CN108379598A - Fibrin ferment intelligent response fluorescence/multi-modal molecular probes of MRI and its preparation method and application - Google Patents

Fibrin ferment intelligent response fluorescence/multi-modal molecular probes of MRI and its preparation method and application Download PDF

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CN108379598A
CN108379598A CN201810121529.7A CN201810121529A CN108379598A CN 108379598 A CN108379598 A CN 108379598A CN 201810121529 A CN201810121529 A CN 201810121529A CN 108379598 A CN108379598 A CN 108379598A
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lasg
fitc
fibrin ferment
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CN108379598B (en
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王亚斌
曹丰
杨瑞晨
侯毅
许梦琪
杨宁
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Chinese PLA General Hospital
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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10—Organic compounds
    • A61K49/14—Peptides, e.g. proteins

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Abstract

The invention belongs to biomedicine technical fields, and in particular to a kind of multi-modal molecular probe FITC LASG Fe of fibrin ferment intelligent response fluorescence/MRI3O4And its preparation method and application.The fluorescent quenching probe is to thrombin substrate small peptide LASG：Fluorescent molecular FITC is connected on Leu Arg Ser Lys Ser Arg Ser Phe Gln Val Ser Asp Glu Gln Tyr Pro Asp Ala Thr Asp Glu, is then coupled with ferroferric oxide nano granules by reacting.Ferriferrous oxide particles have certain absorption to FITC radiofluorescences.The probe can be applied in terms of the targets identification of drug, using animal model living imaging, the drug for screening targeting fibrin ferment.

Description

Fibrin ferment intelligent response fluorescence/multi-modal molecular probes of MRI and preparation method thereof and Using
Technical field
The invention belongs to biomedicine technical fields, and in particular to a kind of fibrin ferment intelligent response fluorescence/MRI is multi-modal Molecular probe FITC-LASG Fe3O4And its preparation method and application.
Background technology
Coronary atherosclerotic heart disease is to seriously threaten the killer of human health.For the anti-freezing of patients with coronary heart disease Treatment has become that current medical field is extremely significant and challenging field.Fibrin ferment is a kind of in human body anti-freezing, is promoted solidifying Coagulation cascade reaction in important role biological activity protein.Fibrin ferment is a kind of serine protease and blood Main effects protease in coagulation cascade reaction, shows the characteristic for promoting solidifying and anti-freezing.Fibrin ferment is by being answered in factor The nonactive factor closed in object is generated under the action of Xa factor (FXa) factor by way of protein cleavage.Work as cycle When exposed extravascular tissue is in contact with tissue factor, fibrin ferment can organizationally be assembled coagulation factor.Fibrin ferment passes through Blood platelet, catalysis fibre proteinogen is activated to be converted into fibrin, promotion clot is stable and the induction in thrombotic diseases has Central role.
LASG is a kind of solid-phase polypeptide (Leu-Arg-Ser-Lys-Ser-Arg-Ser-Phe-Gln- of standard preparation gained Val-Ser-Asp-Glu-Gln-Tyr-Pro-Asp-Ala-Thr-Asp-Glu),
The substrate that can be used as fibrin ferment forms 2 segment polypeptides after being cut by Thrombin specificity.Using LASG as carrier, structure It is a kind of to fibrin ferment intelligent response fluorescence/multi-modal molecular probes of MRI, monitoring, tracer in vivo thrombin activity situation can To be widely used in targets identification field, also there is considerable value to design fibrin ferment target medicine, be expected to realize blood The accurate diagnosis and treatment of bolt integration.
Invention content
The object of the present invention is to provide a kind of multi-modal molecular probe FITC- of fibrin ferment intelligent response fluorescence/MRI LASG- Fe3O4And its preparation method and application, to solve not can be used for monitoring the intelligence of internal thrombin activity in real time now The problem of responding fluorescence probe.
A kind of multi-modal molecular probe FITC-LASG-Fe of fibrin ferment intelligent response fluorescence/MRI3O4, fluorescent quenching shape State structure such as following formula：
Wherein,
Reaction equation is as follows：
The fibrin ferment intelligent response fluorescence/multi-modal molecular probe preparation methods of MRI include the following steps：
1) oil phase ferroso-ferric oxide core granule is prepared by raw material of ferric acetyl acetonade
2) maleic amide ligand PEG is prepared as primary raw material using polyethylene glycol (PEG), Malaysia aminocaproic acid
3) oil phase ferroso-ferric oxide core granule carries out ligand exchange reaction, obtains water phase Fe3O4-PEG
4) LASG small peptides are connect with FITC molecules obtains water phase LASG-FITC
5) fluorescence small peptide LASG-FITC modifies nano particle and obtains Fe3O4-LASG-FITC
The step (1) the specific steps are：By 4mmol ferric acetyl acetonades, 20mmol oleic acid, 20mmol octadecylenes, 10mmol octadecyl alcolols, 40ml diphenyl ether mix in three-necked flask, are passed through 20 nitrogen 20min and drain air.Flask connection is negative Pressure device heats simultaneously, when system temperature reaches 80 DEG C, keeps thermotonus 30min.Nitrogen is then passed to, continues to heat, until 220 DEG C, bubble is generated in system, until 250 DEG C, system bumping.Maintenance system temperature is reacted under 254 DEG C, nitrogen protection 30min.Reaction was completed, obtains oil phase iron particle.
The step (2) the specific steps are：It is small to weigh Malaysia aminocaproic acid 0.56g and 11.6ml oxalyl chlorides room temperature reaction 5 When.Then reaction system is connected into cold-trap and negative pressure device, 5ml anhydrous methylene chlorides is added, until obtaining powder.With 20ml bis- Chloromethanes dissolves, spare.5g amino-polyethyleneglycols (PEG) are dissolved with 90.8ml dichloromethane, 385 microlitres of triethylamines are added, Reserve liquid is added dropwise with constant pressure funnel in the system.Ice-water bath reaction is overnight.Products therefrom is precipitated with ice ether, after suction filtration Obtain maleic amide PEG.Phosphorous acid 0.6g, water 7.24ml, hydrochloric acid 0.36ml are mixed, are heated to 90 DEG C, maleic amide is added PEG products.It measures 0.97ml methanol and above-mentioned system is added dropwise with constant pressure funnel, be warming up to 110 DEG C of reaction 90min.Collect production Object, revolving 2h remove solvent.Then it is dissolved with absolute ethyl alcohol, is filtered 3 times with ice ether precipitation, be put into baking oven and be dried in vacuo Night obtains maleic amide ligand PEG.
The step (3) the specific steps are：Oil phase ferriferrous oxide particles 2ml is taken, is dissolved with 2ml tetrahydrofurans.Take horse Carry out amide PEG ligand 1 .6g, is dissolved with 8ml tetrahydrofurans.It is mixed in both above-mentioned in flask, 40 DEG C of oil bath stirrings 12 are small When.N-hexane precipitation is added, n-hexane volume is about 5-10 times of acquired solution.It shakes up rear 5000r/min to centrifuge 5 minutes, abandon Clear liquid.Tetrahydrofuran dissolving precipitation is added, continuously adds n-hexane, step as above is washed 3 times.It is put into vacuum drying oven overnight. Gained is precipitated with 2ml water dissolutions.
The step (4) the specific steps are：0.05gLASG small peptide powder is taken, with 10ml water dissolutions.0.01gFITC is added Powder adjusts PH to 9 or so with sodium dihydrogen phosphate.Tinfoil is wrapped up, shaking table reaction is overnight.The step (5) the specific steps are：It will Water phase nano particle obtained by step (3) is mixed with the fluorescence small peptide obtained by step (4), wraps up tinfoil, and shaking table reacts 2h.Gained Reaction solution is added in 3000Da super filter tubes, 5000r/min centrifugations 8 minutes, 5-8 times, until gained ultrafiltrate is without color.It collects Liquid phase probe in super filter tube, (is denoted as FITC-LASG-Fe3O4)。
In addition, the present invention provides fibrin ferment intelligent response fluorescence/multi-modal molecular probe FITC-LASG-Fe of MRI3O4 Application in terms of detecting thrombin activity
LASG:Leu-Arg-Ser-Lys-Ser-Arg-Ser-Phe-Gln-Val-Ser-Asp-Glu-Gln-Tyr-Pro- Asp-Ala-Thr-Asp-Glu。
The fibrin ferment intelligent response fluorescence/multi-modal molecular probe FITC-LASG-Fe of MRI3O4Blood coagulation is targeted for screening The drug biologic applications of enzyme.
The fibrin ferment intelligent response fluorescence/multi-modal molecular probe FITC-LASG-Fe of MRI3O4Animal model live body at Picture, the in real time application in terms of fibrin ferment is surveyed in physical examination.
The invention discloses a kind of multi-modal molecular probe FITC-LASG-Fe of fibrin ferment intelligent response fluorescence/MRI3O4 And its preparation method and application.
The fluorescent quenching probe is to thrombin substrate small peptide LASG：
Leu-Arg-Ser-Lys-Ser-Arg-Ser-Phe-Gln-Val-Ser-Asp-Glu-Gln-Tyr-Pro-Asp- Fluorescent molecular FITC is connected on Ala-Thr-Asp-Glu, is then coupled with ferroferric oxide nano granules by reacting.Four oxidations Three iron particles have certain absorption to FITC radiofluorescences.
Compared with the existing technology, benefit of the invention is that：The present invention provides a kind of fibrin ferment intelligence by MOLECULE DESIGN The energy response fluorescence/multi-modal molecular probes of MRI, is denoted as FITC-LASG-Fe3O4, and the study tour probe in vivo, body Outside with the interaction of fibrin ferment, by its change in fluorescence, comprehensive analysis activity and and medicine of the probe in detection fibrin ferment The application of activity aspect after object effect.The probe there are height selection evident characteristics, core polypeptide LASG to make fibrin ferment It for thrombin substrate, is broken after endonuclease reaction, fluorescent molecular FITC and Fe3O4Particle detaches, and sends out 518nm wavelength fluorescents. The fluorescence intensity of the probe and being proportionate property of thrombin activity, while the thrombin activity under different pharmaceutical effect can be detected Strong and weak variation.The probe can be applied in terms of the targets identification of drug, using animal model living imaging, for screening Target the drug of fibrin ferment.The probe as a kind of targeting tool molecule carrier, targets identification field and analysis drug effect, Drug encapsulation design aspect has important application value and foreground.The present invention, which solves, to be lacked currently on the market about fibrin ferment The problem of method of inspection that Activity determination, tracing in vivo are analyzed, there are significant application value and market prospects.
The present invention finally determines fibrin ferment intelligent response fluorescence/multi-modal molecular probes of MRI by repeatedly exploring Synthetic route and preparation method thereof, this method preparation method and last handling process are simple, high income, and price is low.And the preparation The LASG small peptides being applied in the process can be used as drug encapsulation shell, there is the application of new aspect.
Description of the drawings
Fig. 1 FITC-LASG-Fe3O4Phenogram, A.FITC-LASG-Fe3O4Transmission electron microscope picture；B.FITC-LASG- Fe3O4Particle diameter distribution；C.FITC-LASG-Fe3O4Zeta potential；D.FITC-LASG-Fe3O4Infrared absorption spectrum.
Fig. 2 FITC-LASG-Fe3O4With fluorescence imaging outside fibrin ferment reactant.
Fig. 3 FITC-LASG-Fe3O4In mouse thrombus model in-vivo imaging.
Specific implementation mode
The fibrin ferment intelligent response fluorescence/multi-modal molecular probe preparation methods of MRI include the following steps：
1) oil phase ferroso-ferric oxide core granule is prepared by raw material of ferric acetyl acetonade
2) maleic amide ligand PEG is prepared as primary raw material using polyethylene glycol (PEG), Malaysia aminocaproic acid
3) oil phase ferroso-ferric oxide core granule carries out ligand exchange reaction, obtains water phase Fe3O4-PEG
4) LASG small peptides are connect with FITC molecules obtains water phase LASG-FITC
5) fluorescence small peptide LASG-FITC modifies nano particle and obtains Fe3O4-LASG-FITC
The step (1) the specific steps are：By 4mmol ferric acetyl acetonades, 20mmol oleic acid, 20mmol octadecylenes, 10mmol octadecyl alcolols, 40ml diphenyl ether mix in three-necked flask, are passed through 20 nitrogen 20min and drain air.Flask connection is negative Pressure device heats simultaneously, when system temperature reaches 80 DEG C, keeps thermotonus 30min.Nitrogen is then passed to, continues to heat, until 220 DEG C, bubble is generated in system, until 250 DEG C, system bumping.Maintenance system temperature is reacted under 254 DEG C, nitrogen protection 30min.Reaction was completed, obtains oil phase iron particle.
The step (2) the specific steps are：It is small to weigh Malaysia aminocaproic acid 0.56g and 11.6ml oxalyl chlorides room temperature reaction 5 When.Then reaction system is connected into cold-trap and negative pressure device, 5ml anhydrous methylene chlorides is added, until obtaining powder.With 20ml bis- Chloromethanes dissolves, spare.5g amino-polyethyleneglycols (PEG) are dissolved with 90.8ml dichloromethane, 385 microlitres of triethylamines are added, Reserve liquid is added dropwise with constant pressure funnel in the system.Ice-water bath reaction is overnight.Products therefrom is precipitated with ice ether, after suction filtration Obtain maleic amide PEG.Phosphorous acid 0.6g, water 7.24ml, hydrochloric acid 0.36ml are mixed, are heated to 90 DEG C, maleic amide is added PEG products.It measures 0.97ml methanol and above-mentioned system is added dropwise with constant pressure funnel, be warming up to 110 DEG C of reaction 90min.Collect production Object, revolving 2h remove solvent.Then it is dissolved with absolute ethyl alcohol, is filtered 3 times with ice ether precipitation, be put into baking oven and be dried in vacuo Night obtains maleic amide ligand PEG.
The step (3) the specific steps are：Oil phase ferriferrous oxide particles 2ml is taken, is dissolved with 2ml tetrahydrofurans.Take horse Carry out amide PEG ligand 1 .6g, is dissolved with 8ml tetrahydrofurans.It is mixed in both above-mentioned in flask, 40 DEG C of oil bath stirrings 12 are small When.N-hexane precipitation is added, n-hexane volume is about 5-10 times of acquired solution.It shakes up rear 5000r/min to centrifuge 5 minutes, abandon Clear liquid.Tetrahydrofuran dissolving precipitation is added, continuously adds n-hexane, step as above is washed 3 times.It is put into vacuum drying oven overnight. Gained is precipitated with 2ml water dissolutions.
The step (4) the specific steps are：0.05g LASG small peptide powder is taken, with 10ml water dissolutions.0.01g is added FITC powder adjusts PH to 9 or so with sodium dihydrogen phosphate.Tinfoil is wrapped up, shaking table reaction is overnight.Step (5) specific steps For：Water phase nano particle obtained by step (3) is mixed with the fluorescence small peptide obtained by step (4), wraps up tinfoil, shaking table reaction 2h.Gained reaction solution is added in 3000Da super filter tubes, 5000r/min centrifugations 8 minutes, 5-8 times, until gained ultrafiltrate is without face Color.Liquid phase probe in super filter tube is collected, (is denoted as FITC-LASG-Fe3O4)。
Below to the multi-modal molecular probe FITC-LASG-Fe of fibrin ferment intelligent response fluorescence/MRI of the present invention3O4Table Sign and the experimentation of the fluorescence monitoring sensitive to fibrin ferment, result illustrate.
(1) absorption spectrum of probe.Concentration and probe concentration is set as (Fe：0.05mg/ml), spectrophotometer is tested.It can detect To the coupling of nanometer Fe core granule and LASG-FITC fluorescent polypeptides.
(2) the MR imaging parameters of probe.Concentration and probe concentration is set as (Fe:0.05,0.1,0.5,1,5mmol/L), NMR imaging Detection T2-map imagings (7T) in instrument.The MR imagings of iron particle nano-probe show as T2 as signal lowers.
(3) probe sem image, grain size.A concentration of (Fe of probe dilution：0.05mg/ml), 20 microlitres of drops are taken with sample injector On copper mesh, air-dry.Scanning electron microscope observation.Gained probe Fe core granules are more uniform, and grain size is about 7.9nm.
(4) probe zeta current potentials and hydration droplet measurement.By probe dilution to a concentration of (Fe:0.05mg/ml), it is added to " enter to " .05mg/ml needles be diluted to it is a concentration of (, in results sample pond, empty bubble in sample cell.Sample cell is sent into zeta Potential detector detects zeta current potentials and hydration grain size respectively.Nano-probe zeta current potentials are 6.45mV, with fluorescent polypeptide idol Grain size is 76nm after connection.
(5) interaction of probe and fibrin ferment.It is (Fe to take concentration and probe concentration:0.03mg/ml), with fibrin ferment 0.4U/ml, Reacted in 37 DEG C of water-baths, wave spectrum radiated respectively at different time points detection FITC, obtain time of probe thrombin action according to Rely curve.Fibrin ferment 0.4U/ml, concentration and probe concentration is taken to be respectively set to (Fe:0.25,0.1,0.5,1,2.5,5,10mg/ml), 37 2h is reacted in DEG C water-bath, detects FITC fluorescence emission spectras, and the concentration and probe concentration for obtaining probe thrombin action relies on curve.It visits A concentration of (Fe of needle:0.5mg/ml), concentration of thrombin gradient be set as 0,0.1,0.4,2,10,50U/ml, it is anti-in 37 DEG C of water-baths 2h is answered, FITC fluorescence emission spectras are detected, the concentration of thrombin for obtaining probe thrombin action relies on curve.Concentration and probe concentration is (Fe:0.5mg/ml), 1U fibrin ferments are added in first Kong Buzuo specially treated, second hole, and 1U fibrin ferments are added in third hole And 0.01g bivalirudins, 2h is reacted in 37 DEG C of water-baths, detects FITC fluorescence emission spectras, bivalirudin is observed by probe To the inhibiting effect of fibrin ferment.
Imaging research probe in detecting thrombin activity in Mice Body.Make mouse carotid thrombosis model：Along ApoE mouse Neck makees 1.5cm notch, and separating mouse unilateral side arteria carotis communis is infiltrated on fritter filter paper in 0.02mol/L liquor ferri trichloridis, Filter paper is wrapped into arteria carotis communis, continues 2min, filter paper is taken out, sews up the incision.After thrombus model, items coagulation indexes have in vivo It is significantly raised.Experiment mice is divided into 3 groups.First group, without thrombus model group, probe (dosage is same as above) tail vein injection is entered general In logical Mice Body.Second group is thrombus probe groups, by intelligent response probe (probe dosage：Fe 0.5mg) enter from tail vein injection In thrombus model Mice Body.Second group is set as control probe group, and the LASG small peptides in former probe are replaced with cysteine, FITC Fluorescence is quenched by Fe particles always, loses the effect of intelligent response.This is noted without intelligent response functional probe (dosage is same as above) It injects in thrombus model Mice Body.Before injection, injection after 5min, 15min, 30min, 1h, 2h time point acquisition it is small Fluorescence imaging data (Fig. 3) in mouse body.It can be found that fibrin ferment intelligent response fluorescence/multi-modal molecular probes of MRI can be examined Measure the raising of thrombin activity in thrombus model mouse body.

Claims (10)

1. a kind of multi-modal molecular probe FITC-LASG-Fe of fibrin ferment intelligent response fluorescence/MRI3O4, fluorescent quenching state Structure such as following formula：
Wherein,
2. the multi-modal molecular probe FITC-LASG-Fe of fibrin ferment intelligent response fluorescence/MRI described in claim 13O4System Preparation Method, reaction equation are as follows：
3. preparation method according to claim 2, includes the following steps：
1) oil phase ferroso-ferric oxide core granule is prepared by raw material of ferric acetyl acetonade；
2) maleic amide ligand PEG is prepared as primary raw material using polyethylene glycol (PEG), Malaysia aminocaproic acid；
3) oil phase ferroso-ferric oxide core granule carries out ligand exchange reaction, obtains water phase Fe3O4-PEG；
4) LASG small peptides are connect with FITC molecules obtains water phase LASG-FITC；
5) fluorescence small peptide LASG-FITC modifies nano particle and obtains Fe3O4-LASG-FITC。
4. preparation method according to claim 3, it is characterised in that：The step (1) the specific steps are：By 4mmol second Acyl acetone iron, 20mmol oleic acid, 20mmol octadecylenes, 10mmol octadecyl alcolols, 40ml diphenyl ether mix in three-necked flask, are passed through 20 nitrogen 20min drain air；Flask is connected into negative pressure device, is heated simultaneously, when system temperature reaches 80 DEG C, keeps temperature anti- Answer 30min；Nitrogen is then passed to, continues to heat, until 220 DEG C, bubble is generated in system, until 250 DEG C, system bumping；Maintain body It is that temperature reacts 30min under 254 DEG C, nitrogen protection；Reaction was completed, obtains oil phase iron particle.
5. preparation method according to claim 3, it is characterised in that：The step (2) the specific steps are：Weigh Malaysia ammonia Base caproic acid 0.56g is reacted at room temperature 5 hours with 11.6ml oxalyl chlorides；Then reaction system is connected into cold-trap and negative pressure device, be added 5ml anhydrous methylene chlorides, until obtaining powder；It is dissolved with 20ml dichloromethane, it is spare；By 5g amino-polyethyleneglycols (PEG) with 90.8ml dichloromethane dissolves, and 385 microlitres of triethylamines are added, reserve liquid is added dropwise with constant pressure funnel in the system；Ice water Bath reaction is overnight；Products therefrom is precipitated with ice ether, and maleic amide PEG is obtained after suction filtration；By phosphorous acid 0.6g, water 7.24ml, Hydrochloric acid 0.36ml mixing, is heated to 90 DEG C, maleic amide PEG products is added；0.97ml methanol is measured with constant pressure funnel dropwise to add Enter above-mentioned system, is warming up to 110 DEG C of reaction 90min；Product is collected, revolving 2h removes solvent；Then it is dissolved with absolute ethyl alcohol, with Ice ether precipitation filters 3 times, is put into baking oven and is dried in vacuum overnight, obtains maleic amide ligand PEG.
6. preparation method according to claim 3, it is characterised in that：The step (3) the specific steps are：Take four oxygen of oil phase Change three iron particle 2ml, is dissolved with 2ml tetrahydrofurans；Maleic amide PEG ligand 1 .6g are taken, are dissolved with 8ml tetrahydrofurans；It will be upper It states the two to be mixed in flask, 40 DEG C of oil baths are stirred 12 hours；N-hexane precipitation is added, n-hexane volume is about acquired solution 5- 10 times；It shakes up rear 5000r/min to centrifuge 5 minutes, abandons supernatant；Tetrahydrofuran dissolving precipitation is added, continuously adds n-hexane, such as Upper step is washed 3 times；It is put into vacuum drying oven overnight；Gained is precipitated with 2ml water dissolutions.
7. preparation method according to claim 3, it is characterised in that：The step (4) the specific steps are：It takes 0.05gLASG small peptide powder, with 10ml water dissolutions；0.01gFITC powder is added, PH to 9 or so is adjusted with sodium dihydrogen phosphate；Packet Tinfoil is wrapped up in, shaking table reaction is overnight；The step (5) the specific steps are：By the water phase nano particle and step obtained by step (3) (4) the fluorescence small peptide mixing obtained by, wraps up tinfoil, and shaking table reacts 2h；Gained reaction solution is added in 3000Da super filter tubes, 5000r/min centrifugations 8 minutes, 5-8 times, until gained ultrafiltrate is without color；Liquid phase probe in super filter tube is collected, FITC- is denoted as LASG-Fe3O4。
8. the multi-modal molecular probe FITC-LASG-Fe of fibrin ferment intelligent response fluorescence/MRI described in claim 13O4It is examining Survey the application in terms of thrombin activity
LASG:Leu-Arg-Ser-Lys-Ser-Arg-Ser-Phe-Gln-Val-Ser-Asp-Glu-Gln-Tyr-Pro-Asp- Ala-Thr-Asp-Glu。
9. the multi-modal molecular probe FITC-LASG-Fe of fibrin ferment intelligent response fluorescence/MRI described in claim 13O4For The drug biologic applications of screening targeting fibrin ferment.
10. the multi-modal molecular probe FITC-LASG-Fe of fibrin ferment intelligent response fluorescence/MRI described in claim 13O4 Animal model living imaging, the in real time application in terms of fibrin ferment is surveyed in physical examination.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113045672A (en) * 2021-02-09 2021-06-29 甘肃省科学院传感技术研究所 Magnetic fluorescent composite probe for detecting matrix metalloproteinase-2 and preparation method and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007149948A2 (en) * 2006-06-20 2007-12-27 The Gov. Of The Usa As Represented By The Secretary Of The Department Of Health And Human Services Compositions and methods for diagnosis and treatment of tumors
CN102085381A (en) * 2011-01-25 2011-06-08 上海师范大学 Ferroferric oxide-silicon dioxide-thymine nanoparticles and preparation method and application thereof
CN105833299A (en) * 2016-02-03 2016-08-10 中国人民解放军总医院 Preparation method of MRI/optical bimodal nanoprobe for atherosclerotic vulnerable plaque diagnosis
WO2016151297A1 (en) * 2015-03-20 2016-09-29 The University Court Of The University Of Edinburgh Optical probe for thrombin
CN106581698A (en) * 2016-12-21 2017-04-26 中国人民解放军总医院 Preparation method for ultrasonic fluorescence bimodal nano-probe for recognizing unstable plaque of atherosclerosis

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007149948A2 (en) * 2006-06-20 2007-12-27 The Gov. Of The Usa As Represented By The Secretary Of The Department Of Health And Human Services Compositions and methods for diagnosis and treatment of tumors
CN102085381A (en) * 2011-01-25 2011-06-08 上海师范大学 Ferroferric oxide-silicon dioxide-thymine nanoparticles and preparation method and application thereof
WO2016151297A1 (en) * 2015-03-20 2016-09-29 The University Court Of The University Of Edinburgh Optical probe for thrombin
CN105833299A (en) * 2016-02-03 2016-08-10 中国人民解放军总医院 Preparation method of MRI/optical bimodal nanoprobe for atherosclerotic vulnerable plaque diagnosis
CN106581698A (en) * 2016-12-21 2017-04-26 中国人民解放军总医院 Preparation method for ultrasonic fluorescence bimodal nano-probe for recognizing unstable plaque of atherosclerosis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
LONG MA,ET AL: "Polyethylenimine-coated Fe3O4 nanoparticles effectively quench fluorescent DNA, which can be developed as a novel platform for protein detection", 《NANOSCALE》 *
宋晟: "多功能医学显影探针的自组装途径构建及生物医学应用研究", 《中国博士学位论文全文数据库 医药卫生科技辑》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113045672A (en) * 2021-02-09 2021-06-29 甘肃省科学院传感技术研究所 Magnetic fluorescent composite probe for detecting matrix metalloproteinase-2 and preparation method and application thereof

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