CN106267243B - A kind of molecular probe and its preparation method and application - Google Patents
A kind of molecular probe and its preparation method and application Download PDFInfo
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- CN106267243B CN106267243B CN201610659738.8A CN201610659738A CN106267243B CN 106267243 B CN106267243 B CN 106267243B CN 201610659738 A CN201610659738 A CN 201610659738A CN 106267243 B CN106267243 B CN 106267243B
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
- A61K49/0036—Porphyrins
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0054—Macromolecular compounds, i.e. oligomers, polymers, dendrimers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0013—Luminescence
- A61K49/0017—Fluorescence in vivo
- A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
- A61K49/0056—Peptides, proteins, polyamino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/22—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations
- A61K49/221—Echographic preparations; Ultrasound imaging preparations ; Optoacoustic imaging preparations characterised by the targeting agent or modifying agent linked to the acoustically-active agent
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The present invention provides a kind of molecular probe and its preparation method and application, the molecular probe includes water-solubility carrier molecule, responsiveness polypeptide and signaling molecule；Wherein, the sequence of the responsiveness polypeptide is (Xaa)m(Thr‑Phe‑Gly‑Phe)(Xaa)nLys(Xaa)i, wherein Xaa is expressed as arbitrary amino acid, and i, n and m independently are 0-4；The water-solubility carrier molecule and signaling molecule are connected with amido bond with responsiveness polypeptide.Molecular probe of the invention not only has good specificity and signal stabilization, but also enriches cell autophagy detection means, has broad application prospects.
Description
Technical field
The invention belongs to molecular probe technology field, it is related to a kind of molecular probe and its preparation method and application.
Background technique
Cell autophagy is that cell transports the protein and organelle of intracellular impaired, denaturation or aging by lysosome
The process of digestion degradation is carried out to lysosome.Under normal physiological conditions, cell autophagy is conducive to cell and keeps homeostasis；Occurring
Stress when, cell autophagy prevents the accumulation of toxic or carcinogenic injury protein matter and organelle, inhibits cell carcinogenesis, maintains cell
Interior ambient stable and metabolic balance.Cell autophagy is in biology growing development, cell differentiation and to the answer party of environmental stress
Face is extremely crucial, it takes part in the processes such as cells survival, death, pathogen removing, antigen presentation, and with cancer, infection, nerve
A variety of diseases such as degenerative disease are related.Therefore, secrets of life can not only be further appreciated that by studying its mechanism of action, simultaneously
Also new thinking is provided for the treatment of some diseases.
Used detection means, such as TEM, LC3 immuno-precipitation, GFP-LC3 fluorescence detection are studied for cell autophagy
The methods of real-time, quantitative detection data cannot be but provided, and these methods may not apply to tissue biopsy level.Cause
This, develop a kind of new material for cell autophagy internal vitro detection for carry out the basic mechanism study of cell autophagy with
And the diagnosing and treating of the horizontal autophagy related disease of tissue biopsy is of great significance.
Existing research at present is the nano-probe based on FRET effect, but not due to the fluorescent molecule sensitivity of its label
Good specificity is not strong and nano particle influences the autophagy process of cell, affects its extensive use.103275697 A of CN is public
A kind of Di-pyrene amphiphilic fluorescent probe is opened, which can detect metal ion, fast response time, high sensitivity, response range
Width, but it can not be applied to the detection to cell.
Aggregation-induced emission phenomenon (Aggregation-induced emission, AIE) overcomes conventional fluorescent material
In coherent condition the shortcomings that fluorescent quenching, the sensitivity of bioprobe is significantly improved, is had extensively in fields such as bio-imagings
General application prospect.But the molecular probe with specificly-response for the imaging of internal cell autophagy is also rarely reported, thus
AIE feature based on fluorescent molecule, research and development bio-safety performance is high, sensitive and high specificity molecular probe conduct is external in vivo
The diagnostic imaging agent of cell autophagy will provide possibility for the diagnosing and treating of realization autophagy related disease.
Photoacoustic imaging technology is a kind of new bio medical imaging procedure of non-invasive.When pulsed laser irradiation to biology
When tissue, the photo-absorption region of tissue will generate ultrasonic signal, can reconstruct the light absorption in tissue by detecting photoacoustic signal
Distributed image.Photoacoustic imaging combine highly selective and pure ultrasonic tissue in the imaging of pure optical texture be imaged in deep penetration characteristic
The organization chart picture of high-resolution and high contrast can be obtained in advantage.Therefore, it is based on photoacoustic imaging technology, researches and develops biological safety
The preparation that high, sensitive and specific good photoacoustic contrast agent is detected as in-vivo tissue position cell autophagy level will be cancer
The diagnosis and treatment of disease provide effective solution, have important clinical meaning.
Summary of the invention
In view of the deficiencies of the prior art, the purpose of the present invention is to provide a kind of molecular probe and preparation method thereof and answer
With.
In order to achieve that object of the invention, the invention adopts the following technical scheme:
On the one hand, the present invention provides a kind of molecular probe, which is characterized in that the molecular probe includes water-solubility carrier point
Son, responsiveness polypeptide and signaling molecule；Wherein, the sequence of the responsiveness polypeptide is (Xaa)m(Thr-Phe-Gly-Phe)
(Xaa)nLys(Xaa)i, wherein Xaa is expressed as arbitrary amino acid, and i, n and m independently are 0-4；The water-solubility carrier molecule and
Signaling molecule is connected with amido bond with responsiveness polypeptide.
Wherein, the Xaa can be Ala, Arg, Asp, Cys, Glu, Gly, His, Ile, Leu, Lys, Phe, Ser,
Thr, Trp, Try, Pro, or Met.
Wherein, the i can be 0,1,2,3 or 4；N can be 0,1,2,3 or 4；The m can be 0,1,2,3 or 4.
In the present invention, the responsiveness polypeptide is specifically responded to cell autophagy marker enzyme ATG4, in normal environment, point
Sub- probe keeps the unimolecule state of unstressed configuration or low photoacoustic signal, and when cell autophagy activation, polypeptide is cut in molecular probe
Signaling molecule is cut and releases, the aggregation characteristic of signaling molecule assigns the characteristic of fluorescence probe or photoacoustic signal enhancing.
In the present invention, the position that the responsiveness polypeptide is cut is (Thr-Phe-Gly-Phe) sweet ammonia in conserved sequence
Sour carboxyl.
Preferably, the length of the responsiveness polypeptide is 5-17 amino acid, for example, can be 5,6,7,8,9,10,11,
12,13,14,15,16 or 17, preferably 6-12 amino acid, further preferably 7 amino acid.
Preferably, the signaling molecule for the fluorescence signal molecule with aggregation inducing effect (AIE) and/or has aggregation
The fat-soluble signaling molecule for causing photoacoustic signal to enhance.
Preferably, the fluorescence signal molecule with aggregation inducing effect (AIE) is double pyrene analog derivatives and/or four benzene
Ethylene compounds, preferably double pyrenes.
Double pyrene compound structural formulas are as follows:
The tetraphenyl ethylene class structural formula of compound is as follows:
Preferably, the fat-soluble signaling molecule that there is aggregation photoacoustic signal to be caused to enhance is the porphin for being conjugated cyclic structure
Oxazoline derivates, preferably purpurin 18.
The purpurin 18 structural formula is as follows:
Preferably, the water-solubility carrier molecule is dendroid class supermolecule and/or hyperbranched class molecule, preferably four generations
Dendrimer.
Preferably, the molecular probe has the structure as shown in following formula I:
Second aspect, the present invention provide a kind of preparation method of molecular probe described in first aspect, comprising the following steps:
(1) the responsiveness polypeptide is synthesized on resin not soluble in water using Fmoc solid phase synthesis process；
(2) responsiveness polypeptide is connected with the signaling molecule and water-solubility carrier molecule.
Preferably, resin not soluble in water described in step (1) is Wang resin.
Preferably, the modification density of the Wang resin be 0.3-0.4mM, such as can be 0.3mM, 0.31mM,
0.32mM, 0.35mM, 0.36mM, 0.38mM or 0.4mM, preferably 0.35mM.
Preferably, the specific steps of step (1) include:
(a) aminoterminal of first amino acid is protected with Fmoc, c-terminus is fixed on resin；
(b) it is protected with the Fmoc that the DMF solution of hexahydropyridine sloughs aminoterminal, next lysine is using side-chain amino group
The amino acid of Dde protection, carboxyl 4- methyl morpholine and benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester
DMF solution is activated, and carries out condensation reaction with first amino acid of deprotection；
(c) aminoterminal of remaining amino acid is protected with Fmoc, is repeated step (b), the condensation until completing all amino acid.
Preferably, the step (2) specifically includes: by the signaling molecule activated carboxylic, and being connected to responsiveness polypeptide
Aminoterminal；By lysine side chain amino-reactive in responsiveness polypeptide, and it is connected to the carrier molecule c-terminus, then from tree
It is removed on rouge.
Preferably, in the responsiveness polypeptide lysine side chain amino-reactive containing 0.2% hydrazine hydrate DMF solution in into
Row.
Preferably, the removing from resin is in 4- methyl morpholine and benzotriazole-N, N, N', N'- tetramethylurea six
It is carried out in the DMF solution of fluorophosphoric acid ester.
The third aspect, the present invention provide the molecular probe of one kind as described in relation to the first aspect for internal cell in vitro autophagy enzyme
The detection of ATG4 is assessed for the horizontal real-time quantitative of internal cell in vitro autophagy.
Preferably, the cell is tumour cell.
Compared with the existing technology, the invention has the following advantages:
Molecular probe of the invention has good water-soluble and stability, can be entered in the form of free diffusing and endocytosis thin
Portion intracellular, and the activity that intensity reflects autophagy marker enzyme intracellular is changed by signal, and then horizontal to internal cell in vitro autophagy
It is quantitatively evaluated.Therefore molecular probe of the invention not only has good specificity and signal stabilization, but also enriches
Cell autophagy detection means, has broad application prospects.
Detailed description of the invention
Fig. 1 is the nuclear magnetic spectrum (1H NMR) of molecule described in embodiment 1；
Fig. 2 is the fluorescence spectrum of molecule monomer described in embodiment 1 and aggregation；
Fig. 3 be molecule described in embodiment 1 various concentration molecular probe under cell autophagy marker enzyme ATG4 effect
Fluorescence spectrum；
Fig. 4 is fluorescence letter of the molecule described in embodiment 1 in the MCF-7 cell model of rapamycin active cell autophagy
Number；
Fig. 5 be molecule described in embodiment 1 in the MCF-7 cell model of rapamycin active cell autophagy in real time at
Picture；
Fig. 6 be molecule described in embodiment 1 in the MCF-7 cell model of rapamycin active cell autophagy to cell from
Bite horizontal quantitative assessment；
Fig. 7 be molecule described in embodiment 1 MCF-7 cell autophagy activate model (rapamycin, two hours hungry) and
Inhibit the fluorescence imaging in model (3 methyl purines, Bava Lip river mycin)；
Fig. 8 is fluorescence imaging of the molecule described in embodiment 1 in living body level in cell autophagy activation model；
Fig. 9 is photoacoustic imaging of the molecule described in embodiment 2 in living body level in cell autophagy activation model.
Specific embodiment
The technical scheme of the invention is further explained by means of specific implementation.Those skilled in the art should be bright
, the described embodiments are merely helpful in understanding the present invention, should not be regarded as a specific limitation of the invention.
Embodiment 1: the preparation of molecular probe
1, by taking double pyrene molecule probes of AIE effect as an example, responsiveness polypeptide sequence are as follows:
Gly-Lys-Gly-Ser-Phe-Gly-Phe-Thr-Gly。
Peptide systhesis is synthesized using Fmoc solid-phase synthesis: (1) the Wang resin of 0.35mM modification density is selected in synthesis,
In first amino acid (glycine) C-terminal be fixed on resin, N-terminal is protected by Fmoc, with the hexahydropyridine of 20% (v/v)
DMF solution sloughs Fmoc protection, then with ninhydrin method of testing test deprotection result；
(2) by the carboxyl of next amino acid the 4- methyl morpholine of 0.4M and benzotriazole-N, N, the N' of 0.4M,
The DMF solution of N'- tetramethylurea hexafluorophosphoric acid ester activates, and is added to take off in de-protected resin and react 2 hours, wherein second
The lysine that a amino acid uses side-chain amino group to protect for Dde makees double pyrene molecules after the reaction for completing all sequences amino acid
It repeats the above steps for the last one amino acid and is coupled at the N-terminal of polypeptide；
(3) side-chain amino group that 0.2% hydrazine hydrate removes second amino acid (lysine) is protected, by four generation carboxyl branches
Shape molecule coupling labeled is on lysine side chain amino groups, then with the trifluoroacetic acid solution containing 2.5% water and 2.5% tri isopropyl silane
Synthetic polypeptide is removed from resin, while removing the side chain protection of other amino acid, by trifluoroacetic acid rotary evaporation
Method removal, then product is precipitated with anhydrous ether, is washed and is dried, and molecular probe of the present invention is obtained.
The chemical structure of double pyrene molecule probes of the obtained AIE effect of the above method is as follows:
Its nuclear magnetic spectrum is as shown in Figure 1, as can be seen from the figure the peak in dashed box is the double pyrene molecules and responsiveness of synthesis
The structure of polypeptide, four generation dendrimer structures have arrow to mark, illustrate the molecular probe designed.
The monomer for the molecular probe being prepared and the fluorescence spectrum of aggregation and in cell autophagy ATG4 cysteine enzyme
Fluorescence signal variation under effect, result difference is as shown in Figure 2 and Figure 3, by Fig. 2 and Fig. 3 as it can be seen that molecular probe of the invention
It is quenched with monomer fluorescence and generates aggregation and visible signal enhancing characteristic under enzyme effect.
2, cellular level cell autophagy imaging experiment:
Selection MCF-7 cell is cell autophagy cell model.2×105A cell is seeded in dedicated cell copolymerization first
In burnt culture dish and containing 10% 37 DEG C of PBS DMEM and 5%CO2Under conditions of, it is incubated for 15 hours；Add 1 μM
Cell autophagy inducer rapamycin, be maintained at 37 DEG C and 5%CO2Under conditions of, it is incubated for 1 hour；By 100 μ g/mL
Molecular probe is added in culture dish, and co-focusing imaging is carried out after 2 hours.Imaging is micro- using Zeiss LSM710 laser co-focusing
Mirror.Exciting light is 405nm, collects 450-600nm wave band.Figure 4, it is seen that being detected in the cell of rapamycin treatment
There is stronger fluorescence signal, illustrates that the cell is in the state of autophagy activation；Do not have in the cell Jing Guo rapamycin treatment
It detects fluorescence signal, illustrates that the cell is in the inactive state of autophagy.Additionally by application cell novel autophagy derivant, I
Have detected cell autophagy activation front and back real-time status.Cell of its result as shown in institute Fig. 5, before rapamycin treatment
In do not detect fluorescence signal, but after rapamycin addition, with the extension of time, detecting intracellular fluorescence
Signal gradually increases, and illustrates that rapamycin has activated cell autophagy, and change over time and enhance.
Can we demonstrate molecular probe provide the quantitative information of cell autophagy level.Cell is established using rapamycin
The horizontal different cell model of autophagy, the cell fluorescence signal of 0 μM of rapamycin treatment are set to 100%.Its result such as Fig. 6 institute, institute
Show, with the increase of rapamycin concentrations, the fluorescence signal intensity that detects is from 100% (0 μM) enhancing to 154% (0.1 μ
M), 268% (0.5 μM), 350% (1.0 μM), illustrate that molecular probe is capable of providing the quantitative information of cell autophagy level.
3, molecular probe specificity is imaged
Selection EBSS buffer (starvation), rapamycin are cell autophagy inducer, and 3 methyl purines of selection, Bava Lip river are mould
Element is that cell autophagy inhibitor establishes cell autophagy activation with MCF-7 cell incubation respectively and inhibits model.100 μ g/mL molecules
Probe is added in culture dish, and co-focusing imaging is carried out after 2 hours.Imaging uses Zeiss LSM710 laser confocal microscope.
Exciting light is 405nm, collects 450-600nm wave band.Cell autophagy is swashed by confocal laser scanning microscope molecular probe
Living and unactivated state response.
As a result as shown in fig. 7, being detected in the cell autophagy activation model of two hours of rapamycin and Nature enemy
Stronger fluorescence signal；Inhibit not detect fluorescence in model in the cell autophagy of 3 methyl purines, the processing of Bava Lip river mycin
Signal.As a result illustrate that molecular probe has specificly-response to cell autophagy state of activation.
4, living body horizontal cell autophagy imaging experiment
Selection 2dpf zebrafish embryo is living sample.2dpf zebrafish embryo is induced in the cell autophagy containing 1 μM first
It is incubated for 24 hours in the water of breeding fish of agent rapamycin, 100 μ g/mL molecular probes is added in water of breeding fish, are copolymerized after 4 hours
Coke imaging, determines that molecular probe can be used for the imaging of living body horizontal cell autophagy.
As a result as shown in figure 8, the cell autophagy in physiological saline processing inhibits not detect fluorescence in zebra fish model
Signal, but stronger fluorescence signal is detected in the cell autophagy of rapamycin treatment activation model, illustrate that molecular probe can
For living body horizontal cell autophagy imaging experiment.
Embodiment 2: living body horizontal cell autophagy photoacoustic imaging experiment
It is detected in mouse tumor model with 18 molecular probe of purpurine that there is aggregation to generate enhancing photoacoustic signal characteristic
For cell autophagy imaging.Its molecular probe chemical structure is as follows:
6-7 weeks BALB/c nude mice is chosen, in rear leg intramuscular injection 1 × 107A U87 glioma cell establishes mouse back leg
Portion's tumor model.Tumour mould is activated by the 100 μ L cell autophagy of cell autophagy inducer rapamycin that tail vein injects 1 μM
Type is later 100 μ g/mL molecular probe, 200 μ L by tail vein implantation concentration, chooses 0.25,0.5,1,2,4,6,7,9,12
With 24 hours timing nodes, the imaging to tumor locus cell autophagy level was put in observation in different times.
As a result as shown in figure 9, the mouse tumor position of physiological saline processing, any time section all do not detect that fluorescence is believed
Number, illustrate that the mouse tumor position cell autophagy level of physiological saline processing is very low；In the mouse tumor portion of rapamycin treatment
Position, 0.25 hour when, detect faint fluorescence signal, and as time went on, fluorescence signal intensity also enhances therewith.7 hours
When, signal strength reaches maximum, but later as time change fluorescence signal intensity dies down therewith.Illustrate rapamycin treatment
Mouse tumor position cell is in the state of autophagy activation, and detection effect is best when molecular probe injects 7 hours, later tumour portion
The molecular probe of position may be removed gradually.
The Applicant declares that the present invention is explained by the above embodiments, the enzyme of the invention based on double pyrenes responds self assembly
Polypeptide nano material and its preparation method and application, but the present invention is not limited to the above embodiments, that is, does not mean that the present invention
Above-described embodiment, which must be relied on, to be implemented.It should be clear to those skilled in the art, any improvement in the present invention,
Addition, selection of concrete mode of equivalence replacement and auxiliary element to raw material selected by the present invention etc., all fall within of the invention
Within protection scope and the open scope.
Claims (12)
1. a kind of molecular probe, which is characterized in that the molecular probe includes water-solubility carrier molecule, responsiveness polypeptide and signal
Molecule；Wherein, the sequence of the responsiveness polypeptide is Gly-Lys-Gly-Ser-Phe-Gly-Phe-Thr-Gly；It is described water-soluble
Property carrier molecule and signaling molecule are connected with amido bond with responsiveness polypeptide；
The water-solubility carrier molecule is four generation dendrimers；
The four generations dendrimer is connected on the lysine side chain in responsiveness polypeptide；
The signaling molecule is double pyrenes or purpurin 18.
2. molecular probe according to claim 1, which is characterized in that the molecular probe has the structure as shown in following formula I:
3. the preparation method of molecular probe according to claim 1 or 2, which comprises the following steps:
(1) the responsiveness polypeptide is synthesized on resin not soluble in water using Fmoc solid phase synthesis process；
(2) responsiveness polypeptide is connected with the signaling molecule and water-solubility carrier molecule.
4. preparation method according to claim 3, which is characterized in that resin not soluble in water described in step (1) is
Wang resin.
5. the preparation method according to claim 4, which is characterized in that the modification density of the Wang resin is 0.3-
0.4mM。
6. the preparation method according to claim 4, which is characterized in that the modification density of the Wang resin is 0.35mM.
7. preparation method according to claim 3, which is characterized in that the specific steps of step (1) include:
(a) aminoterminal of first amino acid is protected with Fmoc, c-terminus is fixed on resin；
(b) it is protected with the Fmoc that the DMF solution of hexahydropyridine sloughs aminoterminal, next lysine is Dde using side-chain amino group
The DMF of the amino acid of protection, carboxyl 4- methyl morpholine and benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester is molten
Liquid is activated, and carries out condensation reaction with first amino acid of deprotection；
(c) aminoterminal of remaining amino acid is protected with Fmoc, is repeated step (b), the condensation until completing all amino acid.
8. preparation method according to claim 3, which is characterized in that the step (2) specifically includes: by the signal point
Sub- activated carboxylic, and it is connected to the aminoterminal of responsiveness polypeptide；By lysine side chain amino-reactive in responsiveness polypeptide, and connect
It is removed in the carrier molecule c-terminus, then from resin.
9. preparation method according to claim 8, which is characterized in that lysine side chain amino is living in the responsiveness polypeptide
Change and is carried out in the DMF solution containing 0.2% hydrazine hydrate.
10. preparation method according to claim 8, which is characterized in that the removing from resin in 4- methyl morpholine and
It is carried out in the DMF solution of benzotriazole-N, N, N', N'- tetramethylurea hexafluorophosphoric acid ester.
11. molecular probe according to claim 1 or 2 in the detection reagent for preparing internal cell in vitro autophagy enzyme ATG4 or
Application in the preparation of the internal horizontal real-time quantitative assessment of cell in vitro autophagy.
12. purposes according to claim 11, which is characterized in that the cell is tumour cell.
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