WO2021190112A1 - 一种多肽-量子点复合探针、制备方法和应用 - Google Patents

一种多肽-量子点复合探针、制备方法和应用 Download PDF

Info

Publication number
WO2021190112A1
WO2021190112A1 PCT/CN2021/072873 CN2021072873W WO2021190112A1 WO 2021190112 A1 WO2021190112 A1 WO 2021190112A1 CN 2021072873 W CN2021072873 W CN 2021072873W WO 2021190112 A1 WO2021190112 A1 WO 2021190112A1
Authority
WO
WIPO (PCT)
Prior art keywords
polypeptide
quantum dot
dot composite
targeting
probe
Prior art date
Application number
PCT/CN2021/072873
Other languages
English (en)
French (fr)
Inventor
肖建喜
蔡向东
Original Assignee
兰州大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 兰州大学 filed Critical 兰州大学
Publication of WO2021190112A1 publication Critical patent/WO2021190112A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
    • C09K11/88—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing selenium, tellurium or unspecified chalcogen elements
    • C09K11/881—Chalcogenides
    • C09K11/883—Chalcogenides with zinc or cadmium
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/02—Use of particular materials as binders, particle coatings or suspension media therefor
    • C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
    • 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"

Definitions

  • the invention belongs to the field of semiconductor quantum dot materials, and specifically relates to a polypeptide-quantum dot composite probe, a preparation method and an application.
  • Semiconductor quantum dots are a new type of nanomaterial composed of elements of group II-VI or group III-V. Because of its special optical properties, compared with traditional fluorescent dyes, it has good light stability, strong fluorescence intensity, The wide range of excitation spectrum, symmetrical and narrow half-width of emission spectrum, long fluorescence lifetime, diverse and adjustable colors, high quantum yield, etc., have broad application prospects in biological imaging.
  • Peptide quantum dots are a new type of biomarker probe formed by connecting peptide molecules on the surface of quantum dots, and are currently a hot research topic.
  • CdTe quantum dots can cover almost the entire visible spectrum due to their strong quantum confinement effect, and have been widely used in electronic devices and sensors.
  • CdTe quantum dots cause cytotoxicity due to the release of toxic Cd 2+ ions in cell experiments, which greatly limits their application in biological detection.
  • the application of CdTe quantum dots in in vivo imaging requires that they have good optical performance, smaller particle size and lower toxicity. Therefore, the development of new CdTe quantum dots that are soluble in water, high fluorescence intensity, small particle size, and low toxicity is essential to develop their biomedical applications.
  • the fat-soluble quantum dots CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe containing Zn are the most widely used in biomarkers.
  • biomarking There are generally two methods for biomarking: 1 First replace the ligands on the surface of the fat-soluble quantum dots with sulfhydryl small molecules, and then connect them to biomolecules such as peptides through electrostatic adsorption or coupling agents.
  • the introduction of linking groups on biomolecules, the Zn atoms on the surface of quantum dots can be It forms a metal affinity coordination effect with the linking group, and can directly connect the protein or polypeptide containing the linking group to the Zn atom on the surface of the quantum dot CdSe/ZnS.
  • the peptide quantum dots prepared by this method have better stability, it depends on the affinity of the connecting group to the Zn atoms on the surface of the quantum dot CdSe/ZnS, which has certain limitations.
  • this method requires multiple steps to be realized, and the process is relatively complicated: 1 Preparation of hydrophobic semiconductor quantum dots by organic phase synthesis; 2 Further modification by modifiers such as difunctional sulfhydryl ligands, silica and polymers, Make them hydrophilic and biocompatible; 3 bind polypeptide molecules to quantum dots.
  • the method also has the following defects: 1
  • 1 The organic phase synthesis requires the use of a large amount of organic solvents and organometallic reagents, the reaction temperature is high and the toxicity is high; 2
  • 2 The additional complex modification process causes the particle size of the quantum dots to increase and the fluorescence quantum yield decreases , Is not conducive to biological applications; 3
  • 3 The subsequent polypeptide modification process is cumbersome and complicated, with many side reactions, low modification efficiency, poor stability of the obtained probe, and the modification process easily destroys the structure of the targeted polypeptide.
  • the method of synthesizing quantum dots in aqueous phase using sulfhydryl groups as surface modification reagents has attracted widespread attention.
  • the sulfhydryl groups mainly include ⁇ -thioglycerol, thioglycolic acid, mercaptopropionic acid, cysteine, and acetylcysteine.
  • Chinese patent CN103897699B discloses a kind of thioglycerol as a surface modifier, mixing cadmium solution, zinc solution, tellurium solution and polypeptide solution at 90-100 °C constant temperature heating for 1-48 hours and then cooling to obtain polypeptide-quantum dot nanocomposite Solution.
  • this method does not modify the linking group on the polypeptide, which will cause the problem of unstable binding between the polypeptide molecule and the surface of the quantum dot.
  • polyhistidine produces chelation with the metal cations of the quantum dot shell through the side chain imidazole group, which can serve as a bridge for the directional connection of quantum dots and peptides without interfering with the functions of proteins and quantum dots. It is a promising direction for the development of quantum dot biomarkers.
  • a functional polyethylene glycol modified tail containing polyhistidine ( His 6 ) peptide, and the peptide-CdTe quantum dot nanoprobe was synthesized by a one-step method in water phase.
  • connection method of the peptide segment must adopt the hydrazone reaction of aldehyde and amine. The process is complicated, and the competition between hydrazone formation and hydrazone formation reaction is fierce. In the path of product formation, it is also easy to decompose into the original substrate, which leads to the rupture of the linking group and poor stability.
  • Chinese patent CN104231035B discloses a kind of histidine-N-lactam acid anhydride as a polyamino acid monomer, and a polypeptide with a terminal amino group as an initiator to initiate the ring-opening polymerization of histidine-N-lactam acid anhydride to achieve poly
  • the histidine chain segment is covalently coupled with the functional polypeptide, and the polyhistidine side chain imidazole group is chelated with the quantum dot shell to form a quantum dot-polypeptide complex.
  • the preparation process of the method is complicated, and one or more organic solvents such as dimethylformamide, tetrahydrofuran, acetonitrile, dichloromethane, chloroform, methanol, ethanol, toluene, etc. are required to be introduced, which easily leads to residual organic solvents and is relatively toxic.
  • organic solvents such as dimethylformamide, tetrahydrofuran, acetonitrile, dichloromethane, chloroform, methanol, ethanol, toluene, etc. are required to be introduced, which easily leads to residual organic solvents and is relatively toxic.
  • the present invention has unexpectedly discovered through a large number of experimental studies that glutathione is used as a surface modifier for quantum dots; a short chain of charged amino acids with a Cys end as a linking group is used to modify the targeting polypeptide (Cys-X)- Targeting domain: The Te solution and the Cd solution are mixed and heated to reflux, and the targeting polypeptide (Cys-X)-Targeting domain solution is slowly added dropwise, and refluxed for 15-300 minutes to prepare a polypeptide-quantum dot composite probe.
  • the method uses glutathione as the surface modifier of quantum dots, which is compatible with Cys in the targeted polypeptide, and the prepared polypeptide-quantum dot composite probe has good stability; and glutathione is a naturally synthesized peptide in the human body. Good biocompatibility and low toxicity.
  • the method has mild conditions, is environmentally friendly, and has a simple process, and the prepared polypeptide-quantum dot composite probe can be used for in vivo imaging.
  • the purpose of the present invention is to provide a polypeptide-quantum dot composite probe, the polypeptide-quantum dot composite probe comprising quantum dots CdTe coated with glutathione on the surface and a targeting polypeptide (Cys-X ) -Targeting domain, the Targeting domain is a targeting polypeptide sequence, the Cys-X is a linking group, the X is a charged amino acid, and the X is connected to the N-terminus and/or C-terminus of the Targeting domain, The Cys is combined with quantum dots CdTe.
  • the polypeptide-quantum dot composite probe comprising quantum dots CdTe coated with glutathione on the surface and a targeting polypeptide (Cys-X ) -Targeting domain
  • the Targeting domain is a targeting polypeptide sequence
  • the Cys-X is a linking group
  • the X is a charged amino acid
  • the X is connected to the N-terminus and/or C-terminus
  • the X is Asp or Glu.
  • the X is Glu.
  • the Targeting domain includes one or more of protein targeting peptides, cell targeting peptides, and metal ion targeting peptides.
  • the Targeting domain is a protein targeting peptide.
  • the Targeting domain is a diseased collagen targeting peptide or a type I collagen targeting peptide.
  • the Targeting domain is a cell targeting peptide.
  • the Targeting domain is to target cells by targeting organelles.
  • the organelle is endoplasmic reticulum or cell nucleus.
  • the Targeting domain is to target the cell by targeting the cell receptor on the cell.
  • the cell receptor is an integrin.
  • the glutathione is L-glutathione and/or D-glutathione.
  • Another object of the present invention is to provide a method for preparing a polypeptide-quantum dot composite probe, the method comprising the following steps:
  • step (3) After cooling the polypeptide-quantum dot composite probe solution described in step (3), add 1-2 times the volume of isopropanol to precipitate the probe, collect the probe by centrifugation, wash it with isopropanol, and air dry. Obtain polypeptide-quantum dot composite probe.
  • the concentration of the targeting polypeptide (Cys-X)-Targeting domain in the step (3) is 2.5-10 mg/ml.
  • the molar ratio of Cd to Te in the step (3) is 1:0.6-0.3.
  • Another object of the present invention is to provide an application of a polypeptide-quantum dot composite probe in the preparation of protein imaging reagents, cell receptor imaging reagents or organelle imaging reagents.
  • the beneficial effects of the present invention are: 1
  • the polypeptide-quantum dot composite probe provided by the present invention uses glutathione as the surface modifier of quantum dots, and uses the charged amino acid short chain with Cys end as the linking group to modify the polypeptide sequence.
  • Targeting polypeptide (Cys-X)-Targeting domain, the linking group can be compatible with glutathione modified on the surface of quantum dots;
  • the polypeptide-quantum dot composite probe provided by the present invention can be used in complex cell culture media It does not damage and can complete dyeing within a few hours, and has good stability;
  • the glutathione used in the present invention is a naturally synthesized peptide in the human body, with low biocompatibility and low toxicity, and can be used for in vivo imaging; 4provided by the present invention
  • the preparation method of the polypeptide-quantum dot composite probe is simple, the conditions are mild, and it can be synthesized in one step without further quantum dot modification.
  • Figure 2 Staining diagrams of diseased collagen targeting peptide-quantum dot composite probes with different emission wavelengths on mouse diseased bladder tissue;
  • Figure 3 Specific staining diagram of diseased collagen targeting peptide-quantum dot composite probe
  • Figure 4 The staining image of type I collagen targeting peptide-quantum dot composite probe on mouse tail tissue
  • Figure 5 Staining diagram of the nuclear targeting peptide-quantum dot composite probe on HeLa cells
  • Figure 7 Staining image of the endoplasmic reticulum targeting peptide-quantum dot composite probe on HeLa cells.
  • a polypeptide-quantum dot composite probe includes a plurality of the polypeptide-quantum dot composite probes
  • reference to “the polypeptide-quantum dot composite probe” refers to one or more Various polypeptide-quantum dot composite probes and their equivalents known to those skilled in the art, and so on.
  • Quantum dots can also be called semiconductor nanocrystals or artificial atoms. They are semiconductor crystals containing any number of electrons between 100 and 1,000 and having a size of about 2 nm to 10 nm. They are mainly composed of II- in the periodic table of elements. Group VI (such as CdTe, CdS, CdSe, etc.) or Group III-V (InP, InAs) and Group IV-VI (such as PbS, PbSe) elements.
  • Group VI such as CdTe, CdS, CdSe, etc.
  • Group III-V InP, InAs
  • Group IV-VI such as PbS, PbSe
  • coating refers to that the surface of the quantum dot CdTe is modified with glutathione, and the linking group Cys-X in the targeting polypeptide binds the targeting polypeptide and the quantum dot.
  • polypeptide refers to two or more amino acid residues connected to each other by peptide bonds or modified peptide bonds.
  • the term applies to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers that contain modified residues and non-naturally occurring amino acids. Of amino acid polymers.
  • the reflux time of composite probes with different wavelengths is as follows: 530nm probe Needle reflow for 15 minutes, 545nm probe reflow for 30 minutes, 560nm probe reflow for 45 minutes, 575nm probe reflow for 60 minutes, 585nm probe reflow for 70 minutes, 600nm probe reflow for 90 minutes, 620nm probe reflow for 120 minutes, 635nm probe reflow for 150 minutes, and 645nm probe reflow 180min, 680nm probe refluxed for 240min.
  • nuclear targeting peptide-quantum dot composite probe solutions with different emission wavelengths
  • nuclear targeting peptide-quantum dot composite probes After the solution is cooled, 2 times the volume of isopropanol is added to precipitate the probe, the probe is collected by centrifugation, washed with isopropanol and air-dried to obtain nuclear targeting peptide-quantum dot composite probes with different emission wavelengths.
  • a polypeptide-quantum dot probe solution with a concentration of 1 ⁇ g/ml was prepared, and 2ml of the solution was taken to measure the fluorescence emission spectrum.
  • the excitation wavelength is 365nm, and the emission wavelength scanning range is 450-700nm.
  • curve 1 is a type I collagen targeting peptide-quantum dot composite probe with an emission wavelength of 530 nm
  • curve 3 is a type I collagen targeting peptide-quantum dot composite probe with an emission wavelength of 560 nm
  • Curve 10 is the type I collagen targeting peptide-quantum dot composite probe with emission wavelength of 680nm
  • curve 2 is the lesion collagen targeting peptide-quantum dot composite probe with emission wavelength of 545nm
  • curve 4 is the lesion with emission wavelength 575nm
  • curve 6 is the diseased collagen targeting peptide-quantum dot composite probe with emission wavelength of 600nm
  • curve 7 is the diseased collagen targeting peptide-quantum dot composite probe with emission wavelength of 620nm Needle
  • curve 9 is the diseased collagen targeting peptide-quantum dot composite probe with emission wavelength of 645nm
  • curve 8 is the nuclear targeting peptide-quantum dot composite probe with emission wavelength of 6
  • the staining results of different emission wavelength lesion collagen targeting peptide-quantum dot composite probes on damaged mouse bladder tissue are shown in Figure 2, where a and e are the 530nm emission wavelength lesion collagen targeting peptide-quantum dot composite probe
  • the staining results, b and f are the staining results of the lesioned collagen targeting peptide-quantum dot composite probe with an emission wavelength of 545nm
  • c and g are the staining results of the lesioned collagen targeting peptide-quantum dot composite probe with an emission wavelength of 585nm
  • d and h are the staining results of the collagen targeting peptide-quantum dot composite probe with 620nm emission wavelength.
  • the above staining results show that the collagen targeting peptide-quantum dot composite probe with different emission wavelengths can stain the mouse bladder. organization.
  • tissue samples from different parts of the mouse Take tissue samples from different parts of the mouse to prepare frozen sections to 4 ⁇ m; after the sections are cleaned, add blocking solution to the tissue section, and then aspirate the liquid after placing it for 30 minutes. Take 100 ⁇ L of 0.1mg/ml probe solution for staining and incubate at 4°C for 6h; After removing the staining solution; wash 3 times with 1x PBS, and wash the unbound probe with the washing solution; drop the mounting solution, add a cover glass, and use a fluorescence microscope to observe and take pictures;
  • the staining result, b is the staining result of the diseased collagen targeting peptide-quantum dot composite probe on the normal tail tissue of mice
  • c is the staining result of the mouse diseased tail tissue by the quantum dots not labeled with the targeting peptide
  • d is The staining result of diseased collagen targeting peptide-quantum dot composite probe on mouse diseased ear tissue
  • e is the staining result of diseased collagen targeting peptide-quantum dot composite probe on mouse normal ear tissue
  • f is not Staining results of targeted peptide-labeled quantum dots on diseased ear tissue of mice.
  • the staining results show that the diseased tissue can be stained by the diseased collagen targeting peptide-quantum dot composite probe, but cannot be stained by the unlabeled quantum dot; meanwhile, the diseased collagen targeting peptide-quantum dot composite probe does not stain the normal tissue .
  • the above results indicate that the polypeptide-quantum dot composite probe prepared by this method not only provides the inherent excellent luminescence properties of the quantum dot, but also gives the probe superior target recognition ability.
  • the staining result of type I collagen targeting peptide-quantum dot composite probe on mouse tail tissue is shown in Figure 4, where a is the type I collagen targeting peptide-quantum dot composite probe with 585nm emission wavelength.
  • the staining image of tail tissue b is the staining image of type I collagen targeting peptide-quantum dot composite probe with 600nm emission wavelength on mouse tail tissue.
  • the above staining image shows that type I collagen targeting peptides with different emission wavelengths -Quantum dot composite probes can stain mouse tail tissues.
  • HeLa cells were fixed with 3.7% paraformaldehyde for 30 minutes and treated with 0.05% Triton X-100 for 2 minutes. Wash once with PBS. A 0.01 mg/ml nuclear targeting peptide-quantum dot composite probe was stained at room temperature for 2 hours, and DAPI stained for 5 minutes. The cells were washed 3 times with PBS. Use a fluorescence microscope to observe and take pictures.
  • HeLa cells were stained with 0.01 mg/ml integrin targeting peptide-quantum dot composite probe at room temperature for 6 hours, and the cells were washed 3 times with PBS. Fix with 3.7% paraformaldehyde for 30 minutes and wash once with PBS. DAPI staining for 5 min, washing cells with PBS 3 times. Use a fluorescence microscope to observe and take pictures.
  • the staining results of integrin targeting peptide-quantum dot composite probe with 620nm emission wavelength on HeLa cells are shown in Figure 6, where a is the staining image of integrin targeting peptide-quantum dot composite probe on cells, and b is DAPI The staining image of the cell nucleus, c is the superimposed staining image of the DAPI and integrin targeting peptide-quantum dot composite probe on the cell.
  • the above staining results indicate that the integrin targeting peptide-quantum dot composite probe targets the integrin in the cell membrane.
  • Endoplasmic reticulum targeting peptide-quantum dot composite probe stains HeLa cells
  • HeLa cells were stained with 0.01 mg/ml endoplasmic reticulum targeting peptide-quantum dot composite probe at room temperature for 2 hours, and the cells were washed 3 times with PBS. Fix with 3.7% paraformaldehyde for 30 minutes and wash once with PBS. DAPI staining for 5 min, washing cells with PBS 3 times. Use a fluorescence microscope to observe and take pictures.
  • the staining result of the endoplasmic reticulum targeting peptide-quantum dot composite probe on HeLa cells with 620nm emission wavelength is shown in Figure 7, where a is the staining image of the endoplasmic reticulum targeting peptide-quantum dot composite probe on the cells, and b is The staining image of the endoplasmic reticulum targeting peptide-quantum dot composite probe and DAPI at the same time.
  • the above staining results indicate that the probe targets the endoplasmic reticulum.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Materials Engineering (AREA)
  • Organic Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Inorganic Chemistry (AREA)
  • Optics & Photonics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

一种多肽-量子点复合探针，以谷胱甘肽为量子点表面修饰剂，以末端为Cys的带电荷的氨基酸短链为连接基团修饰多肽序列获得靶向多肽(Cys-X)-Targeting domain。多肽-量子点复合探针的制备方法：将Te溶液与Cd溶液混合加热至回流，缓慢滴加合成的靶向多肽(Cys-X)-Targeting domain溶液，回流15-300min。多肽-量子点复合探针以谷胱甘肽为量子点表面修饰剂，能够与靶向多肽中的Cys兼容，制备的多肽-量子点复合探针稳定性良好；谷胱甘肽为人体中自然合成的肽，生物相容性好、毒性低；并且该方法条件温和、绿色环保、工艺简单，不需进一步的量子点修饰即可一步合成，制备的多肽-量子点复合探针可用于体内成像。

Description

一种多肽-量子点复合探针、制备方法和应用
本申请要求申请日为2020年3月27日、申请号为CN202010226767.1、发明名称为“一种多肽-量子点复合探针、制备方法和应用”的中国在先申请的优先权，该在先申请的全部内容均已在本申请中体现。
技术领域
本发明属于半导体量子点材料领域，具体涉及一种多肽-量子点复合探针、制备方法和应用。
背景技术
半导体量子点，是一种由Ⅱ-Ⅵ族或Ⅲ-Ⅴ族元素组成的新型纳米材料，由于其具有特殊的光学性能，与传统的荧光染料相比，具有光稳定性好、荧光强度强、激发光谱范围宽、发射光谱半峰宽对称且狭窄、荧光寿命长、颜色多样可调、量子产率高等优点，在生物成像方面具有广阔的应用前景。
多肽量子点是在量子点表面连接多肽分子形成的一种新型生物标记探针，是目前研究的热点。其中，CdTe量子点由于其强大的量子限制效应，它的荧光几乎可以覆盖整个可见光谱范围，已经被广泛应用于电子设备以及传感器等领域。然而，CdTe量子点在细胞实验中因释放出有毒Cd 2+离子而引起细胞毒性，极大的限制了其在生物检测中的应用。同时，CdTe量子点体内成像中的应用，要求其具备良好的光学性能、较小的粒径和较低的毒性。因此，开发新型可溶于水、高荧光强度、小粒径、低毒性的CdTe量子点，对开拓它们的生物医学应用至关重要。
目前含有Zn的脂溶性量子点CdSe/ZnS、CdTe/ZnS、CdSe/ZnSe或CdTe/ZnSe在生物标记中应用最为广泛。其在进行生物标记时，一般有两种方法：①先通过巯基小分子取代脂溶性量子点表面的配体，然后通过静电吸附或偶联剂与多肽等生物分子连接。但是，静电吸附受环境影响大、稳定性差；而偶联一般需要引入化学试剂，反应效率低、副反应多、毒性较大；②在生物分子上引入连接基团，量子点表面的Zn原子可以和连接基团形成金属亲和协调作用，能够将含有连接基团的蛋白质或多肽直接接到量子点CdSe/ZnS表面的Zn原子上。虽然这种方法制备的多肽量子点稳定性较好，但是，它依赖连接基团与量子点CdSe/ZnS表面的Zn原子的亲和力来实现，具有一定的局限性。并且，该方法需要多个步骤才能实现，过程比较复杂：①通过有机相合成方法制备疏水性的半导体量子点；②通过双官能团的巯基配体、二氧化硅和聚合物等修饰剂进一步修饰，使它们具有亲水性和生物相容性；③将多肽分子结 合到量子点上。该方法还存在如下缺陷：①有机相合成需要使用大量的有机溶剂和有机金属试剂，反应温度高且毒性大；②额外复杂的修饰过程，导致量子点的粒径变大、荧光量子产率降低，不利于生物应用；③后续的多肽修饰过程繁琐复杂、副反应多、修饰效率低、所得探针稳定性差，且修饰过程容易破坏靶向多肽的结构。
为了克服上述不足，以巯基为表面修饰试剂的水相合成量子点的方法受到广泛关注。所述的巯基主要包括α-硫代甘油、巯基乙酸、巯基丙酸、半胱氨酸以及乙酰半胱氨酸等。其中，中国专利CN103897699B公开了一种以硫代甘油为表面修饰剂，将镉溶液、锌溶液、碲溶液以及多肽溶液混合90-100℃恒温加热1-48h后冷却获得多肽-量子点纳米复合物溶液。然而该方法未在多肽上修饰连接基团，会导致多肽分子与量子点表面结合不稳定的问题。
最新研究发现，聚组氨酸通过侧链咪唑基团与量子点壳层的金属阳离子产生螯合作用，可作为量子点与多肽的定向连接的桥梁，且不会干扰蛋白质和量子点的功能，是发展量子点生物标记有潜力的方向。例如，文献(贾静,功能化多肽量子点的制备及其生物医学应用[D].南方医科大学,2013.)中设计了一种功能性聚乙二醇修饰的尾部含有聚组氨酸(His 6)的多肽，并以一步法水相合成了多肽-CdTe量子点纳米探针，但是，天然蛋白质中很少含有聚组氨酸，并且聚组氨酸作为量子点生物标记桥梁与功能性肽段连接方式须采用醛和胺的腙化反应，其过程复杂，成腙与成脎反应竞争激烈。产品成脎路径中，还易分解为原底物，从而导致连接基团断裂，稳定性差。中国专利CN104231035B公开了一种以组氨酸-N-内羧酸酐为聚氨基酸单体，以具有末端氨基的多肽为引发剂，引发组氨酸-N-内羧酸酐的开环聚合，实现聚组氨酸链段与功能性多肽的共价偶联，并以聚组氨酸侧链咪唑基团与量子点壳层螯合连接，形成量子点-多肽复合物。该方法制备过程复杂，需要引入二甲基甲酰胺、四氢呋喃、乙腈、二氯甲烷、三氯甲烷、甲醇、乙醇、甲苯等一种或者几种有机溶剂，容易导致有机溶剂残留，毒性较大。
基于此，本发明通过大量的实验研究意外发现，以谷胱甘肽作为量子点表面修饰剂；用末端为Cys的带电荷的氨基酸短链为连接基团修饰靶向多肽(Cys-X)-Targeting domain；将Te溶液与Cd溶液混合加热至回流，缓慢滴加靶向多肽(Cys-X)-Targeting domain溶液，回流15-300min，制备了多肽-量子点复合探针。该方法以谷胱甘肽为量子点表面修饰剂，能够与靶向多肽中的Cys兼容，制备的多肽-量子点复合探针稳定性良好；并且谷胱甘肽为人体中自然合成的肽，生物相容性好、毒性低。该方法条件温和、绿色环保、工艺简单，制备的多肽-量子点复合探针可用于体内成像。
发明内容
针对上述技术问题，本发明的目的在于提供一种多肽-量子点复合探针，所述多肽-量子点复合探针包括表面包覆谷胱甘肽的量子点CdTe和靶向多肽(Cys-X)-Targeting domain，所述Targeting domain为靶向多肽序列，所述Cys-X为连接基团，所述X为带电荷的氨基酸，所述X与Targeting domain的N端和/或C端连接，所述Cys与量子点CdTe结合。
优选地，所述的X为Asp或Glu。
优选地，所述的X为Glu。
优选地，所述Targeting domain包括蛋白质靶向肽、细胞靶向肽、金属离子靶向肽中的一种或几种。
优选地，所述Targeting domain为蛋白质靶向肽。
优选地，所述Targeting domain为病变胶原蛋白靶向肽或Ⅰ型胶原蛋白靶向肽。
优选地，所述Targeting domain为细胞靶向肽。
优选地，所述Targeting domain是通过靶向细胞器来靶向细胞。
优选地，所述的细胞器为内质网或细胞核。
优选地，所述Targeting domain是通过靶向细胞上的细胞受体来靶向细胞。
优选地，所述细胞受体为整合素。
优选地，所述谷胱甘肽为L-谷胱甘肽和/或D-谷胱甘肽。
本发明的另一目的在于提供一种多肽-量子点复合探针的制备方法，所述方法包括以下步骤：
(1)将谷胱甘肽与氯化镉溶解于超纯水中，得Cd溶液，所得溶液由氢氧化钠调节pH到10-13，脱氧搅拌待用；
(2)称取碲粉和硼氢化钠，脱氧后加入脱氧超纯水，于50-70℃反应得Te溶液；
(3)将Te溶液加入到Cd溶液中，100℃加热至回流，缓慢滴加1-2mL的靶向多肽(Cys-X)-Targeting domain溶液，回流15-300min，获得多肽-量子点复合探针溶液；
(4)将步骤(3)所述的多肽-量子点复合探针溶液冷却后，加入1-2倍体积的异丙醇沉淀探针，离心收集探针并由异丙醇洗涤后风干，即得多肽-量子点复合探针。
优选地，所述步骤(3)中靶向多肽(Cys-X)-Targeting domain的浓度为2.5-10mg/ml。
优选地，所述步骤(3)中Cd与Te的摩尔比为1:0.6-0.3。
本发明的另一目的在于提供一种多肽-量子点复合探针在制备蛋白质成像试剂，细胞受体成像试剂或细胞器成像试剂中的应用。
本发明的有益效果是：①本发明提供的多肽-量子点复合探针以谷胱甘肽为量子点表面修饰剂，以末端为Cys的带电荷的氨基酸短链为连接基团修饰多肽序列获得靶向多肽 (Cys-X)-Targeting domain，所述连接基团能够与量子点表面修饰的谷胱甘肽兼容；②本发明提供的多肽-量子点复合探针可以在成分复杂的细胞培养基中数小时不破坏并能完成染色，具有良好的稳定性；③本发明使用的谷胱甘肽为人体中自然合成的肽，生物相容性、毒性低，可用于体内成像；④本发明提供的多肽-量子点复合探针的制备方法简单、条件温和、不需进一步的量子点修饰即可一步合成。
附图说明
图1不同发射波长的多肽-量子点探针的发射光谱；
图2不同发射波长的病变胶原蛋白靶向肽-量子点复合探针对小鼠病变膀胱组织的染色图；
图3病变胶原蛋白靶向肽-量子点复合探针特异性染色图；
图4 I型胶原蛋白靶向肽-量子点复合探针对小鼠尾组织的染色图；
图5细胞核靶向肽-量子点复合探针对HeLa细胞的染色图；
图6整合素靶向肽-量子点复合探针对HeLa细胞的染色图；
图7内质网靶向肽-量子点复合探针对HeLa细胞的染色图。
具体实施方式
为使本发明实现的技术手段、创作特征、达成目的与功效易于明白了解，下面结合具体实施方式，进一步阐述本发明。但本发明的保护范围并不局限于以下实施例所述。
定义
除非另外定义，否则本发明使用的所有技术和科学术语具有的含义与所述方法所属领域的普通技术人员通常理解的含义相同。尽管与本文所述的那些类似或等同的任何方法和组合物也可用于实践或测试所述方法和组合物，但现在描述具有代表性的示例方法和组合物。也应了解本发明所用的术语仅出于描述特定实施方案的目的，并且不意图限制本发明的将仅由随附权利要求限制的范围。
必须注意，除非上下文另外明确规定，否则如本文以及随附权利要求中所用，单数形式“一个/一种”和“所述”包括复数个(种)指示物。举例来说，提及“一种多肽-量子点复合探针”包括多种所述多肽-量子点复合探针，提及“所述多肽-量子点复合探针”是提及一种或多种多肽-量子点复合探针及其为本领域技术人员所知的等效物，依此类推。
“包含(包括)”意指“包括但不限于”，并且不意图排除例如其它组分、整数等。特定来说，在陈述为“包括金属离子靶向肽”，明确涵盖包括所列内容(除非包括限制 性术语诸如“由...组成”)，这意味着不意图排除例如未列于所述雷人中的其它组分。
在提供范围值的情况下，应理解，该范围的上限和下限之间的每个中间值以及在所述范围内的任何其他所述或中间值包含在所述方法和组合物内。这些较小范围的上限和下限可以独立地包括在所述较小范围内，并且也包括在所述方法和组合物内，受所述范围中任何特别排除的限制。在所述范围包括一个或两个限制的情况下，排除那些所包括的限制之一或两者的范围也包括在方法和组合物中。
应当理解，为了清楚起见，在单独的实施方式的上下文中描述的某些特征也可以在单个实施方式中组合提供。相反，为简洁起见，在单个实施方式的上下文中描述的方法和组合物的各种特征也可单独提供或以任何合适的次组合提供。
对于本领域技术人员在阅读本公开内容时将显而易见的是，本文描述和表示出的每个单独实施方式具有可以容易地与任何其他实施例的特征分离或组合的独立组件和特征，而不脱离本方法的范围或精神。任何列举的方法可以按照所述事件的顺序或以逻辑上可能的任何其他顺序进行。
术语“量子点”又可称为半导体纳米微晶体或人造原子，它们是包含100至1,000之间的任何数量的电子并且大小为约2nm至10nm的半导体晶体，主要由元素周期表中II-VI族(如CdTe，CdS，CdSe等)或III-V族(InP，InAs)及IV-VI族(如PbS，PbSe)元素组成。
术语“包覆”是指量子点CdTe的表面修饰有谷胱甘肽，所述靶向多肽中的连接基团Cys-X将靶向多肽和量子点结合。
术语“多肽”，“肽”和“蛋白质”在本文中可互换使用，是指通过肽键或修饰的肽键彼此连接的两个或更多个氨基酸残基。该术语适用于氨基酸聚合物，其中一个或更多个氨基酸残基是相应天然存在的氨基酸的人工化学模拟物，也适用于天然存在的氨基酸聚合物，它们含有经修饰的残基和非天然存在的氨基酸聚合物。
实施例1病变胶原蛋白靶向肽-量子点复合探针的制备
1.病变胶原蛋白靶向肽-量子点复合探针的制备
称取90mg谷胱甘肽与22.7mg氯化镉溶解于30ml超纯水中，得Cd溶液，所得溶液由1M氢氧化钠调节pH到11.5，脱氧搅拌待用；称取12.75mg碲粉和10mg硼氢化钠，脱氧后加入1ml脱氧超纯水，于65℃反应得Te溶液；将0.5mlTe溶液加入到Cd溶液中，100℃加热回流，其中不同发生波长复合探针的回流时间如下：530nm探针回流15min，545nm探针回流30min，560nm探针回流45min，575nm探针回流60min，585nm探针回流70min，600nm 探针回流90min，620nm探针回流120min，635nm探针回流150min，645nm探针回流180min，680nm探针回流240min。回流至指定时间后，缓慢滴加1ml含10mg病变胶原蛋白靶向肽CE-(GPO) 8-NH 2的溶液，回流15min，分别获得不同发射波长的多肽-量子点复合探针溶液；多肽-量子点复合探针溶液冷却后，加入2倍体积的异丙醇沉淀探针，离心收集探针并由异丙醇洗涤后风干，即得不同发射波长的病变胶原蛋白靶向肽-量子点复合探针。
实施例2 I型胶原蛋白靶向肽-量子点复合探针的制备
称取90mg谷胱甘肽与22.7mg氯化镉溶解于30ml超纯水中，得Cd溶液，所得溶液由1M氢氧化钠调节pH到12.0，脱氧搅拌待用；称取12.75mg碲粉和10mg硼氢化钠，脱氧后加入1ml脱氧超纯水，于65℃反应得Te溶液；将0.55mlTe溶液加入到Cd溶液中，100℃加热回流，回流时间如下：530nm探针回流15min，545nm探针回流30min，560nm探针回流45min，575nm探针回流60min，585nm探针回流70min，600nm探针回流90min，620nm探针回流120min，635nm探针回流150min，645nm探针回流180min，680nm探针回流240min。回流至指定时间后，缓慢滴加1ml含10mg I型胶原蛋白靶向肽CE-Ahx-LRELHLNNN-NH 2的溶液，回流15min，获得不同发射波长的I型胶原蛋白靶向肽-量子点复合探针溶液；多肽-量子点复合探针溶液冷却后，加入2倍体积的异丙醇沉淀探针，离心收集探针并由异丙醇洗涤后风干，即得不同发射波长的I型胶原蛋白靶向肽-量子点复合探针。
实施例3细胞核靶向肽-量子点复合探针的制备
称取90mg谷胱甘肽与22.7mg氯化镉溶解于30ml超纯水中，得Cd溶液，所得溶液由1M氢氧化钠调节pH到12.0，脱氧搅拌待用；称取12.75mg碲粉和10mg硼氢化钠，脱氧后加入1ml脱氧超纯水，于65℃反应得Te溶液；将0.5mlTe溶液加入到Cd溶液中，100℃加热回流，回流时间如下：530nm探针回流15min，545nm探针回流30min，560nm探针回流45min，575nm探针回流60min，585nm探针回流70min，600nm探针回流90min，620nm探针回流120min，635nm探针回流150min。回流至指定时间后，缓慢滴加1.5ml含7mg TAT靶向肽的溶液，回流15min，获得不同发射波长的细胞核靶向肽-量子点复合探针溶液；细胞核靶向肽-量子点复合探针溶液冷却后，加入2倍体积的异丙醇沉淀探针，离心收集探针并由异丙醇洗涤后风干，即得不同发射波长的细胞核靶向肽-量子点复合探针。
实施例4整合素靶向肽-量子点复合探针的制备
称取90mg谷胱甘肽与22.7mg氯化镉溶解于30ml超纯水中，得Cd溶液，所得溶液由1M氢氧化钠调节pH到12.5，脱氧搅拌待用；称取12.75mg碲粉和10mg硼氢化钠，脱氧后 加入1ml脱氧超纯水，于65℃反应得Te溶液；将0.6mlTe溶液加入到Cd溶液中，100℃加热回流，回流时间如下：530nm探针回流15min，545nm探针回流30min，560nm探针回流45min，575nm探针回流60min，585nm探针回流70min，600nm探针回流90min，620nm探针回流120min，635nm探针回流150min。回流至指定时间后，缓慢滴加1ml含5mg RGD靶向肽的溶液，回流15min，获得不同发射波长的整合素靶向肽-量子点复合探针溶液；整合素靶向肽-量子点复合探针溶液冷却后，加入2倍体积的异丙醇沉淀探针，离心收集探针并由异丙醇洗涤后风干，即得不同发射波长的整合素靶向肽-量子点复合探针。
实施例5内质网靶向肽-量子点复合探针的制备
称取90mg谷胱甘肽与22.7mg氯化镉溶解于30ml超纯水中，得Cd溶液，所得溶液由1M氢氧化钠调节pH到12.5，脱氧搅拌待用；称取12.75mg碲粉和10mg硼氢化钠，脱氧后加入1ml脱氧超纯水，于65℃反应得Te溶液；将0.6mlTe溶液加入到Cd溶液中，100℃加热回流，回流时间如下：530nm探针回流15min，545nm探针回流30min，560nm探针回流45min，575nm探针回流60min，585nm探针回流70min，600nm探针回流90min，620nm探针回流120min，635nm探针回流150min。回流至指定时间后，缓慢滴加1ml含5mg ER靶向肽的溶液，回流15min，获得不同发射波长的内质网靶向肽-量子点复合探针溶液；多肽-量子点复合探针溶液冷却后，加入2倍体积的异丙醇沉淀探针，离心收集探针并由异丙醇洗涤后风干，即得不同发射波长的内质网靶向肽-量子点复合探针。
实施例6不同发射波长的多肽-量子点探针发射光谱测定
配制浓度为1μg/ml的多肽-量子点探针溶液，取2ml溶液测定荧光发射光谱。激发波长为365nm，发射波长扫描范围为450-700nm。结果如图1所示，其中曲线1为发射波长530nm的I型胶原蛋白靶向肽-量子点复合探针，曲线3为发射波长560nm的I型胶原蛋白靶向肽-量子点复合探针；曲线10为发射波长680nm的I型胶原蛋白靶向肽-量子点复合探针；曲线2为发射波长为545nm的病变胶原蛋白靶向肽-量子点复合探针；曲线4为发射波长575nm的病变胶原蛋白靶向肽-量子点复合探针；曲线6为发射波长600nm的病变胶原蛋白靶向肽-量子点复合探针；曲线7为发射波长620nm的病变胶原蛋白靶向肽-量子点复合探针；曲线9为发射波长645nm的病变胶原蛋白靶向肽-量子点复合探针；曲线8为发射波长635nm的细胞核靶向肽-量子点复合探针；曲线5为发射波长585nm的整合素靶向肽-量子点复合探针。以上结果说明本方法制备的多肽-量子点复合探针发射波长可覆盖绿光到近红外波长范围。
实施例7组织染色
1.不同发射波长病变胶原蛋白靶向肽-量子点复合探针对损伤小鼠膀胱组织的染色
取损伤小鼠膀胱组织样品制备冰冻切片至4μm；切片清洗后，在组织切片上加封闭液，放置30min后吸走液体，取0.1mg/ml上述病变胶原蛋白靶向肽-量子点复合探针溶液100μL进行染色，4℃孵育6h；吸去染色液后；使用1x PBS洗涤3次，洗液清洗未结合的探针；滴封固液，加盖玻片，使用荧光显微镜观察拍照。
不同发射波长病变胶原蛋白靶向肽-量子点复合探针对损伤小鼠膀胱组织的染色结果如图2所示，其中a和e为530nm发射波长病变胶原蛋白靶向肽-量子点复合探针的染色结果，b和f为545nm发射波长病变胶原蛋白靶向肽-量子点复合探针的染色结果，c和g为585nm发射波长病变胶原蛋白靶向肽-量子点复合探针的染色结果，d和h为620nm发射波长病变胶原蛋白靶向肽-量子点复合探针的染色结果，上述染色结果表明，不同发射波长病变胶原蛋白靶向肽-量子点复合探针均能染色损伤小鼠膀胱组织。
2.病变胶原蛋白靶向肽-量子点复合探针对病变组织的特异性染色
取小鼠不同部位组织样品制备冰冻切片至4μm；切片清洗后，在组织切片上加封闭液，放置30min后吸走液体，取0.1mg/ml探针溶液100μL进行染色，4℃孵育6h；吸去染色液后；使用1x PBS洗涤3次，洗液清洗未结合的探针；滴封固液，加盖玻片，使用荧光显微镜观察拍照；
530nm发射波长的病变胶原蛋白靶向肽-量子点复合探针对各种组织染色结果如图3所示，其中a为病变胶原蛋白靶向肽-量子点复合探针对小鼠病变尾巴组织的染色结果，b为病变胶原蛋白靶向肽-量子点复合探针对小鼠正常尾巴组织的染色结果，c为未被靶向肽标记的量子点对小鼠病变尾巴组织的染色结果，d为病变胶原蛋白靶向肽-量子点复合探针对小鼠病变耳组织的染色结果，e为病变胶原蛋白靶向肽-量子点复合探针对小鼠正常耳组织的染色结果，f为未被靶向肽标记的量子点对小鼠病变耳组织的染色结果。染色结果表明，病变组织能够被病变胶原蛋白靶向肽-量子点复合探针染色，但不能被未标记量子点染色；同时，病变胶原蛋白靶向肽-量子点复合探针未对正常组织染色。以上结果说明，本方法制备的多肽-量子点复合探针在提供量子点固有优良发光性质的同时，赋予了探针优越的靶向识别能力。
3. I型胶原蛋白靶向肽-量子点复合探针对小鼠尾组织的染色
取小鼠尾巴组织样品制备冰冻切片至4μm；切片清洗后，在组织切片上加封闭液，放置30min后吸走液体，取0.1mg/ml探针溶液100μL进行染色，4℃孵育6h；吸去染色液后；使 用1x PBS洗涤3次，洗液清洗未结合的探针；滴封固液，加盖玻片，使用荧光显微镜观察拍照。
I型胶原蛋白靶向肽-量子点复合探针对小鼠尾组织的染色结果如图4所示，其中a为585nm发射波长的I型胶原蛋白靶向肽-量子点复合探针对小鼠尾组织的染色图，b为600nm发射波长的I型胶原蛋白靶向肽-量子点复合探针对小鼠尾组织的染色图，上述染色图表明，不同发射波长的I型胶原蛋白靶向肽-量子点复合探针均能染色小鼠尾组织。
实施例8细胞染色
1.细胞核靶向肽-量子点复合探针对HeLa细胞染色
HeLa细胞由3.7％多聚甲醛固定30min，0.05％Triton X-100处理2min。由PBS洗涤1次。取0.01mg/ml的细胞核靶向肽-量子点复合探针于室温染色2h，DAPI染色5min。PBS洗涤细胞3次。使用荧光显微镜观察拍照。
530nm发射波长的细胞核靶向肽-量子点复合探针对HeLa细胞染色结果如图5所示，其中a为DAPI对细胞核的染色图，b为细胞核靶向肽-量子点复合探针对细胞核的染色图，c为DAPI和细胞核靶向肽-量子点复合探针对细胞核的叠加染色图。上述染色结果表明，探针靶向染色细胞核。
2.整合素靶向肽-量子点复合探针对HeLa细胞染色
HeLa细胞由0.01mg/ml整合素靶向肽-量子点复合探针于室温染色6h，PBS洗涤细胞3次。3.7％多聚甲醛固定30min，由PBS洗涤1次。DAPI染色5min，PBS洗涤细胞3次。使用荧光显微镜观察拍照。
620nm发射波长的整合素靶向肽-量子点复合探针对HeLa细胞的染色结果如图6所示，其中a为整合素靶向肽-量子点复合探针对细胞的染色图，b为DAPI对细胞核的染色图，c为DAPI和整合素靶向肽-量子点复合探针对细胞的叠加染色图。上述染色结果表明，整合素靶向肽-量子点复合探针靶向于细胞膜中整合素。
3.内质网靶向肽-量子点复合探针对HeLa细胞染色
HeLa细胞由0.01mg/ml内质网靶向肽-量子点复合探针于室温染色2h，PBS洗涤细胞3次。3.7％多聚甲醛固定30min，由PBS洗涤1次。DAPI染色5min，PBS洗涤细胞3次。使用荧光显微镜观察拍照。
620nm发射波长的内质网靶向肽-量子点复合探针对HeLa细胞染色结果如图7所示，其中a为内质网靶向肽-量子点复合探针对细胞的染色图，b为内质网靶向肽-量子点复合探针和DAPI同时对细胞的染色图。上述染色结果表明，探针靶向染色内质网。
以上所述仅为本发明的个别示范性实施案例的细节，对于本领域的技术人员来说，本发明在实际应用过程中根据具体的制备条件可以有各种更改和变化，并不用于限制本发明。凡在本发明的精神和原则之内，均应包含在本发明的保护范围之内。

Claims (16)

  1. 一种多肽-量子点复合探针，其特征在于，所述多肽-量子点复合探针包括表面包覆谷胱甘肽的量子点CdTe和靶向多肽(Cys-X)-Targeting domain；所述Targeting domain为靶向多肽序列；所述Cys-X为连接基团，所述X为带电荷的氨基酸，所述X与Targeting domain的N端和/或C端连接，所述Cys与量子点CdTe结合。
  2. 如权利要求1所述的多肽-量子点复合探针，其特征在于，所述的X为Asp或Glu。
  3. 如权利要求2所述的多肽-量子点复合探针，其特征在于，所述的X为Glu。
  4. 如权利要求1所述的多肽-量子点复合探针，其特征在于，所述Targeting domain包括蛋白质靶向肽、细胞靶向肽、金属离子靶向肽中的一种或几种。
  5. 如权利要求4所述的多肽-量子点复合探针，其特征在于，所述Targeting domain为蛋白质靶向肽。
  6. 如权利要求5所述的多肽-量子点复合探针，其特征在于，所述Targeting domain为病变胶原蛋白靶向肽或Ⅰ型胶原蛋白靶向肽。
  7. 如权利要求4所述的多肽-量子点复合探针，其特征在于，所述Targeting domain为细胞靶向肽。
  8. 如权利要求7所述的多肽-量子点复合探针，其特征在于，所述Targeting domain是通过靶向细胞器来靶向细胞。
  9. 如权利要求8所述的多肽-量子点复合探针，其特征在于，所述的细胞器为内质网或细胞核。
  10. 如权利要求7所述的多肽-量子点复合探针，其特征在于，所述Targeting domain是通过靶向细胞上的细胞受体来靶向细胞。
  11. 如权利要求10所述的多肽-量子点复合探针，其特征在于，所述细胞受体为整合素。
  12. 如权利要求1所述的多肽-量子点复合探针，其特征在于，所述谷胱甘肽为L-谷胱甘肽和/或D-谷胱甘肽。
  13. 如权利要求1-12任一所述多肽-量子点复合探针的制备方法，其特征在于，所述方法包括以下步骤：
    (1)将谷胱甘肽与氯化镉溶解于超纯水中，得Cd溶液，所得溶液由氢氧化钠调节pH到10-13，脱氧搅拌待用；
    (2)称取碲粉和硼氢化钠，脱氧后加入脱氧超纯水，于50-70℃反应得Te溶液；
    (3)将Te溶液加入到Cd溶液中，100℃加热至回流，缓慢滴加1-2mL的靶向多肽(Cys-X)-Targeting domain溶液，回流15-300min，获得多肽-量子点复合探针溶液；
    (4)将步骤(3)所述的多肽-量子点复合探针溶液冷却后，加入1-2倍体积的异丙醇沉淀探 针，离心收集探针并由异丙醇洗涤后风干，即得多肽-量子点复合探针。
  14. 如权利要求13所述的方法，其特征在于，所述步骤(3)中靶向多肽(Cys-X)-Targeting domain的浓度为2.5-10mg/ml。
  15. 如权利要求13所述的方法，其特征在于，所述步骤(3)中Cd与Te的摩尔比为1:0.6-0.3。
  16. 如权利要求1-12任一所述多肽-量子点复合探针在制备蛋白质成像试剂、细胞受体成像试剂或细胞器成像试剂中的应用。
PCT/CN2021/072873 2020-03-27 2021-01-20 一种多肽-量子点复合探针、制备方法和应用 WO2021190112A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202010226767.1 2020-03-27
CN202010226767.1A CN111410962B (zh) 2020-03-27 2020-03-27 一种多肽-量子点复合探针、制备方法和应用

Publications (1)

Publication Number Publication Date
WO2021190112A1 true WO2021190112A1 (zh) 2021-09-30

Family

ID=71487771

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/072873 WO2021190112A1 (zh) 2020-03-27 2021-01-20 一种多肽-量子点复合探针、制备方法和应用

Country Status (2)

Country Link
CN (1) CN111410962B (zh)
WO (1) WO2021190112A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114920798A (zh) * 2022-05-09 2022-08-19 国家纳米科学中心 一种在伤口部位原位构筑的自组装材料及其制备方法和应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111410962B (zh) * 2020-03-27 2021-02-02 兰州大学 一种多肽-量子点复合探针、制备方法和应用
CN113512089B (zh) * 2021-06-30 2023-06-13 兰州大学 一种水溶性量子点的多肽稳定剂及应用
CN117384246B (zh) * 2023-12-11 2024-05-10 北京大学第三医院（北京大学第三临床医学院） 一种多肽及含该多肽的水凝胶、其制备方法与应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1793283A (zh) * 2005-12-08 2006-06-28 上海交通大学 含半胱氨酸多肽辅助合成高发光碲化镉量子点的方法
US20120107800A1 (en) * 2010-09-17 2012-05-03 Subramanian Tamil Selvan Cell-targeting nanoparticles and uses thereof
CN110231327A (zh) * 2019-03-11 2019-09-13 兰州大学 一种靶向识别胶原蛋白的多肽拉曼探针及其制备和成像方法
CN111410962A (zh) * 2020-03-27 2020-07-14 兰州大学 一种多肽-量子点复合探针、制备方法和应用

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103830745B (zh) * 2014-03-11 2017-05-17 南通大学 一种基于量子点的多功能纳米siRNA载体系统的制备及其应用
CN106568757A (zh) * 2016-11-10 2017-04-19 常州大学 一种检测结肠癌肿瘤的量子点靶向探针试剂盒

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1793283A (zh) * 2005-12-08 2006-06-28 上海交通大学 含半胱氨酸多肽辅助合成高发光碲化镉量子点的方法
US20120107800A1 (en) * 2010-09-17 2012-05-03 Subramanian Tamil Selvan Cell-targeting nanoparticles and uses thereof
CN110231327A (zh) * 2019-03-11 2019-09-13 兰州大学 一种靶向识别胶原蛋白的多肽拉曼探针及其制备和成像方法
CN111410962A (zh) * 2020-03-27 2020-07-14 兰州大学 一种多肽-量子点复合探针、制备方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAN, SIHAI: "Preparation of Quantum Dots Fluorescent Probes and Their Applications in Cellular and in Vivo Imaging", CHINESE DOCTORAL THESIS ZHEJIANG UNIVERSITY, no. 2013/08, 12 June 2012 (2012-06-12), CN, pages 1 - 94, XP009531095 *
HE HUA, FENG MIN, HU JING, CHEN CUIXIA, WANG JIQIAN, WANG XIAOJUAN, XU HAI, LU JIAN R.: "Designed Short RGD Peptides for One-Pot Aqueous Synthesis of Integrin-Binding CdTe and CdZnTe Quantum Dots.", APPLIED MATERIALS & INTERFACES, vol. 4, no. 11, 29 October 2012 (2012-10-29), US, pages 6362 - 6370, XP055853643, ISSN: 1944-8244, DOI: 10.1021/am3020108 *
JIA, JING: "Synthesis of Peptide-programmed Quantum Dots for Biomedical Application", CHINA MASTER’S THESES FULL-TEXT DATABASE, no. 2014/03, 20 May 2013 (2013-05-20), pages 1 - 83, XP009531064 *
MING-ZHEN ZHANG; RONG-NA YU; JUN CHEN; ZHI-YA MA; YUAN-DI ZHAO: "Targeted quantum dots fluorescence probes functionalized with aptamer and peptide for transferrin receptor on tumor cells", NANOTECHNOLOGY, vol. 23, no. 48, 485104, 9 November 2012 (2012-11-09), GB, pages 1 - 11, XP020233864, ISSN: 0957-4484, DOI: 10.1088/0957-4484/23/48/485104 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114920798A (zh) * 2022-05-09 2022-08-19 国家纳米科学中心 一种在伤口部位原位构筑的自组装材料及其制备方法和应用

Also Published As

Publication number Publication date
CN111410962B (zh) 2021-02-02
CN111410962A (zh) 2020-07-14

Similar Documents

Publication Publication Date Title
WO2021190112A1 (zh) 一种多肽-量子点复合探针、制备方法和应用
JP6619048B2 (ja) 官能化発色性ポリマードットおよびその生物共役体
Jiang et al. Gold–carbon dots for the intracellular imaging of cancer-derived exosomes
CN103920889B (zh) 一种基于巯基聚乙二醇的金纳米簇制备方法
JPH05508015A (ja) 蛍光プローブとしての蛍光性ポルフィリン、および蛍光性フタロシアニン―ポリエチレングリコール、ポリオール、およびサッカライド誘導体
JP2007506084A (ja) ナノ粒子コンジュゲート及びその製造方法
CN102725299A (zh) 光致发光纳米粒子及其制备方法与应用
TWI392869B (zh) 功能化量子點及其製備方法
Song et al. Synthesis of fluorescent silica nanoparticles and their applications as fluorescence probes
CN111057539A (zh) 一种电荷排斥作用诱导的单链胶原多肽探针及制备方法
CN111303868B (zh) 近红外发光多肽自组装金纳米材料及其制备方法与应用
CN111175283A (zh) 一种靶向识别胶原蛋白的多肽拉曼探针及其制备方法
Wei et al. Orange-emissive carbon quantum dots for ligand-directed Golgi apparatus-targeting and in vivo imaging
WO2013025347A1 (en) Compact multifunctional ligand to enhance colloidal stability of nanoparticles
Zhang et al. Organic-to-aqueous phase transfer of Zn–Cu–In–Se/ZnS quantum dots with multifunctional multidentate polymer ligands for biomedical optical imaging
CN101113457A (zh) 富精氨酸多肽-金纳米粒子细胞传输载体合成方法
CN110885675A (zh) 纳米荧光探针及制备方法与其在检测高尔基体中hno的应用
Zhu et al. Immuno-affinitive supramolecular magnetic nanoparticles incorporating cucurbit [8] uril-mediated ternary host-guest complexation structures for high-efficient small extracellular vesicle enrichment
WO2022094912A1 (zh) 一种生物探针及其制备方法和应用
Wang et al. Photoluminescence enhancement by coupling of ovalbumin and CdTe quantum dots and its application as protein probe
CN112899231A (zh) 一种可视化肿瘤细胞检测试剂、试剂盒及其制备方法与应用
WO2020217985A1 (ja) 発光色素含有粒子及び病理診断用標識剤
Chen et al. Bioorthogonal chemistry in metal clusters: a general strategy for the construction of multifunctional probes for bioimaging in living cells and in vivo
CN110000395A (zh) 一步法合成荧光金属锰纳米簇的方法及其应用
Vibin et al. Biokinetics and in vivo distribution behaviours of silica-coated cadmium selenide quantum dots

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21774734

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21774734

Country of ref document: EP

Kind code of ref document: A1