample to be tested.
And the present invention also provides a kind of non-peptides fluorescence probe butyrylcholine esterase detection field application, it is described
Using including the fluorescence probe for detecting the activity of BuChE, the fluorescence probe is for screening butyrylcholine esterase
Inhibitor or promotor, the fluorescence probe are used for answering for the Kinetic Characterization field of butyrylcholinesterase inhibitor or promotor
With.
Fluorescence probe provided by the present invention for detecting the activity of BuChE is non-peptides fluorescence probe, is had steady
Qualitative feature strong, atom utilization is high, fluorescence intensity is high, storage and transportation condition is flexible.When for detecting the activity of BuChE,
Butyrylcholine esterase sample to be tested can produce fluorogen (Fluorophore) after contacting with the non-peptides fluorescence probe, pass through
The intensity for the fluorescence that detection fluorogen issues under excitation light, realizes the purpose of the activity of BuChE detection.With it is existing
Polypeptide Substrate fluorescence probe is compared, provided in an embodiment of the present invention for detecting the non-peptides fluorescence of the activity of BuChE
Probe has the advantages that specificity height and fluorescence intensity change are significant, therefore, has preferably parent with the butyrylcholine esterase
And power, detection have higher sensitivity, so as to it is fast and convenient, the activity of BuChE is effectively detected.
The kit of detection the activity of BuChE provided by the invention, due to being made using the non-peptides fluorescence probe
Therefore there is easy to use, the high advantage of detection sensitivity for detection probe.
The application method of the kit of detection the activity of BuChE provided by the invention, passes through butyrylcholine esterase mark
After quasi- product obtain butyrylcholine esterase-fluorescence intensity linear equation, the fluorescence for the butyrylcholine esterase sample to be tested that will test
Intensity is substituted into equation and be can be obtained, and method is simple, and accuracy is high.
For non-peptides fluorescence probe provided by the invention in the application of butyrylcholine esterase detection field, application field is extensive,
And the high testing result of accuracy can be obtained.
Detailed description of the invention
Fig. 1 is excitation photoscan provided in an embodiment of the present invention；
Fig. 2 is the launch wavelength scanning figure of fluorogen provided in an embodiment of the present invention；
Fig. 3 is the selection result schematic diagram of non-peptides fluorescence probe and amino acid substrate provided in an embodiment of the present invention；
Fig. 4 is the selection result schematic diagram of non-peptides fluorescence probe and metal ion substrate provided in an embodiment of the present invention；
Fig. 5 is the selection result schematic diagram of non-peptides fluorescence probe provided in an embodiment of the present invention and hydrolysis substrate.
Specific embodiment
In order to which technical problems, technical solutions and advantageous effects to be solved by the present invention are more clearly understood, below in conjunction with
Embodiment, the present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only used to explain
The present invention is not intended to limit the present invention.
In the description of the present invention, it is to be understood that, term " first ", " second " are used for description purposes only, and cannot
It is interpreted as indication or suggestion relative importance or implicitly indicates the quantity of indicated technical characteristic.Define as a result, " the
One ", the feature of " second " can explicitly or implicitly include one or more of the features.In the description of the present invention,
The meaning of " plurality " is two or more, unless otherwise specifically defined.
The embodiment of the invention provides a kind of for detecting the fluorescence probe of the activity of BuChE, the fluorescence probe
For non-peptides fluorescence probe, and the structure of the non-peptides fluorescence probe is as shown in following formula 1:
Fluorescence probe provided in an embodiment of the present invention for detecting the activity of BuChE is non-peptides fluorescence probe,
Have the characteristics that stability is strong, atom utilization is high, fluorescence intensity is high, storage and transportation condition is flexible.For detecting butyrylcholine esterase
When active, butyrylcholine esterase sample to be tested can produce fluorogen after contacting with the non-peptides fluorescence probe, glimmering by detecting
The intensity for the fluorescence that light blob issues under excitation light realizes the purpose of the activity of BuChE detection.With existing polypeptide
Substrate fluorescence probe is compared, the non-peptides fluorescence probe tool provided in an embodiment of the present invention for detecting the activity of BuChE
Have the advantages that specificity height and fluorescence intensity change are significant, therefore, there is better affinity, inspection with the butyrylcholine esterase
Measuring tool has higher sensitivity, so as to it is fast and convenient, the activity of BuChE is effectively detected.
In the embodiment of the present invention, the non-peptides fluorescence probe is for when detecting butyrylcholine esterase, the non-peptides to be glimmering
The benzoyl of light probe is combined as recognition group with the butyrylcholine esterase (BchE).It is described in the embodiment of the present invention
Butyrylcholine esterase generates the principle of fluorogen in conjunction with the non-peptides fluorescence probe are as follows: the benzene of the non-peptides fluorescence probe
Formoxyl generates intermediate state compound in conjunction with butyrylcholine esterase, and excited state molecule endoplasm occurs for the intermediate state compound
Son transfer (ESIPT) generates fluorogen, and then the fluorescence intensity by detection fluorogen under excitation light, the fluorescence intensity
The activity for indicating butyrylcholine esterase in sample to be tested achievees the purpose that detect butyrylcholine esterase.Its reaction equation is as follows
It is shown:
In the embodiment of the present invention, as shown in Figure 1, the wavelength of the exciting light is 325nm, as shown in Fig. 2, the fluorogen
Launch wavelength be 460nm.Wherein, Wavelength indicates that wavelength, Probe indicate non-peptides fluorescence probe, Fluorophore
Indicate fluorogen.
At identical conditions, after by different substrates in conjunction with the non-peptides fluorescence probe, in identical exciting light
With fluorescence intensity under the conditions of launch wavelength, the non-peptides fluorescence probe and amino acid substrate, metal ion substrate, hydrolysis
The selectivity difference of substrate is as in Figure 3-5, and as seen from the figure, non-peptides fluorescence probe is to the butyryl described in the embodiment of the present invention
Cholinesterase has high specificity.
Further, in the embodiment of the present invention, for detect the activity of BuChE fluorescence probe dosage with to
The property for surveying butyrylcholine esterase sample and such probe is related.The dosage of the non-peptides fluorescence probe is required to characterization fourth
The activity of acetylcholinesterase sample to be tested.Preferably, when the non-peptides fluorescence probe is used to detect butyrylcholine esterase, relatively
Every 1 μM of butyrylcholine esterase in sample to be tested, the dosage of the fluorescence probe are 1-100 μM, i.e., the described BuCh ester
The mole dosage ratio of enzyme and the non-peptides fluorescence probe is 1 μM: (1-100) μM.It is described in order to reach optimal detection effect
The dosage of fluorescence probe is preferably 1-10 μM, i.e., the mole dosage ratio of the described butyrylcholine esterase and the non-peptides fluorescence probe
It is 1 μM: (1-10) μM.
The embodiment of the present invention butyrylcholine esterase sample is contacted with non-peptides fluorescence probe after fluorogen fluorescence it is strong
The detection of degree preferably carries out in microplate reader or sepectrophotofluorometer, can be obtained using the method for time dependence detection
The case where butyrylcholine esterase fluorescence spectrum that produces chemiluminescence derivative hydrolyzes in contact process changes over time, obtains glimmering
The value added that luminous intensity changes over time.Further, it is also possible to be protected using multiplicating test and by the way of several control groups are arranged
Demonstrate,prove the reliability of detection.
The embodiment of the invention provides a kind of kits for detecting the activity of BuChE, including as above-mentioned non-peptide class is glimmering
The standard items of light probe and butyrylcholine esterase.
The kit of detection the activity of BuChE provided in an embodiment of the present invention, due to using the non-peptides fluorescence
Probe is as detection probe, therefore, has easy to use, the high advantage of detection sensitivity.
It preferably, further include that concentration is in the kit of detection the activity of BuChE provided in an embodiment of the present invention
The Tris-HCl buffer solution of 50-100mM.Concentration is the Tris-HCl buffer solution of 50-100mM, can give the fourth
Suitable environment is stablized in acetylcholinesterase offer, to improve the accuracy of detection.Further, the Tris-HCl buffering is molten
The pH of liquid is 7.0, and the CaCl containing concentration 10mM2。
Specifically, being visited when detection the activity of BuChE by the butyrylcholine esterase and the non-peptides fluorescence
Needle contact before, first by suitable sample to be tested containing butyrylcholine esterase, 10 μM of non-peptides fluorescence probes respectively with 50-150 μ L
The Tris-HCl buffer mixing, to preferably improve the Stability and veracity of detection.
It is including following the embodiment of the invention provides a kind of application method of kit for detecting the activity of BuChE
Step:
S01., butyrylcholine esterase standard items, butyrylcholine esterase sample to be tested and non-peptides fluorescence probe are provided；
S02. by the butyrylcholine esterase standard items and the non-peptides fluorescence probe hybrid reaction, it is anti-to obtain fluorogen
Answer product；The fluorescence intensity change value of reaction product under detection excitation light stimulus changes with time value, acquisition BuCh
Esterase-fluorescence intensity change value standard curve obtains butyrylcholine esterase-fluorescence intensity change value linear equation；
S03. under testing conditions identical with the butyrylcholine esterase standard items, the butyrylcholine esterase is to be measured
Sample and the non-peptides fluorescence probe hybrid reaction, obtain fluorogen reaction product；Reaction under detection excitation light stimulus produces
The fluorescence intensity change value of object substitutes into the butyrylcholine esterase-fluorescence intensity change value linear equation, calculates described in obtaining
The concentration of butyrylcholine esterase sample to be tested.
The kit of detection the activity of BuChE provided in an embodiment of the present invention is under conditions of subzero 20 DEG C to 4 DEG C
It saves.
The application method of the kit of detection the activity of BuChE provided in an embodiment of the present invention, passes through BuCh
After esterase standard items obtain butyrylcholine esterase-fluorescence intensity linear equation, the butyrylcholine esterase sample to be tested that will test
Fluorescence intensity substitute into equation and can be obtained, method is simple, and accuracy is high.
Specifically, in above-mentioned steps S01, structure, non-peptides fluorescence probe and the butyryl gallbladder of the non-peptides fluorescence probe
The amount ratio of the action principle of alkali esterase and non-peptides fluorescence probe and butyrylcholine esterase is as described above, in order to save a piece
Width, details are not described herein again.
In above-mentioned steps S02, by the butyrylcholine esterase standard items or the butyrylcholine esterase sample to be tested and institute
The step of stating non-peptides fluorescence probe hybrid reaction, reaction needs to carry out in reaction buffer, it is further preferred that in concentration
To be carried out in the Tris-HCl buffer of 50-100mM, so that the butyrylcholine esterase standard items or the butyrylcholine esterase
Sample to be tested is reacted with the non-peptides fluorescence probe stable and uniform, improves accuracy in detection.
It is described in order to guarantee the activity of butyrylcholine esterase standard items, butyrylcholine esterase sample to be tested in detection process
The condition of hybrid reaction are as follows: temperature is 25-37 DEG C, pH value 6-8, time 5-30min.It is further preferred that the mixing
The condition of reaction are as follows: temperature is 25-30 DEG C, pH value 6.5-8.0, time 10-25min.
It should be understood that hybrid reaction described in the embodiment of the present invention carries out under the conditions of being protected from light, and to hybrid reaction
Container is preheated, to keep the bioactivity of sample to be tested.Preferably, the container of the hybrid reaction is black flat-bottom hole
Plate, cuvette or ELISA Plate, and before contact be preheated to each component for participating in hybrid reaction and reaction vessel suitable anti-
Temperature is answered, preheating temperature to be achieved is preferably 25-37 DEG C.
In the embodiment of the present invention, the wavelength of the exciting light is 325nm, the fluorogen under the excitation light stimulus
The launch wavelength of the fluorescence of reaction product is 460nm.
In above-mentioned steps S03, under testing conditions identical with the butyrylcholine esterase standard items, by the butyryl gallbladder
Alkali esterase sample to be tested and the non-peptides fluorescence probe hybrid reaction, obtain fluorogen reaction product；Detection excitation light stimulus
Under reaction product fluorescence intensity change value, substitute into the butyrylcholine esterase-fluorescence intensity change value linear equation, count
Calculate the concentration for obtaining the butyrylcholine esterase sample to be tested.
As a particular preferred embodiment, the application method of the kit of the detection the activity of BuChE, packet
Include following steps:
The DMSO solution of the non-peptides fluorescence probe of 10mM is configured, and is kept in dark place at subzero 20 DEG C to 4 DEG C.By butyryl
The cholinesterase standard items Tris-HCl of 0.1M (contains 10mM CaCl2, pH=7.0) and buffer solution, it is configured to 100U/
The butyrylcholine esterase standard solution of ml, and gradient dilution is carried out with 10 times of dilution.Butyrylcholine esterase will be contained
100mM Tris-HCl (containing 10mM CaCl2, pH=7.0) buffer solution of 100 μ L of sample to be tested.
The Tris-HCl of 0.2mL (is contained into 10mM CaCl respectively2, pH=7.0) and buffer, for detecting BuCh
The non-peptides fluorescence probe based on ESIPT mechanism, butyrylcholine esterase sample to be measured and the butyrylcholine esterase standard of esterase
Product are incorporated in black ELISA Plate.It is detected using the method for detecting the activity of BuChE described in the embodiment of the present invention to be measured
The activity of butyrylcholine esterase in sample and butyrylcholine esterase standard items.
Standard curve is drawn with the fluorescence intensity change value of the butyrylcholine esterase standard items of the gradient dilution detected, is led to
Cross the activity for comparing and determining butyrylcholine esterase in detected sample.
And the embodiment of the invention provides a kind of non-peptides fluorescence probe answering in butyrylcholine esterase detection field
With the application includes the fluorescence probe for detecting the activity of BuChE, and the fluorescence probe is for screening butyryl
Anticholinesterase or promotor, the fluorescence probe is for butyrylcholinesterase inhibitor or the Kinetic Characterization of promotor
The application in field.
Application of the non-peptides fluorescence probe provided in an embodiment of the present invention in butyrylcholine esterase detection field, application field
Extensively, and the high testing result of accuracy can be obtained.
As a kind of specific embodiment, using the fluorescence probe for detecting the activity of BuChE.Specifically,
Including being detected for detecting the activity of BuChE to individual the activity of BuChE using the fluorescence probe,
It also include being detected to the enzyme system containing the activity of BuChE.In the embodiment of the present invention, the non-polypeptide fluorescence is visited
Needle can be also used for one of elastoser, trypsase, chymotrypsin and carboxypeptidase or a variety of enzymatic activitys
Measurement.Wherein, when in sample to be tested contain in elastoser, trypsase, chymotrypsin and carboxypeptidase determine one
When kind, the non-polypeptide fluorescent probe molecule provided by the embodiment of the present invention can be used for measuring known kind contained in sample
The butyrylcholine esterase of class activity (i.e. according to probe and the operative condition of target come compared with its selective size, judge to be measured
Whether enzyme is BChE)；When containing one in elastoser, trypsase, chymotrypsin and carboxypeptidase in sample to be tested
When planting but not can determine that specially any, is also detected using the non-polypeptide fluorescence probe, obtain the fourth of unknown type
Acetylcholinesterase is catalyzed the Michaelis constant that the non-polypeptide fluorescence probe also hydrolyzes, thus butyryl gallbladder contained in judgement sample
The type (by the Michaelis constant of acquisition, contrasted with standard items and learn its type) of alkali esterase；In addition, the embodiment of the present invention mentions
The non-polypeptide fluorescence probe supplied can also be applied to the total of a variety of butyrylcholine esterases contained in test sample simultaneously
Activity, to analyze total activity in sample containing butyrylcholine esterase.
As another embodiment, the fluorescence probe is for screening butyrylcholinesterase inhibitor or promotor.Make
For a specific embodiment, the butyrylcholine esterase containing certain concentration, certain density butyrylcholine esterase are inhibited respectively
Agent or solution of organic compound and 50-100mM Tris-HCl (contain 10mM CaCl2, pH=7.0) and it is placed in 30 DEG C of thermostatted waters
It is preheated in slot；Meanwhile 96 orifice plate of black flat-bottom being placed in 30 DEG C of incubators and is preheated.Then, make suitable have been warmed up
Tris-HCl (contains 10mM CaCl2, pH=7.0) and buffer solution, the various butyrylcholinesterase inhibitors of same volume and suitable
The fluorescence probe of amount is contacted with butyrylcholine esterase.And with microplate reader or Full Featured sepectrophotofluorometer to the glimmering of sample
Luminous intensity carries out the monitoring of time dependence, observes its fluorescence intensity level, then draw standard curve meter with software (ORIGINLAB)
Calculate the correlation values such as IC50.
It is worth noting that, when the kit of the embodiment of the present invention is for butyrylcholinesterase inhibitor or the sieve of promotor
When selecting, since the cholinesterase activity in reaction system can all change after having added unknown inhibitor, if inhibitor is always
It is effective, lack then substrate in system is hydrolyzed, it therefore, can be by the BuCh ester inhibitor before detection with suitable
When ratio or viscosity make an addition in reaction mixture.Suitable butyrylcholine esterase standard items starting reaction, reaction is then added
Condition be 25-37 DEG C, time 5-30min.
It is illustrated combined with specific embodiments below.
Soybean trypsin inhibitor used in following embodiment of the present invention is 10063 purchased from Mayan company's numbering,
Silica gel column chromatography is Xin Weier Products.Microplate reader is purchased from Molecular Devices company, model SpectraMax M5.
Fluorophore-part is according to document (Journal of Organic Chemistry, 78 (22), 11184- in structure shown in formula (1)
11193；2013) method recorded in is prepared.
The source of enzyme used in following embodiment and parameter are as follows:
Acetylcholinesterase is purchased from a-Aldrich (C1682) Rate activity >=1,500U/mg
Butyrylcholine esterase is purchased from Sigma-Aldrich (C1057) Rate activity >=900U/mg
Bovine serum albumin is 98% purchased from lark prestige scientific & technical corporation (109636) purity
Porcine pancreatic elastase is purchased from Mayan company (10095) Rate activity >=30U/mg
Trypsase is purchased from Mayan company (10020) Rate activity >=250U/mg
Human neutrophil elastase is purchased from Mayan company (E8140) Rate activity >=50U/mg
Butyrylcholine esterase is purchased from Mayan company (10001) Rate activity >=1500U/mg
Carboxypeptidase is purchased from Worthington (CLS005304) Rate activity >=170U/mg
Fluorescence intensity change rate in following embodiment (Δ F/ Δ t) is calculated according to following formula:
Wherein, F2Indicate t2The fluorescence intensity of moment sample, F1Indicate t1The fluorescence intensity of moment sample.
Ultravioletvisible absorption value change rate (Δ A/ Δ t) is calculated according to following formula:
Wherein, A2Indicate t2Ultravioletvisible absorption value of the moment sample at 325nm, A1Indicate t1Moment, sample was in 325nm
The ultravioletvisible absorption value at place.
Embodiment 1
The present embodiment is for illustrating that compound shown in formula provided by the invention (1) detects the activity of BuChE
Method.
1mg butyrylcholine esterase sample is dissolved in 100mM Tris-HCl (containing 10mM CaCl2, pH=7.0) and buffering
In liquid and gradient dilution is carried out, the butyrylcholine esterase standard solution of various concentration is prepared by the concentration provided in table 1, will be matched
The butyrylcholine esterase standard solution for setting acquisition, which is placed in 30 DEG C of thermostatic water baths, to be preheated；96 orifice plate of black flat-bottom is placed in simultaneously
It is preheated in 30 DEG C of incubators.Then, the butyrylcholine esterase standard solution for respectively taking 40 μ L warmed-up is in the black flat-bottom of preheating
In 96 orifice plates, 120 μ L 100mM Tris-HCl are added (containing 10mM CaCl2, pH=7.0) and buffer, and feminine gender is set
The control group buffer of 160 μ l (only plus), under the conditions of being protected from light, by the non-peptides probe of structure shown in 40 μ L formulas (1) respectively with
Butyrylcholine esterase standard items and control group Buffer fluid contacts, with microplate reader to each butyrylcholine esterase standard solution and control
Group solution fluorescence intensity after the contact under the exciting light of 325nm carries out kinetic scans, obtains the fluorescence in 5-10 minutes
As a result the change rate of intensity is listed in table 1.
Table 1
It can be seen that by the result of embodiment 1 and detect butyryl using non-polypeptide fluorescence probe described in the embodiment of the present invention
Cholinesterase activity can carry out the detection of simple and sensitive to the activity of the butyrylcholine esterase in sample, it is sensitive to improve detection
Degree and affinity.
Embodiment 2
The present embodiment is for illustrating using non-peptides fluorescence probe shown in formula (1) to elastoser, BuCh ester
The method that enzyme, trypsase, carboxypeptidase activity are measured.
Each butyrylcholine esterase sample is dissolved in 100mM Tris-HCl (containing 10mM CaCl respectively2, pH=7.0)
It in buffer and is diluted, is made into the enzyme solutions that concentration is 0.01mg/L；96 orifice plate of black flat-bottom 30 DEG C are placed in simultaneously to incubate
It educates in case and preheats.Then, the enzyme solutions for respectively taking 40 μ L warmed-up add 120 μ L in 96 orifice plate of black flat-bottom of preheating
100mM Tris-HCl (contains 10mM CaCl2, pH=7.0) and buffer, and negative control group is set and (only adds the buffering of 160 μ l
Liquid), under the conditions of being protected from light, the probe of structure shown in 40 μ L formulas (1) is contacted with enzyme solutions and control group respectively, with microplate reader pair
Enzyme standard solution and control group the solution fluorescence intensity after the contact under the exciting light of 325nm carry out kinetic scans, obtain
As a result the change rate of fluorescence intensity in 5-10 minutes is listed in table 2.
Table 2
Can be seen that by the result of embodiment 2 can be to a variety of butyryl gallbladders in method provided by the embodiment of the present invention
Alkali esterase carries out effective, quick and easy active testing, provided by the present invention to be detected using non-peptides fluorescence probe
Method, fluorescence intensity change is obvious, and the sensitivity of detection and affinity are high, and the variation of fluorescence intensity is more when as fluorescence probe
Obviously, the sensitivity of detection is higher.
Test case 1
This test case is used to illustrate the specificity of the activity of BuChE detection method provided by the invention.
Respectively by 1mg sample to be tested (acetylcholinesterase, butyrylcholine esterase, bovine serum albumin(BSA), elastoser,
Chymotrypsin, trypsase and carboxypeptidase) it is dissolved in 100mM Tris-HCl (containing 10mM CaCl2, pH=7.0) v buffer
Middle configuration concentration is placed in 30 DEG C of thermostatic water baths for the solution of the sample to be tested of 0.01mg/L and preheats；Simultaneously by black flat-bottom 96
Orifice plate is placed in 30 DEG C of incubators and preheats.Then, the solution for the sample to be tested for respectively taking 40 μ L warmed-up is in the black flat-bottom of preheating
In 96 orifice plates, 120 μ L 100mM Tris-HCl are added (containing 10mM CaCl2, pH=7.0) and buffer, it is being protected from light condition
Under, the non-peptides probe of structure shown in 10 μ L formulas (1) is contacted with each sample to be tested respectively, while being arranged and not adding sample to be tested
Control group, detected under the exciting light of 325nm with microplate reader various samples to be tested in 10 minutes be catalyzed it is non-shown in 10 μ L formulas (1)
The relative speed of peptides fluorescence probe hydrolysis, as a result as shown in Figure 5, it is seen then that the non-peptides fluorescence probe hydrolysis ability is obvious
Be higher than other kinds of enzyme, therefore, the non-peptides fluorescence probe based on ESIPT mechanism has the special of height to butyrylcholine esterase
One property.
Embodiment 3
The present embodiment is used to illustrate the screening technique of butyrylcholinesterase inhibitor according to the present invention.
1mg butyrylcholine esterase sample is dissolved in 100mM Tris-HCl (containing 10mM CaCl2, pH=7.0) and buffering
The butyrylcholine esterase standard solution to be measured that butyrylcholine esterase concentration is 0.05mg/mL is configured in liquid, by BuCh ester
Enzyme inhibitor tacrine is dissolved in 1mL 100mM Tris-HCl (containing 10mM CaCl2, pH=7.0) and it is configured in buffer
The inhibitor solution that concentration gradient is 0.1,1,4,7,10,20 μM dissolves the fluorescence probe of structure shown in 0.01mmol formula (1)
(contain 10mM CaCl in 10mL 100mM Tris-HCl2, pH=7.0) and it is configured to the fluorescence that concentration is 1mM in buffer visits
Needle solution, respectively will configuration obtain butyrylcholine esterase standard solution, butyrylcholinesterase inhibitor tacrine solution and
Fluorescence probe solution is placed in 30 DEG C of thermostatic water baths and preheats；96 orifice plate of black flat-bottom is placed in 30 DEG C of incubators simultaneously and is preheated.
Then, respectively by the butyrylcholinesterase inhibitor tacrine solution of 40 each concentration gradients of μ L, 100 μ L 100mM Tris-HCl
(contain 10mM CaCl2, pH=7.0) and buffer and 40 μ l fluorescence probes (final concentration of 5 μM) solution is added to black flat-bottom 96
It is mixed into experimental group in orifice plate, and sets up 140 μ L 100mM Tris-HCl (containing 10mM CaCl2, pH=7.0) buffer and
The control group of 40 μ L fluorescence probe solution mixing is added rapidly in experimental group and control group with the volley of rifle fire respectively under the conditions of being protected from light
Enter the starting reaction of 20 μ L butyrylcholine esterase standard solutions.Finally with microplate reader respectively to experimental group and control group fluorescence intensity
Variation and the variation of ultravioletvisible absorption value detected, be as a result listed in table 3.
Table 3
It can be seen that non-peptides fluorescence probe provided by the present invention can be used in BuCh by the result of embodiment 3
The screening of esterase inhibitor.Also, it is noted that the screening of butyrylcholinesterase inhibitor provided in an embodiment of the present invention
Method, the larger progress for being easy to detect of the variation range of fluorescence signal, and the UV, visible light of polypeptide Substrate fluorescence probe is inhaled
The change rate of receipts value small (need to be accurate to 2 significant digits number) is not easy to the progress of detection, provided by the embodiment of the present invention
Non- peptides fluorescence probe based on ESIPT mechanism has higher Atom economy.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (10)
1. a kind of for detecting the fluorescence probe of the activity of BuChE, which is characterized in that the fluorescence probe is non-peptides
Fluorescence probe, and the structure of the non-peptides fluorescence probe is as shown in following formula 1:
2. as described in claim 1 for detecting the fluorescence probe of the activity of BuChE, which is characterized in that described non-peptide
Class fluorescence probe for when detecting butyrylcholine esterase, the benzoyl of the non-peptides fluorescence probe as recognition group, with
The butyrylcholine esterase combines.
3. as described in claim 1 for detecting the fluorescence probe of the activity of BuChE, which is characterized in that described non-peptide
Class fluorescence probe for when detecting butyrylcholine esterase, the butyrylcholine esterase and the non-peptides fluorescence probe mole with
Amount is than being 1 μM: (1-100) μM.
4. a kind of kit for detecting the activity of BuChE, which is characterized in that including as described in claim any one of 1-3
The standard items of non-peptides fluorescence probe and butyrylcholine esterase.
5. the kit of detection the activity of BuChE as claimed in claim 4, which is characterized in that further include that concentration is
The Tris-HCl buffer solution of 50-100mM.
6. a kind of application method of the kit of detection the activity of BuChE as claimed in claim 4, which is characterized in that
The following steps are included:
Butyrylcholine esterase standard items, butyrylcholine esterase sample to be tested and non-peptides fluorescence probe are provided；
By the butyrylcholine esterase standard items and the non-peptides fluorescence probe hybrid reaction, fluorogen reaction product is obtained；
The fluorescence intensity change value of reaction product under detection excitation light stimulus changes with time value, and acquisition butyrylcholine esterase-is glimmering
Intensity variation value standard curve obtains butyrylcholine esterase-fluorescence intensity change value linear equation；
Under testing conditions identical with the butyrylcholine esterase standard items, by the butyrylcholine esterase sample to be tested and institute
Non- peptides fluorescence probe hybrid reaction is stated, fluorogen reaction product is obtained；The fluorescence of reaction product under detection excitation light stimulus
Strength Changes value substitutes into the butyrylcholine esterase-fluorescence intensity change value linear equation, calculates and obtains the BuCh
The concentration of esterase sample to be tested.
7. the application method of the kit of detection the activity of BuChE as claimed in claim 6, which is characterized in that described
The wavelength of exciting light is 325nm, and the launch wavelength of the fluorescence of the fluorogen reaction product under the excitation light stimulus is
460nm。
8. the application method of the kit of detection the activity of BuChE as claimed in claim 6, which is characterized in that described
The condition of hybrid reaction are as follows: temperature is 25-37 DEG C, pH value 6-8, time 5-30min.
9. the application method of the kit of detection the activity of BuChE as claimed in claim 6, which is characterized in that by institute
State butyrylcholine esterase standard items or the butyrylcholine esterase sample to be tested and the non-peptides fluorescence probe hybrid reaction
Step carries out in the Tris-HCl buffer that concentration is 50-100mM.
10. a kind of peptides fluorescence probe non-as described in claim any one of 1-3 is in the application of butyrylcholine esterase detection field,
It is characterized in that, the application includes the fluorescence probe for detecting the activity of BuChE, the fluorescence probe is used for
Screen butyrylcholinesterase inhibitor or promotor, the fluorescence probe is dynamic for butyrylcholinesterase inhibitor or promotor
The application in mechanics characterization field.
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| CN111592504A (en) * | 2020-06-12 | 2020-08-28 | 青岛科技大学 | Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof |
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| CN111592504A (en) * | 2020-06-12 | 2020-08-28 | 青岛科技大学 | Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof |
| CN111592504B (en) * | 2020-06-12 | 2022-04-19 | 青岛科技大学 | Fluorescent probe for detecting butyrylcholine esterase activity and synthetic method and application thereof |
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