CN108192597B - Half cyanines class fluorescence probe of near-infrared and its preparation method and application for detecting butyrylcholine esterase - Google Patents
Half cyanines class fluorescence probe of near-infrared and its preparation method and application for detecting butyrylcholine esterase Download PDFInfo
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Abstract
The present invention relates to the detection fields of butyrylcholine esterase, disclose a kind of half cyanines class fluorescence probe of near-infrared and its preparation method and application for detecting butyrylcholine esterase, specifically, the half cyanines class fluorescence probe of near-infrared has structure shown in formula (1).The method includes in organic solvent, formula (2) compound represented being contacted with Cyclopropyl carbonyl chloride in the presence of necleophilic reaction condition and acid binding agent.Using include: detection butyrylcholine esterase activity in application, a kind of active detection method and detection kit of butyrylcholine esterase.Half cyanines class probe compound of near-infrared of the invention is for related with carboxypeptidase y Activity determination in application, high sensitivity, good, fluorescence intensity the variation of selectivity are easy to detect greatly, and stability is higher, using more convenient, price is less expensive.
Description
Technical field
The present invention relates to the detection fields of butyrylcholine esterase, and in particular, to a kind of half cyanines class fluorescence probe of near-infrared,
The preparation method of the half cyanines class fluorescence probe of near-infrared and application and a kind of fourth in the activity of detection butyrylcholine esterase
The active detection method of acetylcholinesterase and the detection kit of the activity of BuChE.
Background technique
Butyrylcholine esterase (BChE) is mainly present in organism with a kind of isodynamic enzyme of acetylcholinesterase, it be by
A kind of nonspecific esterase of liver synthesis, is immediately released into blood plasma, in serum after being synthesized in liver due to the enzyme
BChE activity is the sensitive indexes for measuring Hepatocyte matter synthesis function.With horizontal continuous of detection technique and clinic diagnosis
It improves, butyrylcholine esterase is in addition to extending also to malignant tumour, nervous system disease, the heart for assessing liver reserve function
In the detection and diagnosis field of numerous diseases such as vascular diseases, metabolic syndrome.In addition, measuring serum choline in clinic
Esterase active is also to assist diagnosis organic phosphorus compound (such as organic phostoxin gas, organophosphorus pesticide) and cocaine poisoning is important
Approach.
There are mainly three types of the methods of existing measurement the activity of BuChE.The first is to use ultraviolet absorption spectroscopy (i.e.
Ellman method) cholinesterase activity in sample to be tested is tested, mainly by with acetylthiocholine or thio butyryl
Choline generates yellow as substrate, thiocholine generated and Ellman reagent (5,5- bis- thiobis -2- nitrobenzoic acid)
Substance.Second is the cholinesterase activity tested in sample to be tested with fluorescent spectrometry, mainly by with thioacetyl
Choline or thio BuCh carry out the activity of indirect monitoring enzyme using the fluorescence probe of identification thiocholine as substrate.The
Three kinds directly with the natural substrate acetylcholine or BuCh of isotope labelling, the directly cholinesterase in measurement sample to be tested
Activity.
In above method, before two kinds belong to indirect method, be easy to cause error；Although the third method is direct side
Method, but operating process is complex, and is required to expensive large-scale instrument and is just able to achieve with testing cost, also causes to safety
Very big pressure.Moreover, above-mentioned three kinds of methods cannot distinguish between butyrylcholine esterase and acetylcholinesterase, because being surveyed
The activity obtained is the gross activity of all cholinesterases.
Therefore, it designs and develops based on small organic molecule as direct substrate type fluorescence probe, and then establishes sensitive, simple
Single, economic specific the activity of BuChE detection method is very urgent and necessary.However so far, both at home and abroad
It is less to be used to detect butyrylcholine esterase (No. EC: 3.1.1.8) active report about specific substrate type fluorescence probe.
Summary of the invention
It is an object of the invention to overcome drawbacks described above in the prior art, provide it is a kind of it is highly selective, highly sensitive,
Property is stable, it is easy to operate, be easy to storage and transportation and low-cost non-peptide substrates type butyrylcholine esterase detection fluorescence probe and its
Preparation method and application.
In order to achieve the above objectives, in a first aspect, the present invention provides a kind of half cyanines class fluorescence probe of near-infrared, feature exists
In the half cyanines class fluorescence probe of near-infrared has structure shown in formula (1):
Second aspect, the present invention provides the preparation method of above-mentioned half cyanines class fluorescence probe of near-infrared, this method is included in
In the presence of necleophilic reaction condition and acid binding agent, in organic solvent, structural compounds shown in formula (2) are connect with Cyclopropyl carbonyl chloride
Touching；
The third aspect, the present invention provide above-mentioned half cyanines class fluorescence probe of near-infrared in the activity of detection butyrylcholine esterase
Application.
Fourth aspect, the present invention provides a kind of active detection methods of butyrylcholine esterase, wherein this method packet
It includes:
Sample to be tested is contacted with half cyanines class fluorescence probe of near-infrared as described above, so that the butyryl gallbladder in sample to be tested
Alkali esterase identified by the cyclopropane carbonyl in the infrared half cyanines class fluorescence probe, the material HCY after being contacted；
The value added of the fluorescence intensity that material HCY after detection contact is issued under excitation light at any time, the fluorescence are strong
The value added of degree at any time indicates the activity of butyrylcholine esterase in sample to be tested；The wavelength of the exciting light is 450-
750nm, the launch wavelength of the fluorescence are 680-800nm.
5th aspect, the present invention provide a kind of detection kit of the activity of BuChE, which includes:
Half cyanines class fluorescence probe of near-infrared as described above, butyrylcholine esterase standard items and reaction buffer.
Half cyanines class fluorescence probe of near-infrared of the invention is when being used to detect the activity of BuChE, with BuCh ester
Enzyme has higher affinity, and the validity of detection is higher.In the activity for detecting butyrylcholine esterase, with use polypeptide
Class fluorescence probe is compared, and has higher sensitivity and selectivity；And synthetic method is simple and quick, fluorescence background value is low.Cause
This, half cyanines class probe compound fluorescence probe of near-infrared provided by the invention is used to detect with the activity of BuChE related
In application, high sensitivity, good, fluorescence intensity the variation of selectivity are big, it is easy to detect, and half cyanines class of near-infrared of the invention is visited
Needle compound is the substrate type fluorescence probe of non-peptides, non-peptides fluorescence probe is higher compared to peptides fluorescence probe stability,
Using more convenient, price is less expensive.Substrate type fluorescence probe is lower compared to suppressive fluorescence probe background values, to cytotoxicity
It is lower.
Other features and advantages of the present invention will the following detailed description will be given in the detailed implementation section.
Detailed description of the invention
The drawings are intended to provide a further understanding of the invention, and constitutes part of specification, with following tool
Body embodiment is used to explain the present invention together, but is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 reflects the near infrared fluorescent probe of structure shown in formula (1) and butyrylcholine esterase acts on front and back in significant wave
Fluorescence intensity change under section.
Fig. 2 is specificity schematic diagram of the half cyanines class fluorescence probe of near-infrared to butyrylcholine esterase of structure shown in formula (1).
Specific embodiment
Detailed description of the preferred embodiments below.It should be understood that described herein specific
Embodiment is merely to illustrate and explain the present invention, and is not intended to restrict the invention.
The endpoint of disclosed range and any value are not limited to the accurate range or value herein, these ranges or
Value should be understood as comprising the value close to these ranges or value.For numberical range, between the endpoint value of each range, respectively
It can be combined with each other between the endpoint value of a range and individual point value, and individually between point value and obtain one or more
New numberical range, these numberical ranges should be considered as specific open herein.
On the one hand, the present invention provides a kind of half cyanines class fluorescence probe of near-infrared, the half cyanines class fluorescence probe of near-infrared tools
There is structure shown in formula (1)；
Wherein, in the half cyanines class fluorescence probe of near-infrared shown in formula (1), using cyclopropane carbonyl as butyrylcholine esterase
Recognition group.
Second aspect, the preparation method the present invention also provides half cyanines class fluorescence probe of near-infrared as described above include:
Wherein, in the presence of necleophilic reaction condition and acid binding agent, in organic solvent, by structural compounds and cyclopropyl shown in formula (2)
Formyl chloride contact；
In the preparation method of half cyanines class fluorescence probe of near-infrared of the present invention, the necleophilic reaction is in formula (2)
Hydroxyl reacted with what Cyclopropyl carbonyl chloride occurred, the condition of the necleophilic reaction include temperature be subzero 5 DEG C to 30 DEG C, the time
For 10-200min, under preferable case, the condition of the necleophilic reaction is that temperature is 0-25 DEG C, time 80-160min.
In the preparation method of half cyanines class fluorescence probe of near-infrared of the present invention, the contact needs to exist in acid binding agent
Under conditions of carry out, the acid binding agent can be any acid that can be generated in absorbing reaction commonly used in the art, avoid contact with
The acid generated in reaction influences the substance of reaction or reaction balance, it is preferable that the acid binding agent is selected from triethylamine, diethylamine, two
Isopropylamine, pyridine, N, N- dimethylamino naphthyridine, 2,6- lutidines, potassium carbonate, cesium carbonate, sodium carbonate and sodium bicarbonate
At least one of, most preferably, the acid binding agent is triethylamine.
In order to improve the effect of haptoreaction mass transfer and heat transfer, haptoreaction of the present invention in organic solvent environment into
Row, the organic solvent can not react to be any under contact conditions of the present invention with reactant of the invention
And the organic solvent of suitable solution environmental can be provided for reaction, the boiling point of the organic solvent is preferably 30-150 DEG C, more
Preferably, the organic solvent be methylene chloride, tetrahydrofuran, chloroform, carbon tetrachloride, acetonitrile, acetone, glycol dimethyl ether,
N,N-Dimethylformamide and 1, at least one of 2- dichloroethanes, most preferably, the organic solvent is methylene chloride.
In the preparation method of half cyanines class fluorescence probe of near-infrared of the invention, tied relative to shown in 1 mM of formula (2)
Structure compound, the dosage of institute's Cyclopropyl carbonyl chloride are 1-2 mMs；The dosage of the acid binding agent is not particularly limited, in order to
The assimilation effect of the acid generated to reaction reached, the dosage of the acid binding agent can be 1-3 mMs, under preferable case,
The dosage of the acid binding agent is 2-3 mMs；Dosage for organic solvent of the invention is also not particularly limited, preferably feelings
Under condition, the dosage of the organic solvent is 20-30 milliliters.
It, can be right with thin-layer chromatography (TLC) in the preparation method of half cyanines class fluorescence probe of near-infrared of the present invention
The carry out situation of reaction is monitored, and when testing result shows that raw material point disappears, shows that reaction terminates.
Preparation method of the invention further includes purifying to catalytic product, and the process of purification may include in 0-
Successively reaction system is washed with 20-60ml water and 20-60ml saturated common salt aqueous solution at a temperature of 25 DEG C, stratification
Separation organic phase is simultaneously dried organic phase with anhydrous sodium sulfate, removes solvent by decompression method and obtains crude product, finally
Chromatography is carried out to crude product with silicagel column and obtains product.
The third aspect, the present invention also provides half cyanines class fluorescence probes of near-infrared as described above in detection BuCh ester
Application in the activity of enzyme.
The application can be related to the application of the activity of BuChE detection for this field is any, for example, the application
Can include but is not limited to: the detection of the activity of BuChE, butyrylcholinesterase inhibitor screening, butyrylcholine esterase promote
It is screened into agent, butyrylcholinesterase inhibitor or the Kinetic Characterization of promotor etc..Application of the present invention simultaneously also can be used
In the diagnosing and treating for detecting related disease with the activity of BuChE, but the present invention is not limited to this.
Preferably, the present invention provides half cyanines class fluorescence probes of near-infrared as described above to prepare for detecting butyryl gallbladder
Application in the active product of alkali esterase.
Based on application as above, fourth aspect, the present invention also provides a kind of detection method of the activity of BuChE,
This method comprises:
Sample to be tested containing butyrylcholine esterase is contacted with half cyanines class fluorescence probe of near-infrared as described above, so that
Butyrylcholine esterase in sample to be tested is identified by the cyclopropane carbonyl in the infrared half cyanines class fluorescence probe, is contacted
Material HCY afterwards；
The value added of the fluorescence intensity that material HCY after detection contact is issued under excitation light at any time, the fluorescence are strong
The value added of degree at any time indicates the activity of butyrylcholine esterase in sample to be tested；The wavelength of the exciting light is 450-
750nm, the launch wavelength of the fluorescence are 680-800nm.
In the present invention, term " value added at any time " refers within the scope of minute, the value added of fluorescence intensity with
The ratio of time, that is, fluorescence intensity advancing the speed at any time.
In the detection method of the activity of BuChE of the present invention, the dosage of half cyanines class fluorescence probe of near-infrared
Range is related with the concentration of butyrylcholine esterase sample to be measured, it is generally the case that the dosage energy of half cyanines class fluorescence probe of near-infrared
The activity for enough reaching characterization butyrylcholine esterase sample to be tested, under preferable case, relative to 1 μ g/mL every in sample to be tested
Butyrylcholine esterase, the dosage of the half cyanines class fluorescence probe of near-infrared can be 0.05-100 μM, optimal in order to reach
The dosage of detection effect, the half cyanines class fluorescence probe of near-infrared is preferably 2-10 μM.
In the detection method of the activity of BuChE provided by the invention, the butyrylcholine esterase sample to be tested with
The condition of half cyanines class fluorescence probe of near-infrared contact includes: that temperature is 25-37 DEG C, pH value 5-9, time 10-30min.It is excellent
In the case of choosing, the condition of the contact includes: that temperature is 25-30 DEG C, pH value 7-8, time 10-25min.
In detection method of the present invention, butyrylcholine esterase sample to be measured and probe (half cyanines class fluorescence of near-infrared
Probe) mixing is preferably contacted under the conditions of being protected from light, and before contact mixing, buffer and container preferably used to mixing
It is preheated, reaction vessel can be black plate, cuvette or ELISA Plate, the preferred cuvette of reaction vessel.Preheating to be reached
Temperature be preferably 25-37 DEG C.
The condition of the detection method of the activity of BuChE of the present invention further includes in 5-100mM phosphate buffer
(pH=7.4) it is carried out in.
The fluorescence of material after contacting to the butyrylcholine esterase sample to be tested with half cyanines class fluorescence probe of near-infrared is strong
The detection of degree value added at any time can carry out in microplate reader or sepectrophotofluorometer, can be used measurement sample to be tested with
Fluorescence intensity changes with time value after the mixing of half cyanines class probe compound of near-infrared, to obtain the enhancement value.Furthermore, it is possible to
Guarantee the reliability of detection using multiplicating test and by the way of several control groups are arranged.
When the half cyanines class fluorescence probe of near-infrared is structure shown in formula (1), method of the present invention can be used for
The determination of activity of special butyrylcholine esterase, wherein when in sample to be tested contain elastoser, trypsase, pancreas curdled milk
When the interference of protease and carboxypeptidase, probe molecule shown in formula (1) provided by the present invention can be unaffected for measuring
The activity of the butyrylcholine esterase of the known concentration contained in sample；Especially when containing acetylcholine ester in sample to be tested simultaneously
When enzyme and butyrylcholine esterase, the differentiation and detection of isodynamic enzyme can be carried out with formula (1) compound；By to fourth in sample to be tested
The kinetic test of acetylcholinesterase can obtain the Michaelis constant of compound hydrolysis shown in butyrylcholine esterase catalysis type (1),
To the activity level of butyrylcholine esterase contained in judgement sample；In addition, chemical combination shown in formula (1) provided by the invention
The content of butyrylcholine esterase that object also can be applied to while contain in test sample, thus to butyryl is contained in sample
The activity level of cholinesterase carries out quantitative analysis.
When the detection method of the activity of BuChE of the present invention is used for butyrylcholinesterase inhibitor or promotion
When the screening of agent, can carry out by the following method, respectively by certain density butyrylcholine esterase, certain density inhibitor or
Promotor is dissolved in above-mentioned buffer, is placed in thermostatic water bath and is preheated to preference temperature (25-37 DEG C)；Reaction is held simultaneously
Device, which is placed in incubator, is preheated to preference temperature (25-37 DEG C).Then, in the reaction vessel by suitable buffering having been warmed up
Solution, a certain amount of butyrylcholinesterase inhibitor or promotor and suitable half cyanines class fluorescence probe of near-infrared as described above
Mixing.Butyrylcholine esterase starting reaction is added under the conditions of being protected from light, and glimmering to sample with microplate reader or sepectrophotofluorometer
The situation of change of luminous intensity is monitored.
5th aspect, the present invention also provides a kind of detection kit of the activity of BuChE, the detection kits
Including half cyanines class fluorescence probe of near-infrared, butyrylcholine esterase standard items and reaction buffer as described above.Wherein, described
Butyrylcholine esterase standard items may serve as control group when detecting or draw standard curve.The reaction buffer can be
Buffer as described above.
Kit provided by the present invention can be applied to detection sample to be tested in butyrylcholine esterase activity (including sieve
Butyrylcholinesterase inhibitor or promotor and the detection of other and the activity of BuChE or butyrylcholine esterase is selected to inhibit
Agent or the relevant field of accelerator function).In addition, this kit can also be used for detecting related doctor with the activity of BuChE
Detection and therapy field are learned, but the present invention is not limited to this.
In kit provided by the present invention, reaction needs to carry out in reaction buffer, it is preferable that reaction buffer is
5-100mM phosphate buffer (pH=7.4), the preservation condition of this kit are to be kept in dark place at subzero 20 DEG C to 4 DEG C.The examination
The specifically used method of agent box can be carried out according to the method for act set forth below:
(1) DMSO (dimethyl sulfoxide) solution of half cyanines class fluorescence probe of 20mM near-infrared is configured, and at subzero 20 DEG C
Or be kept in dark place at 4 DEG C, when use, carries out gradient dilution.
(2) the butyrylcholine esterase standard items phosphate buffer (pH=7.0) of 10mM is dissolved, is configured to 100U/ml
Butyrylcholine esterase standard solution, stored at subzero 20 DEG C or so, gradient dilution carried out with 10 times of dilution when use.
(3) phosphate buffer (pH=7.0) of the 10mM of 100 μ l of the sample to be tested containing butyrylcholine esterase is buffered
Liquid dissolution.
(4) respectively by the phosphate buffer (pH=7.0) of the 10mM of 0.2ml, half cyanines class fluorescence probe of near-infrared, to be measured
Butyrylcholine esterase sample and butyrylcholine esterase standard items are incorporated in black ELISA Plate.Utilize butyryl gallbladder of the present invention
The activity of butyrylcholine esterase in the detection method detection sample to be tested and butyrylcholine esterase standard items of alkali esterase.
(5) standard song is drawn with the fluorescence intensity change value of the butyrylcholine esterase standard items of the gradient dilution detected
Line determines the activity of butyrylcholine esterase in detected sample by comparing.
It, can be by the inhibition when kit of the invention is used for the screening of butyrylcholinesterase inhibitor or promotor
Agent is before detection in appropriate proportions or viscosity makes an addition in reaction mixture.Suitable butyrylcholine esterase standard items are then added
Starting reaction, the condition of reaction are 25-37 DEG C, pH value 5-9, time 10-30min.
The present invention will be described in detail by way of examples below.Iodate thioacetyl gallbladder used in following embodiment
Alkali is the commercially available product for being A5751-1G purchased from Sigma-alderich Reagent Company product number.Soybean trypsin inhibitor purchase
It is 10063 from Mayan company's numbering.Microplate reader is purchased from Molecular Devices company, model SpectraMax M5.
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；
Trypsase is purchased from Mayan company (10020) Rate activity >=250U/mg；
Human neutrophil elastase is purchased from Mayan company (E8140) Rate activity >=50U/mg；
Carboxypeptidase A is purchased from Worthington (CLS005304) Rate activity >=170U/mg；
Protaminase is purchased from Sigma-Aldrich Rate activity >=50U/mg；
Elastoser is purchased from Mayan company Rate activity >=30U/mg；
Chymotrypsin is purchased from Mayan company Rate activity >=1500U/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 t2The ultravioletvisible absorption value of moment sample, A1Indicate t1Moment sample is ultraviolet at 412nm
Visible absorbance value.
Preparation example 1
The present embodiment is used to illustrate the preparation method of half cyanines class fluorescence probe of near-infrared of the present invention.
It, will be shown in formula (2) in the presence of acid binding agent (triethylamine, 2mmol) in organic solvent (methylene chloride, 20mL)
Compound (1mmol) and Cyclopropyl carbonyl chloride (2mmol) at 5 DEG C, maintain the contact of 120min, the object after being contacted
Material.Then the material after successively washing contact with water (50mL) and saturated common salt aqueous solution (50mL), the material after being washed.
The organic phase in material after separating, washing is simultaneously dried with anhydrous sodium sulfate, and decompression removal solvent obtains crude product, will be thick
Product obtains white solid product 0.27g through silica gel column purification.
1H NMR(400MHz,DMSO-d6): δ 8.95 (d, J=4.0Hz, 1H), 8.02 (s, 1H), 7.87 (s, 1H), 7.42
(d, J=8.0Hz, 2H), 7.25 (t, J=6.0Hz, 1H), 6.81 (s, 1H), 6.53 (s, 1H), 6.43 (d, J=8.0Hz,
1H), 5.12 (d, J=8.0Hz, 1H), 4.01 (q, J=6.0Hz, 2H), 2.73 (m, 4H), 1.41 (s, 1H), 1.25 (m,
11H),1.12(m,4H).HRMS calcd for[C31H32NO3]+:466.5841.Found:466.5425.。
Confirm that the white solid is structure shown in formula (1) with NMR and HRMS detection characterization.
Test case 1
This test case is used to illustrate that compound shown in the formula (1) of offer of the invention and butyrylcholine esterase to act on front and back
Fluorescence intensity change.
1mg butyrylcholine esterase sample is dissolved in the butyryl that 10mM phosphate buffer (pH=7.0) prepares 1mg/L concentration
The butyrylcholine esterase standard solution that configuration obtains is placed in 30 DEG C of thermostatic water baths and preheats by cholinesterase standard solution；
96 orifice plate of black flat-bottom is placed in 30 DEG C of incubators simultaneously and is preheated.Then, the butyrylcholine esterase standard for taking 40 μ L warmed-up
Product solution adds 120 μ L 10mM phosphate buffers (pH=7.4) in 96 orifice plate of black flat-bottom of preheating, and feminine gender is arranged
The control group buffer of 160 μ l (only plus), under the conditions of being protected from light, structure shown in formula (1) prepared by 40 μ L, 10 μM of preparation examples 1
Infrared half cyanines class fluorescence probe respectively with butyrylcholine esterase standard items and control group Buffer fluid contacts 5min.With microplate reader pair
Butyrylcholine esterase standard solution and control group the solution fluorescence intensity after the contact under 665nm exciting light carry out dynamics and sweep
It retouches, obtains the fluorescence intensity change of compound shown in formula (1) and butyrylcholine esterase effect front and back, the result is shown in Figure 1.
As seen from Figure 1, the infrared half cyanines class fluorescence probe of structure shown in formula (1) provided by the invention is substrate fourth
There is the launch wavelength of large change, thus, it is easy to detect after acetylcholinesterase effect.
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 10mM phosphate buffer (pH=7.0) and is carried out gradient dilution, is pressed
The concentration provided in table 1 prepares the butyrylcholine esterase standard solution of various concentration, the butyrylcholine esterase that configuration is obtained
Standard 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.With
Afterwards, the butyrylcholine esterase standard solution for respectively taking 40 μ L warmed-up adds 120 μ L in 96 orifice plate of black flat-bottom of preheating
10mM phosphate buffer (pH=7.4), and negative control group (only plus buffer of 160 μ l) is set, under the conditions of being protected from light, by 40
10 μM of preparation examples 1 of μ L preparation formula (1) shown in structure infrared half cyanines class fluorescence probe respectively with butyrylcholine esterase standard items
And control group Buffer fluid contacts.Each butyrylcholine esterase standard solution and control group solution are swashed in 665nm with microplate reader
Fluorescence intensity carries out kinetic scans at 705nm after contact under shining, obtains the change of the fluorescence intensity in 5-10 minutes
As a result rate is listed in table 1.
Comparative example 1
The comparative example is used to illustrate the detection method of the activity of BuChE in the prior art.
Using the substrate water of method detection butyrylcholine esterase standard items catalysis series of concentrations gradient same as Example 1
The case where solution, unlike, substrate probe used is that acetylthiocholine iodide (is purchased from Sigma-Aldrich company, product
Number is A5751-1G), and after excessive DTNB (50 μM, 5,5'- bis- thiobis (2- nitrobenzoic acid)) is added, in wavelength
For docking at 412nm, ultravioletvisible absorption value carries out kinetic scans after touch, obtains the ultravioletvisible absorption in 5-10 minutes
As a result the change rate of value is listed in table 1.
Table 1
It can be seen that by the result of embodiment 1 using method provided by this law, it can be to the elastin laminin in sample
The activity of enzyme carries out the detection of simple and sensitive, and can be seen that through the invention by the result of embodiment 1 and comparative example 1
The method of offer, formula (1) compound represented fluorescence probe and existing iodate used in method provided by the present invention
Acetylthiocholine probe, which is compared, has higher sensitivity and affinity.
Embodiment 2
The present embodiment utilizes the non-infrared half cyanines class fluorescence probe pair of peptides shown in formula provided by the invention (1) for illustrating
The method that the activity of BuChE is measured in sample containing butyrylcholine esterase.
Respectively by butyrylcholine esterase and sample containing butyrylcholine esterase (respectively containing butyrylcholine esterase
Elastin laminin enzyme sample, trypsin sample, protaminase sample and chymotrypsin-like product, wherein butyrylcholine esterase
Weight ratio with other each enzymes is 1:199) it is dissolved in 10mM phosphate buffer (pH=7.4) buffer and is diluted, match
The enzyme solutions (total enzyme concentration) for being 10mg/L at concentration；96 orifice plate of black flat-bottom is placed in 30 DEG C of incubators simultaneously and is preheated.With
Afterwards, the enzyme solutions for respectively taking 40 μ L warmed-up add 120 μ L 10mM phosphate buffers in 96 orifice plate of black flat-bottom of preheating
(pH=7.4) buffer, and negative control group (only plus buffer of 160 μ l) is set, under the conditions of being protected from light, by 40 μ L formulas (1)
The probe of shown structure is contacted with enzyme solutions and control group respectively, is existed with microplate reader to enzyme standard solution and control group solution
Fluorescence intensity carries out kinetic scans at 705nm after contact under the exciting light of 665nm, obtains the fluorescence in 5-10 minutes
As a result the change rate of intensity is listed in table 2.
Comparative example 2
This comparative example is for illustrating using existing acetylthiocholine iodide probe to the sample containing butyrylcholine esterase
The method that the activity of BuChE is measured in product.
(respectively using the sample of method same as Example 2 to butyrylcholine esterase and containing butyrylcholine esterase
For the elastin laminin enzyme sample containing butyrylcholine esterase, trypsin sample, protaminase sample and chymotrypsin-like
Product) in the activity of BuChE be measured, unlike, used substrate probe be acetylthiocholine iodide (purchase
From Sigma-Aldrich company, product number A5751-1G), and after excessive DTNB (50 μM) is added, it is in wavelength
Ultravioletvisible absorption value carries out kinetic scans after touch for docking at 412nm, obtains the ultravioletvisible absorption value in 5-10 minutes
Change rate, be as a result listed in table 2.
Table 2
It can be seen that compared with prior art by the result of embodiment 2 and comparative example 2, method provided by the present invention
In can the butyrylcholine esterase to a variety of sources carry out effective, quick and easy active testing, and with prior art institute
The method used is compared, and the fluorescence intensity change of method provided by the present invention becomes apparent from, and the sensitivity of detection and affinity are more
It is high.
Test case 2
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, elastoser, trypsase, chymotrypsin, carboxylic peptide
Enzyme A, protaminase, bovine serum albumin(BSA), butyrylcholine esterase) it is dissolved in 10mM phosphate buffer (pH=7.0) buffer and matches
The solution for setting the sample to be tested that concentration is 100mg/L, which is placed in 30 DEG C of thermostatic water baths, to be preheated；Simultaneously by 96 orifice plate of black flat-bottom
It 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 96 hole of black flat-bottom of preheating
In plate, 120 μ L 10mM phosphate buffer (pH=7.4) buffers are added, under the conditions of being protected from light, will be tied shown in 40 μ L formulas (1)
The half cyanines class fluorescence probe of near-infrared of structure is contacted with each sample to be tested respectively, while the control group for not adding sample to be tested is arranged,
Various samples to be tested in 10 minutes are detected under the exciting light of 665nm with microplate reader and are catalyzed compound hydrolysis shown in 40 μ L formulas (1)
Relative speed, as a result as shown in Figure 2.
It can be seen that butyrylcholine esterase to used in method provided by the present invention by the result of test case 2
The hydrolysis ability of half cyanines class fluorescence probe of near-infrared shown in formula (1) is significantly higher than other kinds of enzyme, especially significantly high
In isodynamic enzyme acetylcholinesterase.Therefore, half cyanines class fluorescence probe used in method of the invention is very high to the specificity of enzyme,
It can be realized the specific detection of butyrylcholine esterase.
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 configuration concentration in 10mM phosphoric acid acid buffer (pH=7.0) buffer
For the butyrylcholine esterase standard solution to be measured of 1.5mg/mL, inhibitor Tacrine (tacrine) is dissolved in 1mL 10mM
Be configured in phosphoric acid acid buffer (pH=7.0) buffer concentration gradient be 0.004,0.008,0.016,0.032,0.04,
The fluorescence probe of structure shown in 20mmol formula (1) is dissolved in 2mL 10mM phosphoric acid acid buffer by 0.08 μM of inhibitor solution
(pH=7.0) the fluorescence probe solution that concentration is 0.2mM, the butyrylcholine esterase for respectively obtaining configuration are configured in buffer
Standard solution, Tacrine and fluorescence probe solution are placed in 30 DEG C of thermostatic water baths and preheat；96 orifice plate of black flat-bottom is set simultaneously
It is preheated in 30 DEG C of incubators.Then, respectively by the Tacrine solution of 50 each concentration gradients of μ L, 90 μ L 10mM phosphoric acid acid bufferings
Liquid (pH=7.4) buffer and 10 μ l fluorescence probes (final concentration of 10 μM) solution are added in 96 orifice plate of black flat-bottom and are mixed into
Experimental group, and set up pair of 140 μ L 10mM phosphoric acid acid buffer (pH=7.4) buffers and the mixing of 10 μ L fluorescence probe solution
According to group, under the conditions of being protected from light, 50 μ L butyrylcholine esterase standard items are rapidly added with the volley of rifle fire in experimental group and control group respectively
Solution starting reaction.With microplate reader, to each group solution, fluorescence intensity is carried out at 705nm after the contact under the exciting light of 665nm
Kinetic scans obtain the change rate of the fluorescence intensity in 5-10 minutes, are as a result listed in table 3.
Comparative example 3
This comparative example is used to illustrate the screening technique of the butyrylcholinesterase inhibitor using the prior art.
The screening of butyrylcholinesterase inhibitor is carried out using method same as Example 3, unlike, fluorescence is visited
Needle solution is changed to the acetylthiocholine iodide (CAS:1866-15-5) of Sigma-Aldrich Reagent Company, and excessive being added
DTNB reagent (50 μM) after, be that ultravioletvisible absorption value carries out kinetic scans, acquisition the to the docking of the place 412nm after touch in wavelength
The change rate of ultravioletvisible absorption value in 5-10 minutes, is as a result listed in table 3.
Table 3
It can be seen that method provided by the present invention can be used in BuCh by the result of embodiment 3 and comparative example 3
The screening of esterase inhibitor.Also, it is noted that the screening technique of butyrylcholinesterase inhibitor provided by the invention with
It is compared using acetylthiocholine iodide probe, the larger progress for being easy to detect of the variation range of fluorescence signal.Therefore, of the invention
Provided direct detecting method has higher sensitivity.
In addition, half cyanines class fluorescence probe of near-infrared provided by the present invention is BuCh for the accuracy of method
The substrate type probe of esterase can directly be reflected the activity of enzyme after enzyme hydrolysis；And the side Ellman used in comparative example 1-3
Method (making substrate using the acetylthiocholine iodide that Sigma-Aldrich Reagent Company provides) then needs that coupling reagent is added
(DTNB) determination of activity of enzyme can just be carried out.Therefore, half cyanines class fluorescence probe provided by the present invention and its to BuCh
The detection of esterase is more directly and accurate.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail within the scope of the technical concept of the present invention can be with various simple variants of the technical solution of the present invention are made, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that specific technical features described in the above specific embodiments, in not lance
In the case where shield, can be combined in any appropriate way, in order to avoid unnecessary repetition, the present invention to it is various can
No further explanation will be given for the combination of energy.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (10)
1. a kind of half cyanines class fluorescence probe of near-infrared, which is characterized in that the half cyanines class fluorescence probe of near-infrared has formula (1) institute
The structure shown:
2. the preparation method of half cyanines class fluorescence probe of near-infrared described in claim 1, which is characterized in that this method is included in parent
In the presence of nuclear reaction condition and acid binding agent, in organic solvent, formula (2) compound represented is contacted with Cyclopropyl carbonyl chloride；
3. preparation method according to claim 2, wherein relative to structural compounds shown in 1 mM of formula (2), institute
The dosage for stating Cyclopropyl carbonyl chloride is 1-2 mMs, and the dosage of the acid binding agent is 1-3 mMs, the use of the organic solvent
Amount is 20-30 milliliters.
4. preparation method according to claim 2 or 3, wherein the acid binding agent is selected from triethylamine, diethylamine, diisopropyl
Base amine, pyridine, N, in N- dimethylamino naphthyridine, 2,6- lutidines, potassium carbonate, cesium carbonate, sodium carbonate and sodium bicarbonate
It is at least one；The organic solvent is selected from methylene chloride, tetrahydrofuran, chloroform, carbon tetrachloride, acetonitrile, acetone, glycol dinitrate
At least one of ether, N,N-dimethylformamide and 1,2- dichloroethanes.
5. the preparation method according to any one of claim 2-4, wherein the necleophilic reaction condition includes: temperature
It is subzero 5 DEG C to 30 DEG C, time 10-120min.
6. application of the half cyanines class fluorescence probe of near-infrared described in claim 1 in the activity of detection butyrylcholine esterase.
7. a kind of detection method of the activity of BuChE, which is characterized in that this method comprises:
Sample to be tested is contacted with the half cyanines class fluorescence probe of near-infrared described in claim 1, so that the fourth in sample to be tested
Acetylcholinesterase identified by the cyclopropane carbonyl in the infrared half cyanines class fluorescence probe, the material HCY after being contacted；
The value added of the fluorescence intensity that issues under excitation light of material HCY at any time after detection contact, the fluorescence intensity with
The value added of time indicates the activity of butyrylcholine esterase in sample to be tested；The wavelength of the exciting light is 450-750nm, institute
The launch wavelength for stating fluorescence is 680-800nm.
8. detection method as claimed in claim 7, wherein relative to the butyrylcholine esterase of 1 μ g/mL, half cyanines of near-infrared
The dosage of class fluorescence probe is 0.05-100 μM.
9. detection method described in claim 7 or 8, wherein the condition of contact includes: that temperature is 25-37 DEG C, pH value 5-9,
Time is 10-30min.
10. a kind of detection kit of the activity of BuChE, which includes: described in claim 1 close red
Outer half cyanines class fluorescence probe, butyrylcholine esterase standard items and reaction buffer.
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| CN110028503B (en) * | 2019-03-28 | 2022-02-18 | 青岛科技大学 | Fluorescent probe for measuring acetylcholinesterase and preparation method and application thereof |
| CN109929548B (en) * | 2019-04-15 | 2020-10-09 | 济南大学 | Novel near-infrared fluorescent probe for detecting carboxypeptidase A |
| 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 |
| CN115322178A (en) * | 2022-07-27 | 2022-11-11 | 陕西师范大学 | Near-infrared compound, preparation method, butyrylcholinesterase detection kit and application |
| CN115974837B (en) * | 2022-12-21 | 2024-04-09 | 中国消防救援学院 | Ratio type fluorescent probe and preparation method and application thereof |
| CN116425694B (en) * | 2023-04-18 | 2023-11-14 | 吉林大学 | Fluorescent probe LAN-bche for specifically recognizing butyrylcholine esterase and preparation method and application thereof |
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