CN104458685B - A kind of cell membrane imaging agent based on fluorescent nano particle - Google Patents
A kind of cell membrane imaging agent based on fluorescent nano particle Download PDFInfo
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- CN104458685B CN104458685B CN201410706068.1A CN201410706068A CN104458685B CN 104458685 B CN104458685 B CN 104458685B CN 201410706068 A CN201410706068 A CN 201410706068A CN 104458685 B CN104458685 B CN 104458685B
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Abstract
The invention discloses a kind of cell membrane imaging agent based on fluorescent nano particle, it is made up of component A and component B；The component A is that side chain contains biotin molecule unit and the macromolecule with amino of hydrophobic anchor unit；The component B is the fluorescent nano particle for being grafted with avidin molecule.The reagent is based on fluorescent nano particle and many site anchoring techniques, and good biocompatibility, imaging effect are excellent, and its fluorescent grain is on cell membrane without by endocytosis, the irradiation of long-time laser is difficult to be quenched, and can be utilized for long-time cell membrane trace labelling.
Description
Technical field
The invention belongs to biological technical field, more particularly to a kind of cell membrane fluorescence imaging examination based on fluorescent nano particle
Agent.
Background technology
Cell membrane not only in structure as cell border, provide a metastable interior environment for cell, simultaneously
Vital effect is also played to the matter transportation between cell and external environment, energy conversion and information exchanging process.Carefully
After birth imaging technique is marked and spike to cell membrane using fluorescent dye, disclosed as a kind of strong research meanses
Many important biological processes including pinocytosis, exocytosis, cell division and apoptosis.
However, endocytosis phenomenon can not only occur quickly for some traditional lipophilic dyes, imaging time is short；And these have
Machine small molecule dyes, it is easy to be quenched that (its is glimmering after laser excitation is irradiated 5-15 minutes for such as FITC organic fluorescences molecule
Luminous intensity is just quenched significantly), weaken fluorescence intensity, great limitation is brought to research.Nanometer technology is in bioluminescence imaging
The application in field possesses many advantages, especially fluorescence quantum and metalfluorescent nano-cluster, with very high fluorescent quantum production
Rate and photostability, allow them prolonged and carry out fluorescence labeling to cell and tissue and be unlikely to be quenched.However, at present
Fluorescent nano particle contribute to cell overall labeling and the spike of specific cells device greatly, and fail the spy for cell membrane
Opposite sex imaging, main reason is that fluorescent nano particle is easy to by cell endocytic in itself, so as to lose the spy of cell membrane marker
The opposite sex.In order to make full use of the advantage of fluorescent nano particle, a kind of cell membrane fluorometric reagent is developed based on fluorescent nano particle and shown
Obtain particularly important.
The content of the invention
Goal of the invention：It is an object of the invention to provide a kind of cell membrane imaging agent based on fluorescent nano particle, realize
Long-time imaging to cell membrane.
Technical scheme：The invention provides a kind of cell membrane fluorescence imaging reagent based on fluorescent nano particle, by component A
With component B compositions；The component A is that side chain contains biotin molecule unit and the macromolecule with amino of hydrophobic anchor unit,
Wherein hydrophobic anchor unit is collectively constituted by hydrophobic units and polyethylene glycol polymer；The component B divides to be grafted with Avidin
The fluorescent nano particle of son.Component B is connected by avidin molecule with the biotin molecule unit on component A.
As it is another preferably, the biotin molecule include (+) biotin-N- succinimide base esters；Its by with
Amino reactive grafting is on the macromolecule with amino.
As it is another preferably, in component B, the fluorescent nano particle includes fluorescence quantum, fluorescence nano cluster, glimmering
The nano particle of light group mark, and be grafted with the fluorescence quantum of biotin, fluorescence nano cluster, marked by fluorophor
Nano particle.
As it is another preferably, in component A, the macromolecule with amino is glycol-chitosan, and its molecular weight exists
More than 10000, preferably 10000-100000.
As it is another preferably, in component A, the hydrophobic units include cholesterol, phospholipid molecule, alkane or vitamin E；
It is linked on the high molecular amino with amino by polyethylene glycol polymer；The molecular weight of the polyethylene glycol polymer exists
More than 500, preferably 500-5000.
As it is another preferably, in component A, the quantity of the biotin molecule unit repeats single for the macromolecule with amino
The 5-30% of first number；The quantity of the hydrophobic anchor unit is the 10-30% of the macromolecule number of repeat unit with amino.
Beneficial effect：The cell membrane imaging agent that the present invention is provided is based on fluorescent nano particle and many site anchoring techniques,
Good biocompatibility, imaging effect are excellent, and its fluorescent grain is on cell membrane without by endocytosis, the irradiation of long-time laser is difficult to quench
Go out, can be utilized for long-time cell membrane trace labelling.
Specifically, the present invention has advantage following prominent relative to prior art：
(1) unit is chained up by macromolecule in the reagent, makes fluorescent nano particle not in the form of individual particle
Free movement is carried out, it is difficult endocytosis, it is ensured that cell membrane imaging specificity.Meanwhile, in the shitosan macromolecule of the reagent
The positive charge that remaining amino is carried, can occur electrostatic interaction, increase and the parent of cell membrane with negatively charged cell membrane
And power, thus further extend retention time of the dyestuff on cell membrane.
(2) the hydrophobic grappling effect in many sites can improve the anchoring efficiencies of macromolecule grappling reagent, biotin and avidin
High specific, which consolidates combination, can greatly save dyeing time, make the reagent good biocompatibility, imaging effect excellent.
(3) because the luminescence unit of the reagent is fluorescent nano particle (especially quantum dot and fluorescence nano cluster), so
Lower fluorescence is irradiated for a long time to be difficult to be quenched.
(4) reagent can be different by replacing as the cell membrane fluorescence imaging application platform based on fluorescent nano particle
Fluorescent nano particle, it is possible to achieve the fluorescence imaging of multiple color, including near-infrared fluorescence imaging.
Brief description of the drawings
Fig. 1 is the molecular structure of the cell membrane imaging agent based on fluorescent nano particle of embodiment 1；
Fig. 2 is the molecular structure of the cell membrane imaging agent based on fluorescent nano particle of embodiment 2；
Fig. 3 is the cell membrane imaging design sketch using cell membrane imaging agent of the present invention；
Fig. 4 is the cell membrane imaging effect after cell membrane imaging agent of the present invention is irradiated 1 hour under the conditions of most strong exciting light
Fruit is schemed；
Fig. 5 is the nano copper sulfate particle imaging effect figure that FITC is modified；
Fig. 6 is the nano SiO 2 particle imaging effect figure that rhodamine is modified.
Embodiment
Embodiment 1
Cell membrane fluorescence imaging reagent based on fluorescent nano particle, is made up of component A and component B.Component A：
Chitosan-10%cholesterol-10%biotin, component B：avidin-QDs(CdSe@ZnS).
Specifically, the molecular structure of the cell membrane fluorescence imaging reagent based on fluorescent nano particle is shown in Fig. 1, by group
Divide A and component B compositions.Wherein, component A is with glycol-chitosan macromolecule (glycolchitosan) for skeleton, and side chain contains
10% polyethylene glycol 2000-cholesterol (PEG2000-cholesterol) hydrophobic units and 10% biotin molecule
(biotin)；Glycol-chitosan macromolecule, with good biocompatibility and water solubility, the degree of polymerization is about 400, molecular weight
For more than 10000, experiment proves that molecular weight can realize the present invention well in 10000-100000 chitosan；Cholesterol is dredged
Water anchor unit, by PEG2000 of the molecular weight more than 500, (experiment proves that molecular weight can realize this in 500-5000
Goal of the invention) it is connected in amino of chitosan, i.e. NHS-PEG2000-cholesterol, increase is water-soluble.Wherein, component B by
The CdSe@ZnS quantum dots for being grafted with Avidin (avidin) molecule are constituted.The CdSe@ZnS of Avidin (avidin) molecular modification
Quantum dot, amino reaction during by being prepared by quantum dot on the carboxyl and avidin molecule on surface is made.
Cholesterol hydrophobic units insert cell membrane by many sites of hydrophobic interaction, by glycol-chitosan macromolecule
Skeleton is anchored on cell membrane；Then, fluorescence quantum is grafted by the strong bonded of biotin and avidin high-affinity
On macromolecule, quantum dot-labeled cell membrane imaging is realized.
After A549 lung carcinoma cells are adherent in copolymerization Jiao's culture plate, component A (100 μ g/mL) is first added and in incubator
It is incubated 30 minutes, subsequent PBS 1 time adds component B and is incubated in incubator 2 minutes.Dyeing terminates rear PBS 3
It is secondary, observe and take pictures under laser scanning co-focusing microscope, imaging effect is as shown in Figure 3.
Sample excites (excitation wavelength 488nm, about energy 100%, 3mW) Continuous irradiation under the conditions of largest light intensity after dyeing
After one hour, fluorescence signal only somewhat weakens, imaging effect is as shown in Figure 4 still substantially without being quenched.
Above experimental result, which fully demonstrates fluorescent nano particle, is used for the excellent light stability that cell membrane imaging has.
Embodiment 2
Cell membrane fluorescence imaging reagent based on fluorescent nano particle, is made up of component A and component B.Component A：
Chitosan-10%cholesterol-10%biotin, component B1：avidin-biotin-CuS-FITC.
Specifically, the molecular structure of the cell membrane fluorescence imaging reagent based on fluorescent nano particle is shown in Fig. 2, by group
Divide A and component B compositions.Wherein, component A is with glycol-chitosan macromolecule (glycolchitosan) for skeleton, and side chain contains
10% polyethylene glycol 2000-cholesterol (PEG2000-cholesterol) hydrophobic units and 10% biotin molecule
(biotin)；Glycol-chitosan macromolecule, with good biocompatibility and water solubility, the degree of polymerization is about 400, molecule
Measure as more than 10000；Cholesterol hydrophobic side chain, is connected in amino of chitosan by PEG2000 of the molecular weight more than 500,
That is NHS-PEG2000-cholesterol, increase is water-soluble.Wherein, the copper sulphide nano that component B is modified by FITC and biotin
Particle (biotin-CuS-FITC) is constituted, and it is grafted with avidin；Copper sulfide (CuS) nanometer of FITC and biotin modifications
Grain, makes its surface take amino, then pass through amino and FITC reaction by HS-PEG2000-NH2 and copper sulfide reaction first
Reaction with amino and NHS-biotin obtains biotin-CuS-FITC.
Cholesterol hydrophobic units can insert cell membrane by many sites of hydrophobic interaction, by glycol-chitosan high score
Sub- skeleton is anchored on cell membrane；Component B biotin molecule is carried out after strong bonded with avidin molecule, then with high-molecular bone
Avidin strong bonded on frame, so that the nano copper sulfate particle with FITC fluorophors is grafted on macromolecule, it is real
Existing cell membrane imaging.Imaging effect is as shown in Figure 5.
Embodiment 3
The implementation of the present embodiment is similar with the method in embodiment 2, and simply component B is grafting Avidin mixed with sieve
The nano SiO 2 particle of red bright dyestuff, imaging effect is as shown in Figure 6.
Embodiment 4
The implementation of the present embodiment is similar with the method in embodiment 1, and simply component B, which is followed successively by, is grafted with Avidin point
The fluorescence gold nanoclusters or silver nanoclusters of son.
Embodiment 5
The implementation of the present embodiment is similar with the method in embodiment 1, simply NHS-PEG2000-cholesterol points
Cholesterol hydrophobic units in son make phospholipid molecule (1,2- myristoyl phosphatidyl-ethanolamine, DMPE), alkane (ten into successively
Eight carbon atoms, C18) or the hydrophobic side chain such as vitamin E, synthesis obtains three kinds of cell membrane fluorescence imaging reagents.
Embodiment 6
The implementation of the present embodiment is similar with the method in embodiment 1, simply the anchor unit in imaging agents component A
The percentage for accounting for shitosan number of repeat unit is changed to 30%, and the percentage that biotin units account for shitosan number of repeat unit is changed to
5%, obtained imaging agents composition is chitosan-30%cholesterol-5%biotin.
Embodiment 7
The implementation of the present embodiment is similar with the method in embodiment 1, simply the anchor unit in imaging agents component A
The percentage for accounting for shitosan number of repeat unit is changed to 10%, and the percentage that biotin units account for shitosan number of repeat unit is changed to
30%, obtained imaging agents composition is chitosan-10%cholesterol-30%biotin.
Claims (7)
1. a kind of cell membrane fluorescence imaging reagent based on fluorescent nano particle, it is characterised in that：It is made up of component A and component B；
The component A is that side chain contains biotin molecule unit and the macromolecule with amino of hydrophobic anchor unit, wherein hydrophobic grappling
Unit is collectively constituted by hydrophobic units and polyethylene glycol polymer, and the connected mode of the hydrophobic anchor unit is logical for hydrophobic units
Polyethylene glycol polymer is crossed to be linked on the high molecular amino with amino；The component B is be grafted with avidin molecule glimmering
Light nano particle.
2. a kind of cell membrane fluorescence imaging reagent based on fluorescent nano particle according to claim 1, its feature exists
In：The biotin includes (+) biotin-N- succinimide base esters.
3. a kind of cell membrane fluorescence imaging reagent based on fluorescent nano particle according to claim 1, its feature exists
In：In component B, the fluorescent nano particle includes fluorescence quantum, fluorescence nano cluster, the nanometer marked by fluorophor
Grain, and it is grafted with the fluorescence quantum of biotin, fluorescence nano cluster, the nano particle marked by fluorophor.
4. a kind of cell membrane fluorescence imaging reagent based on fluorescent nano particle according to claim 1, its feature exists
In：In component A, the macromolecule with amino is glycol-chitosan, and its molecular weight is more than 10000.
5. a kind of cell membrane fluorescence imaging reagent based on fluorescent nano particle according to claim 1, its feature exists
In：In component A, the hydrophobic units include cholesterol, phospholipid molecule, alkane or vitamin E.
6. a kind of cell membrane fluorescence imaging reagent based on fluorescent nano particle according to claim 1, its feature exists
In：The hydrophobic units are linked on the high molecular amino with amino by polyethylene glycol polymer, the polyethylene glycol high score
The molecular weight of son is more than 500.
7. a kind of cell membrane fluorescence imaging reagent based on fluorescent nano particle according to claim 1, it is characterised in that：
In component A, the quantity of the biotin molecule unit is the 5-30% of the macromolecule number of repeat unit with amino；The hydrophobic anchor
The quantity of order member is the 10-30% of the macromolecule number of repeat unit with amino.
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| CN104888219B (en) * | 2015-06-10 | 2017-11-03 | 东南大学 | A kind of tumour phototherapy reagent coated based on cell membrane and its preparation method and application |
| CN105969334B (en) * | 2016-05-06 | 2018-07-20 | 东南大学 | Fluorescent dyeing reagent, Preparation method and use for detection bacterium and fungi life or death state |
| CN110846286B (en) * | 2019-09-12 | 2022-11-22 | 天津大学 | Method for labeling Sendai virus envelope by using rare earth up-conversion fluorescent particles |
| CN112843256A (en) * | 2019-11-26 | 2021-05-28 | 上海微知卓生物科技有限公司 | Cell capable of tracing in organism, preparation method thereof and application thereof in cell pharmacokinetics research |
| CN111678900B (en) * | 2020-06-30 | 2023-03-21 | 东南大学 | Cell membrane and cell wall fluorescence labeling method based on tyrosinase catalysis and application |
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| CN101643645A (en) * | 2009-07-24 | 2010-02-10 | 华中科技大学 | Calcium phosphate material marked by fluorescein isothiocyanate and preparation method thereof |
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