CN103073427A - Probe for detecting activity of acetylcholinesterase and inhibitor thereof, application and preparation method - Google Patents
Probe for detecting activity of acetylcholinesterase and inhibitor thereof, application and preparation method Download PDFInfo
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- 102000012440 Acetylcholinesterase Human genes 0.000 title claims abstract description 81
- 108010022752 Acetylcholinesterase Proteins 0.000 title claims abstract description 81
- 229940022698 acetylcholinesterase Drugs 0.000 title claims abstract description 81
- 239000000523 sample Substances 0.000 title claims abstract description 63
- 230000000694 effects Effects 0.000 title claims abstract description 20
- 239000003112 inhibitor Substances 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title abstract description 8
- 150000001875 compounds Chemical class 0.000 claims abstract description 22
- 238000000034 method Methods 0.000 claims abstract description 20
- DBLXOVFQHHSKRC-UHFFFAOYSA-N ethanesulfonic acid;2-piperazin-1-ylethanol Chemical compound CCS(O)(=O)=O.OCCN1CCNCC1 DBLXOVFQHHSKRC-UHFFFAOYSA-N 0.000 claims abstract description 15
- 238000006243 chemical reaction Methods 0.000 claims description 50
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 24
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 claims description 18
- 238000001514 detection method Methods 0.000 claims description 16
- 238000013016 damping Methods 0.000 claims description 14
- 239000012530 fluid Substances 0.000 claims description 14
- 239000002243 precursor Substances 0.000 claims description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 12
- 239000002253 acid Substances 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- YLQBMQCUIZJEEH-UHFFFAOYSA-N tetrahydrofuran Natural products C=1C=COC=1 YLQBMQCUIZJEEH-UHFFFAOYSA-N 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 8
- 239000012043 crude product Substances 0.000 claims description 6
- 238000004821 distillation Methods 0.000 claims description 6
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- 239000002904 solvent Substances 0.000 claims description 6
- 238000004587 chromatography analysis Methods 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 5
- FYSNRJHAOHDILO-UHFFFAOYSA-N thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 claims description 5
- ZIBGPFATKBEMQZ-UHFFFAOYSA-N triethylene glycol Chemical compound OCCOCCOCCO ZIBGPFATKBEMQZ-UHFFFAOYSA-N 0.000 claims description 5
- NGNBDVOYPDDBFK-UHFFFAOYSA-N 2-[2,4-di(pentan-2-yl)phenoxy]acetyl chloride Chemical class CCCC(C)C1=CC=C(OCC(Cl)=O)C(C(C)CCC)=C1 NGNBDVOYPDDBFK-UHFFFAOYSA-N 0.000 claims description 2
- 238000004090 dissolution Methods 0.000 claims description 2
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- 238000010926 purge Methods 0.000 claims description 2
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 abstract description 28
- 238000001917 fluorescence detection Methods 0.000 abstract description 2
- 230000035945 sensitivity Effects 0.000 abstract description 2
- CNJMYRYCYNAIOF-UHFFFAOYSA-M trimethyl(2-tetradecanoyloxyethyl)azanium;chloride Chemical compound [Cl-].CCCCCCCCCCCCCC(=O)OCC[N+](C)(C)C CNJMYRYCYNAIOF-UHFFFAOYSA-M 0.000 abstract description 2
- 239000007853 buffer solution Substances 0.000 abstract 1
- MWMPEAHGUXCSMY-UHFFFAOYSA-N pentacosanoic acid Chemical compound CCCCCCCCCCCCCCCCCCCCCCCCC(O)=O MWMPEAHGUXCSMY-UHFFFAOYSA-N 0.000 abstract 1
- 101100515472 Mus musculus Mycl gene Proteins 0.000 description 31
- 239000010453 quartz Substances 0.000 description 19
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 19
- 238000002189 fluorescence spectrum Methods 0.000 description 16
- 239000007788 liquid Substances 0.000 description 11
- 230000000994 depressogenic effect Effects 0.000 description 7
- 229960002362 neostigmine Drugs 0.000 description 6
- ALWKGYPQUAPLQC-UHFFFAOYSA-N neostigmine Chemical compound CN(C)C(=O)OC1=CC=CC([N+](C)(C)C)=C1 ALWKGYPQUAPLQC-UHFFFAOYSA-N 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 238000000862 absorption spectrum Methods 0.000 description 5
- OIPILFWXSMYKGL-UHFFFAOYSA-N acetylcholine Chemical compound CC(=O)OCC[N+](C)(C)C OIPILFWXSMYKGL-UHFFFAOYSA-N 0.000 description 4
- 229960004373 acetylcholine Drugs 0.000 description 4
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- 239000002329 esterase inhibitor Substances 0.000 description 3
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- 229940079593 drug Drugs 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000003987 organophosphate pesticide Substances 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 238000005160 1H NMR spectroscopy Methods 0.000 description 1
- KIUMMUBSPKGMOY-UHFFFAOYSA-N 3,3'-Dithiobis(6-nitrobenzoic acid) Chemical compound C1=C([N+]([O-])=O)C(C(=O)O)=CC(SSC=2C=C(C(=CC=2)[N+]([O-])=O)C(O)=O)=C1 KIUMMUBSPKGMOY-UHFFFAOYSA-N 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010039966 Senile dementia Diseases 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- 239000012230 colorless oil Substances 0.000 description 1
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- 238000013461 design Methods 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to a probe for detecting activity of acetylcholinesterase and an inhibitor thereof, application and a preparation method. The probe is polymerized by 10, 12-diyne pentacosanic acid and a compound shown in a formula I in a weight ratio of 7:3,the vesicle probe is blue and non-fluorescent in the 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution, is red and emits fluorescence (the fluorescence emission wavelength is about 550 nm) after reacting with the myristoyl choline chloride, has high sensitivity and easy operation, and the fluorescence detection method adopting the probe has great application prospect in the field of biomedicine.
Description
Technical field
The present invention relates to a kind of probe for detection of acetylcholinesterase and inhibitor activity thereof, purposes and preparation method.
Background technology
The development of the active Fast Detection Technique of acetylcholinesterase (AChE) is for promoting health and environment protection is significant.
If be used for the treatment of at present the drug main acetylcholinesterase depressant of senile dementia, control acetyl choline content in the patient body by acetylcholine esterase inhibition (AChE) activity, so the exploitation of the active detection agent of acetylcholinesterase (AChE) has vital role for the screening of related drugs.
In addition, some organophosphorus pesticide, carbamate pesticide can produce restraining effect to acetylcholinesterase (AChE) is active, so the development of the active detection agent of acetylcholinesterase (AChE) also can play a significant role for organophosphorus pesticide, carbamate pesticide in the testing environment.
The active detection technique of tradition acetylcholinesterase (AChE) mainly adopts the indirect control and supervision method, the method adopts thiocholine as substrate, acetylcholinesterase (AChE) is hydrolyzed this substrate and generates thiocholine, the latter and dithio nitrobenzoic acid (Ellman) reaction generate yellow product, determine enzymic activity by the formation speed of absorption spectrum monitoring yellow substance.But the method in use tends to the interference of having powerful connections, and sensitivity is lower.
Understand according to the contriver, the detection method of using fluorescent probe has good susceptibility, and angle is started with thus, researches and develops better detection method.
Summary of the invention
Technical problem to be solved by this invention is: overcome the problem that prior art exists, a kind of probe for detection of acetylcholinesterase and inhibitor activity thereof, purposes and preparation method are provided.
The technology of the present invention design is as follows: known to the contriver, MyCl (Myristoylcholine chloride) is cracked into acid and choline under acetylcholinesterase (AChE) effect, produce the probe of fluorescence as long as prepare to react with MyCl.
The technical scheme that the present invention solves its technical problem is as follows:
A kind of probe precursor for detection of acetylcholinesterase and inhibitor activity thereof of the present invention is characterized in that, has suc as formula the structure shown in the I:
A kind of method for preparing above-mentioned probe precursor of the present invention is characterized in that, may further comprise the steps:
The first step, the reaction of 10,12-diine neocerotic acid and sulfur oxychloride is obtained formula II compound;
Second step, the reaction of formula II compound and triethylene glycol is obtained probe precursor shown in the formula I.
Further improving is that the detailed process of the first step is: first 10,12-diine neocerotic acid is dissolved in methylene dichloride, adds sulfur oxychloride again and obtain reaction solution, continue to stir this reaction solution under nitrogen protection until reaction finishes; Then, solvent is removed in underpressure distillation, namely gets formula II compound.
Further improve and be, the detailed process of second step is: first formula II compound dissolution is obtained formula II compound solution in tetrahydrofuran (THF), add to formula II compound solution in the tetrahydrofuran solution that contains triethylene glycol again, obtain reaction solution, under nitrogen protection, continue to stir this reaction solution until reaction finishes; Then, underpressure distillation is removed solvent and is obtained crude product, and with crude product with the chromatography column purifying, namely get probe precursor shown in the formula I.
Further improve and be: in the second step, adopt the methylene dichloride of volume ratio 100:2 in the chromatography column purge process: methyl alcohol is elutriant.
A kind of vesica probe for detection of acetylcholinesterase and inhibitor activity thereof of the present invention is characterized in that, is 10 of 7:3 by weight ratio, and 12-diine neocerotic acid and formula I compound polymerization form.
A kind of method for preparing above-mentioned vesica probe of the present invention is characterized in that, may further comprise the steps:
With 10,12-diine neocerotic acid and formula I compound are dissolved in dimethyl sulfoxide (DMSO) by weight 7:3 and obtain DMSO solution, DMSO solution is added to temperature obtain reaction solution at least 80 ℃ the 4-hydroxyethyl piperazine ethanesulfonic acid damping fluid, the cooling reaction solution, carry out polyreaction with the UV-irradiation reaction solution, namely get the vesica probe.
Further improve and be, before the cooling reaction solution, that reaction solution is ultrasonic and filter with filter.
Further improve is to adopt the 254nm ultraviolet lamp as ultraviolet source.
In addition, the present invention also provides the purposes of aforementioned vesica probe for detection of acetylcholinesterase and inhibitor activity thereof.
Vesica probe of the present invention is blue in 4-hydroxyethyl piperazine ethanesulfonic acid damping fluid and without fluorescence, meet and become redness and send fluorescence (about fluorescent emission wavelength 550nm) after MyCl reacts, can detect acetylcholinesterase (AChE) activity by the fluorescent quenching that adds acetylcholinesterase (AChE) generation afterwards, perhaps can detect by the fluorescence recovery process that adding acetylcholinesterase depressant and acetylcholinesterase (AChE) occur the activity of acetylcholinesterase depressant.
Compare with existing detection method, adopt the fluorescence detection method speed of response of vesica probe of the present invention fast, highly sensitive, have suitable fluorescent emission wavelength (about 550nm), at biomedicine field great application prospect is arranged.
Description of drawings
Fig. 1 is the fluorescence emission spectrogram of the embodiment of the invention 2.
Fig. 2 is the ultraviolet-visible abosrption spectrogram of the embodiment of the invention 2.
Fig. 3 is the colour-change figure of the embodiment of the invention 2.
Fig. 4 is the fluorescence emission spectrogram of the embodiment of the invention 3.
Fig. 5 is the ultraviolet-visible abosrption spectrogram of the embodiment of the invention 3.
Fig. 6 is the colour-change figure of the embodiment of the invention 3.
Fig. 7 is the fluorescence emission spectrogram of the embodiment of the invention 4.
Fig. 8 is the fluorescence emission spectrogram of the embodiment of the invention 5.
Fig. 9 is the suppression efficiency figure of the embodiment of the invention 5.
Embodiment
Below in conjunction with embodiment the present invention is described in further detail.But the invention is not restricted to given example.
The experiment material that relates in the following content and reagent then are commercially available product as not specifying.
The preparation of embodiment 1 formula I probe precursor, vesica probe
One, preparation formula I probe precursor
The first step, with 0.375g (1mmol) 10,12-diine neocerotic acid is dissolved in the 20ml methylene dichloride, then obtain reaction solution to wherein dripping the 2ml sulfur oxychloride, under nitrogen protection, continue to stir this reaction solution until reaction finishes (be generally to stir and spend the night); Then, solvent is removed in underpressure distillation, namely gets the formula II compound that is colorless oil.
Second step, the first step gained formula II compound is dissolved in obtains formula II compound solution in a small amount of tetrahydrofuran (THF) first, drop to formula II compound solution in the 20ml tetrahydrofuran solution that contains the 0.18g triethylene glycol again, obtain reaction solution, under nitrogen protection, continue to stir this reaction solution until reaction finishes (be generally to stir and spend the night); Then, underpressure distillation is removed solvent and is obtained crude product, and with crude product take chromatography column purifying (adopt the methylene dichloride of volume ratio 100:2: methyl alcohol is as elutriant), namely get probe precursor shown in the formula 1, weight 90.5mg, molecular weight are 506.
Formula I probe precursor
1H NMR (CDC
13, 500MHz) δ (ppm): δ 0.86-0.89 (t, 3H, J=13.6Hz), 1.25 (s, 26H), (1.47-1.52 t, 4H, J=20.55Hz), (1.60-1.63 t, 2H, J=14.1Hz), (2.22-2.24 t, 2H, J=13.9Hz), (2.31-2.34 t, 4H, J=15.1), (3.60-3.62 t, 2H, J=9.05), (3.66-3.67 t, 6H, J=1.55Hz), (3.69-3.73 t, 2H, J=22.4), (4.22-4.24 t, 2H, J=9.55).
Formula I probe precursor
13C NMR (CDC
13, 500MHz) δ (ppm): 14.03,19.12,19.14,22.62,24.79,28.27,28.31,28.70,28.80,28.84,28.99,29.02,29.28,29.42,29.54,29.56,29.58,29.63,31.86,34.09,61.71,65.24,65.32,66.83,69.13,70.31,70.36,70.48,76.75,77.00,173.7.
HRMS (EI) m/z 506.3865 (C of formula I probe precursor
31H
54O
5).
Operational path is as follows:
Two, preparation vesica probe
With 10,12-diine neocerotic acid and formula I compound are dissolved in the 1ml dimethyl sulfoxide (DMSO) by weight 7:3 and obtain DMSO solution, DMSO solution was dropped to temperature with syringe in 5 minutes be to obtain reaction solution in 80 ℃ 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4), this moment 10, the ultimate density of 12-diine neocerotic acid is 1mM; Reaction solution was descended ultrasonic 25 minutes at 80 ℃, and filter with 0.8 μ m filter, remove insoluble particle; Reaction solution is put 4 ℃ of lower coolings 12 hours; Adopt 254nm ultraviolet lamp (1mW/cm
2) irradiation reaction solution carried out polyreaction in 10 minutes, namely get blue vesica probe.If no special instructions, this vesica probe is all adopted in following experiment.
Spectral quality and the colour-change of embodiment 2 vesica probes and MyCl reaction
One, the spectral quality of vesica probe and MyCl reaction
The vesica probe of getting respectively nine part of 60 μ l 1mM adds in each quartz container, then adds respectively 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) and dilutes and be settled to 3ml; Then, add respectively 0 μ l, 0.6 μ l, 1.2 μ l, 1.8 μ l, 2.4 μ l, 3 μ l, 3.6 μ l, (final concentration is 0 μ M to the 10mM MyCl of 4.5 μ l, 2 μ M, 4 μ M, 6 μ M, 8 μ M, 10 μ M, 12 μ M, 15 μ M), reaction was measured respectively fluorescence emission spectrum and UV, visible light optical absorption spectra after 5 minutes.Excite with 492nm when fluorescence emission spectrum is measured, exciting with the slit width of launching is 5nm.
Fluorescence emission spectrum as shown in Figure 1, MyCl concentration is in 0-15 μ M scope the time, the fluorescence intensity at about 550nm place strengthens with the increase of MyCl concentration.
The UV, visible light optical absorption spectra as shown in Figure 2, MyCl concentration is in 0-12 μ M scope the time, with the increase of MyCl concentration, the absorbancy at 640nm place reduces gradually, the absorbancy at 545nm place increases gradually.
Two, the colour-change of vesica probe and MyCl reaction
Get respectively the vesica probe of six part of 40 μ l 1mM and put into 96 orifice plates, it is 200 μ M that adding 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) makes each vesica probe ultimate density; In each hole, add respectively 0 μ l, 1 μ l, 2 μ l, 3 μ l, 4 μ l, the 10mM MyCl of 5 μ l, making its ultimate density is 0 μ M, 1 μ M, 5 μ M, 10 μ M, 20 μ M, 50 μ M; React and observe colour-change after 5 minutes.
The result increases with MyCl concentration as shown in Figure 3, and liquid becomes redness by blueness gradually in each hole, and wherein, the MyCl colour-change of 50 μ M is the most obvious, becomes redness fully, and naked eyes can observe directly.
Spectral quality and colour-change that the product of embodiment 3 MyCls and acetylcholinesterase (AChE) reaction mixes with the vesica probe
One, the spectral quality that mixes with the vesica probe of product of MyCl and acetylcholinesterase (AChE) reaction
Get seven part of 3.6 μ l 10mM MyCl and add in each quartz container, and add 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) dilution and be settled at last 3ml; Then, the acetylcholinesterase (AChE) (0U/ml, 0.025U/ml, 0.05U/ml, 0.1U/ml, 0.2U/ml, 0.3U/ml, 0.4U/ml) that adds respectively an amount of (such as 2 μ l) different concns; React after 10 minutes, measure respectively again 60 μ l 1mM vesica probes and add in the quartz container, reacted again afterwards 5 minutes, measure at last fluorescence emission spectrum and UV, visible light optical absorption spectra.Excite with 492nm when fluorescence emission spectrum is measured, exciting with the slit width of launching is 5nm.
Fluorescence emission spectrum as shown in Figure 4, with the increase of acetylcholinesterase (AChE) concentration, the fluorescence intensity of liquid at about 550nm place reduces gradually in the quartz container; When acetylcholinesterase (AChE) when concentration reaches 0.4U/ml, the interior liquid of quartz container is down to minimum in the fluorescence intensity at about 550nm place, the situation when almost only adding the vesica probe with not adding MyCl and acetylcholinesterase (AChE) is consistent.
The UV, visible light optical absorption spectra as shown in Figure 5, with the increase of acetylcholinesterase (AChE) concentration, the absorption peak of liquid at the 545nm place reduces gradually in the quartz container, the absorption peak at the 640nm place strengthens gradually; When acetylcholinesterase (AChE) when concentration reaches 0.4U/ml, the absorption peak of liquid at the 640nm place reaches maximum value in the quartz container, and the situation when almost only adding the vesica probe with not adding MyCl and acetylcholinesterase (AChE) is consistent.
Above presentation of results, the product of MyCl after acetylcholinesterase (AChE) cracking can not react with the vesica probe, also is that the vesica probe only can send fluorescence with the MyCl reaction.
Two, the product colour-change of mixing with the vesica probe of MyCl and acetylcholinesterase (AChE) reaction
Get three quartz containeres, add first 300 μ l, 100 μ M vesica probes in first quartz container, use again 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) to be diluted to 3ml, and observe color.The MyCl that adds first 9 μ l30 μ M in second quartz container, use again 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) dilution, add again 300 μ l, 100 μ M vesica probes, use at last 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) to be diluted to 3ml, and observe color.The MyCl that adds first 9 μ l30 μ M in the 3rd quartz container, use again 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) dilution, the acetylcholinesterase (AChE) that adds afterwards 2U/ml, react and add again 300 μ l, 100 μ M vesica probes after 10 minutes, use at last 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) to be diluted to 3ml, and observe color.
The result as shown in Figure 6, first, liquid is blueness and no significant difference in the 3rd quartz container, liquid takes on a red color in second quartz container.This explanation, MyCl with acetylcholinesterase (AChE) fully after the reaction, can not with vesica probe generation color reaction, also namely only have MyCl itself can with vesica probe generation color reaction, reaction liquid is reddened.This namely can be observed by naked eyes, and effect is very obvious.
The fluorescence emission spectrum that embodiment 4 measures MyCl and acetylcholinesterase (AChE) reaction with fixed time changes
Get six part of 3.6 μ l 10mM MyCl and add in six quartz containeres, add respectively 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) and dilute and be settled to 3ml; Add respectively afterwards the acetylcholinesterase (AChE) of 0.4U/ml, reach respectively 0,2,4,6 when the reaction times, in the time of 8,10 minutes, add 60 μ l 1mM vesica probes, and reacted again 5 minutes, measure at last fluorescence emission spectrum.Excite with 492nm when fluorescence emission spectrum is measured, exciting with the slit width of launching is 5nm.
The result as shown in Figure 7, with the prolongation in reaction times, the fluorescence intensity of liquid at about 550nm place weakens gradually in the quartz container, i.e. fluorescent quenching degree strengthens gradually; When the reaction times was 10 minutes, liquid was the most weak in the fluorescence intensity at about 550nm place in the quartz container, i.e. fluorescent quenching degree is the strongest, and the situation when almost only adding the vesica probe with not adding MyCl and acetylcholinesterase (AChE) is consistent.
This experiment shows, adopts vesica probe of the present invention, can detect well by the variation of fluorescence spectrum acetylcholinesterase (AChE) activity.
The fluorescence emission spectrum of embodiment 5 acetylcholinesterase depressant prostigmin(e) acetylcholine esterase inhibitions (AChE) changes
The acetylcholinesterase (AChE) of getting respectively 2.4 μ l500U/ml adds in the serial quartz container, then (concentration is respectively: 0 to add respectively the acetylcholinesterase depressant prostigmin(e) of an amount of (such as 5 μ l) different concns, 0.5,1,5,10nM), adding respectively 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) damping fluid (10mM pH=7.4) dilutes and is settled to 3ml again; Afterwards, add respectively 3.6 μ l 10mM MyCls, react after 10 minutes, add the vesica probe, and reacted again 5 minutes, measure at last fluorescence emission spectrum.Excite with 492nm when fluorescence emission spectrum is measured, exciting with the slit width of launching is 5nm.
The result of fluorescence emission spectrum as shown in Figure 8, liquid strengthens with the increase of prostigmin(e) concentration in the fluorescence intensity at about 550nm place in the quartz container; When the concentration of prostigmin(e) was 10nM, liquid was the strongest in the fluorescence intensity at about 550nm place in the quartz container, and the situation that does not almost add acetylcholinesterase (AChE) with only adding MyCl and vesica probe is consistent.
The suppression efficiency of calculating prostigmin(e) by fluorescence emission spectrum as shown in Figure 9, and to calculate thus inhibiting rate be IC
50=4.1nM.
This result shows, the acetylcholinesterase depressant prostigmin(e) is the activity of acetylcholine esterase inhibition (AChE) well, make the acetylcholinesterase (AChE) can not the cracking MyCl, like this, the vesica probe can produce fluorescence with the MyCl reaction.
As shown in this experiment, adopt vesica probe of the present invention, can detect well by the variation of fluorescence spectrum the activity of acetylcholinesterase depressant.
Claims (10)
1. the probe precursor for detection of acetylcholinesterase and inhibitor activity thereof is characterized in that, has suc as formula the structure shown in the I:
2. a method for preparing the described probe precursor of claim 1 is characterized in that, may further comprise the steps: the first step, the reaction of 10,12-diine neocerotic acid and sulfur oxychloride is obtained formula II compound;
Second step, the reaction of formula II compound and triethylene glycol is obtained probe precursor shown in the formula I.
3. described method according to claim 2 is characterized in that the detailed process of the first step is: first 10,12-diine neocerotic acid is dissolved in methylene dichloride, adds sulfur oxychloride again and obtain reaction solution, continue to stir this reaction solution under nitrogen protection until reaction finishes; Then, solvent is removed in underpressure distillation, namely gets formula II compound.
4. described method according to claim 2, it is characterized in that, the detailed process of second step is: first formula II compound dissolution is obtained formula II compound solution in tetrahydrofuran (THF), add to formula II compound solution in the tetrahydrofuran solution that contains triethylene glycol again, obtain reaction solution, under nitrogen protection, continue to stir this reaction solution until reaction finishes; Then, underpressure distillation is removed solvent and is obtained crude product, and with crude product with the chromatography column purifying, namely get probe precursor shown in the formula I.
5. described method according to claim 4 is characterized in that, in the second step, adopt the methylene dichloride of volume ratio 100:2 in the chromatography column purge process: methyl alcohol is elutriant.
6. the vesica probe for detection of acetylcholinesterase and inhibitor activity thereof is characterized in that, is 10 of 7:3 by weight ratio, and 12-diine neocerotic acid and formula I compound polymerization form.
7. a method for preparing the described vesica probe of claim 6 is characterized in that, may further comprise the steps:
With 10,12-diine neocerotic acid and formula I compound are dissolved in dimethyl sulfoxide (DMSO) by weight 7:3 and obtain DMSO solution, DMSO solution is added to temperature obtain reaction solution at least 80 ℃ the 4-hydroxyethyl piperazine ethanesulfonic acid damping fluid, the cooling reaction solution, carry out polyreaction with the UV-irradiation reaction solution, namely get the vesica probe.
8. described method according to claim 7 is characterized in that, and is before the cooling reaction solution, that reaction solution is ultrasonic and filter with filter.
9. described method according to claim 7 is characterized in that, adopts the 254nm ultraviolet lamp as ultraviolet source.
10. the described vesica probe of claim 6 is for detection of the purposes of acetylcholinesterase and inhibitor activity thereof.
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KR20080054963A (en) * | 2006-12-14 | 2008-06-19 | 고려대학교 산학협력단 | Polydiacetylene sensor chip comprising aptamer and manufacturing process thereof |
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Cited By (2)
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CN105241882A (en) * | 2015-09-23 | 2016-01-13 | 山东大学 | Applications of liquid crystal sensor in detection of organophosphorus pesticides and carbamate pesticides |
CN108801998A (en) * | 2018-06-13 | 2018-11-13 | 青岛大学 | A method of the ratio fluorescent probe in detecting choline based on copper nano-cluster compound |
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