CN111410962B - Polypeptide-quantum dot composite probe, preparation method and application - Google Patents
Polypeptide-quantum dot composite probe, preparation method and application Download PDFInfo
- Publication number
- CN111410962B CN111410962B CN202010226767.1A CN202010226767A CN111410962B CN 111410962 B CN111410962 B CN 111410962B CN 202010226767 A CN202010226767 A CN 202010226767A CN 111410962 B CN111410962 B CN 111410962B
- Authority
- CN
- China
- Prior art keywords
- quantum dot
- polypeptide
- probe
- dot composite
- composite probe
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
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"
Abstract
The invention provides a polypeptide-quantum dot composite probe, which uses glutathione as a quantum dot surface modifier and uses a charged amino acid short chain with the tail end of Cys as a connecting group to modify a polypeptide sequence to obtain a target polypeptide (Cys-X) -Targeting domain. The invention also provides a preparation method of the polypeptide-quantum dot composite probe, which comprises the following steps: mixing the Te solution and the Cd solution, heating to reflux, slowly adding the synthesized Targeting polypeptide (Cys-X) -Targeting domain solution dropwise, and refluxing for 15-300 min. The polypeptide-quantum dot composite probe disclosed by the invention takes glutathione as a quantum dot surface modifier, can be compatible with Cys in a targeted polypeptide, and has good stability; the glutathione is a peptide naturally synthesized in a human body, and has good biocompatibility and low toxicity; the method is mild in condition, environment-friendly and simple in process, can be synthesized in one step without further quantum dot modification, and the prepared polypeptide-quantum dot composite probe can be used for in vivo imaging.
Description
Technical Field
The invention belongs to the field of semiconductor quantum dot materials, and particularly relates to a polypeptide-quantum dot composite probe, a preparation method and application.
Background
The semiconductor quantum dot is a novel nano material composed of II-VI group or III-V group elements, and has the advantages of good light stability, strong fluorescence intensity, wide excitation spectrum range, symmetrical and narrow emission spectrum half-peak width, long fluorescence life, various and adjustable colors, high quantum yield and the like compared with the traditional fluorescent dye due to the special optical performance, thereby having wide application prospect in the aspect of biological imaging.
The polypeptide quantum dots areA novel biomarker probe formed by connecting polypeptide molecules on the surface of a quantum dot is a hotspot of current research. Among them, CdTe quantum dots can cover almost the entire visible spectrum due to its strong quantum confinement effect, and have been widely used in the fields of electronic devices and sensors. However, CdTe quantum dots release toxic Cd in cell experiments2+The ions cause cytotoxicity, which greatly limits the application of the ions in biological detection. Meanwhile, the application of the CdTe quantum dot in vivo imaging requires that the CdTe quantum dot has good optical performance, smaller particle size and lower toxicity. Therefore, the development of novel CdTe quantum dots which are soluble in water, high in fluorescence intensity, small in particle size and low in toxicity is very important for developing the biomedical applications of the CdTe quantum dots.
At present, fat-soluble quantum dots containing Zn, such as CdSe/ZnS, CdTe/ZnS, CdSe/ZnSe or CdTe/ZnSe, are most widely applied to biomarkers. When the biomarker is used for carrying out biomarker, two methods are generally adopted: firstly, a sulfydryl micromolecule is used for replacing a ligand on the surface of a fat-soluble quantum dot, and then the sulfydryl micromolecule is connected with biomolecules such as polypeptide and the like through electrostatic adsorption or a coupling agent. However, electrostatic adsorption is greatly affected by the environment and has poor stability; coupling generally needs introduction of chemical reagents, and has low reaction efficiency, more side reactions and higher toxicity; secondly, a connecting group is introduced into the biomolecule, Zn atoms on the surface of the quantum dot can form metal affinity coordination with the connecting group, and protein or polypeptide containing the connecting group can be directly connected to the Zn atoms on the surface of the quantum dot CdSe/ZnS. Although the polypeptide quantum dot prepared by the method has good stability, the method is realized by depending on the affinity of a connecting group and a Zn atom on the surface of the quantum dot CdSe/ZnS, and has certain limitation. Moreover, the method needs a plurality of steps to be realized, and the process is relatively complex: preparing hydrophobic semiconductor quantum dots by an organic phase synthesis method; modifying with bifunctional mercapto ligand, silica, polymer and other modifier to make them have hydrophilicity and biocompatibility; and combining the polypeptide molecule to the quantum dot. This method also has the following drawbacks: firstly, organic phase synthesis needs a large amount of organic solvent and organic metal reagent, and the reaction temperature is high and the toxicity is high; secondly, an additional complex modification process causes the particle size of the quantum dots to be increased, the fluorescence quantum yield to be reduced, and the biological application is not facilitated; and the subsequent polypeptide modification process is complicated, has more side reactions, low modification efficiency and poor stability of the obtained probe, and the structure of the targeted polypeptide is easily damaged in the modification process.
In order to overcome the above disadvantages, a method for aqueous phase synthesis of quantum dots using thiol as a surface modification reagent has attracted much attention. The sulfydryl mainly comprises alpha-thioglycerol, thioglycolic acid, mercaptopropionic acid, cysteine, acetylcysteine and the like. The Chinese patent CN103897699B discloses a polypeptide-quantum dot nanocomposite solution which is prepared by mixing a cadmium solution, a zinc solution, a tellurium solution and a polypeptide solution, heating at a constant temperature of 90-100 ℃ for 1-48h, and cooling. However, the method does not modify a connecting group on the polypeptide, and can cause the problem of unstable combination of the polypeptide molecule and the surface of the quantum dot.
The latest research finds that the polyhistidine can be used as a bridge for the directional connection of quantum dots and polypeptide by generating chelation with metal cations of quantum dot shells through side chain imidazole groups, and the function of protein and quantum dots can not be interfered, so that the method is a potential direction for developing quantum dot biomarkers. For example, literature (Jamin, preparation of functionalized polypeptide quantum dots and biomedical applications thereof [ D]Southern medical university, 2013) designed a functional polyethylene glycol-modified tail containing polyhistidine (His)6) The polypeptide-CdTe quantum dot nanoprobe is synthesized by a one-step method, however, natural protein contains little polyhistidine, and the polyhistidine is used as a quantum dot biomarker bridge to be connected with a functional peptide segment by adopting the hydrazone reaction of aldehyde and amine, the process is complex, and the hydrazone formation and the osazone formation reaction compete violently. In the product osazone pathway, it is also prone to decomposition into protosubstrates, leading to cleavage of the linking group and poor stability. Chinese patent CN104231035B discloses a method for initiating ring-opening polymerization of histidine-N-internal carboxylic anhydride by using histidine-N-internal carboxylic anhydride as polyamino acid monomer and polypeptide with terminal amino group as initiator to realize covalent coupling of polyhistidine chain segment and functional polypeptide, and using polyhistidineAnd the acid side chain imidazole group is chelated and connected with the quantum dot shell layer to form the quantum dot-polypeptide compound. The preparation method is complex in process, needs to introduce one or more organic solvents such as dimethylformamide, tetrahydrofuran, acetonitrile, dichloromethane, trichloromethane, methanol, ethanol, toluene and the like, easily causes organic solvent residues, and has high toxicity.
Based on the above, the invention is unexpectedly found through a large amount of experimental researches that glutathione is used as a quantum dot surface modifier; modifying a target polypeptide (Cys-X) -Targeting domain by using a charged amino acid short chain with the tail end of Cys as a connecting group; and mixing the Te solution and the Cd solution, heating to reflux, slowly dropwise adding the Targeting polypeptide (Cys-X) -Targeting domain solution, and refluxing for 15-300min to prepare the polypeptide-quantum dot composite probe. The method takes glutathione as a quantum dot surface modifier, can be compatible with Cys in the targeted polypeptide, and the prepared polypeptide-quantum dot composite probe has good stability; and the glutathione is a peptide naturally synthesized in a human body, and has good biocompatibility and low toxicity. The method is mild in condition, environment-friendly and simple in process, and the prepared polypeptide-quantum dot composite probe can be used for in vivo imaging.
Disclosure of Invention
Aiming at the technical problems, the invention aims to provide a polypeptide-quantum dot composite probe, which comprises a quantum dot CdTe and a Targeting polypeptide (Cys-X) -Targeting domain, wherein the surface of the quantum dot CdTe is coated with glutathione, the Targeting domain is a Targeting polypeptide sequence, the Cys-X is a connecting group, the X is charged amino acid, the X is connected with the N end and/or the C end of the Targeting domain, and the Cys is combined with the quantum dot CdTe.
Preferably, X is Asp or Glu.
Preferably, X is Glu.
Preferably, the Targeting domain comprises one or more of a protein Targeting peptide, a cell Targeting peptide and a metal ion Targeting peptide.
Preferably, the Targeting domain is a protein Targeting peptide.
Preferably, the Targeting domain is a diseased collagen Targeting peptide or a type i collagen Targeting peptide.
Preferably, the Targeting domain is a cell Targeting peptide.
Preferably, the Targeting domain is targeted to the cell by Targeting an organelle.
Preferably, the organelle is the endoplasmic reticulum or nucleus.
Preferably, the Targeting domain is targeted to the cell by Targeting a cellular receptor on the cell.
Preferably, the cellular receptor is an integrin.
Preferably, the glutathione is L-glutathione and/or D-glutathione.
The invention also aims to provide a preparation method of the polypeptide-quantum dot composite probe, which comprises the following steps:
(1) dissolving glutathione and cadmium chloride in ultrapure water to obtain a Cd solution, adjusting the pH of the obtained solution to 10-13 by sodium hydroxide, and deoxidizing and stirring for later use;
(2) weighing tellurium powder and sodium borohydride, deoxidizing, adding deoxidized ultrapure water, and reacting at 50-70 ℃ to obtain a Te solution;
(3) adding the Te solution into the Cd solution, heating to reflux at 100 ℃, slowly dropwise adding 1-2mL of targeted polypeptide (Cys-X) -Targeting domain solution, and refluxing for 15-300min to obtain a polypeptide-quantum dot composite probe solution;
(4) and (3) cooling the polypeptide-quantum dot composite probe solution in the step (3), adding 1-2 times of isopropanol in volume to precipitate the probe, centrifugally collecting the probe, washing the probe by the isopropanol, and air-drying to obtain the polypeptide-quantum dot composite probe.
Preferably, the concentration of the target polypeptide (Cys-X) -Targeting domain in said step (3) is 2.5-10 mg/ml.
Preferably, the molar ratio of Cd to Te in the step (3) is 1: 0.6-0.3.
The invention also aims to provide application of the polypeptide-quantum dot composite probe in preparing a protein imaging reagent, a cell receptor imaging reagent or an organelle imaging reagent.
The invention has the beneficial effects that: the polypeptide-quantum dot composite probe provided by the invention uses glutathione as a quantum dot surface modifier, and uses a charged amino acid short chain with the end of Cys as a connecting group to modify a polypeptide sequence to obtain a target polypeptide (Cys-X) -Targeting domain, wherein the connecting group can be compatible with the glutathione modified on the surface of the quantum dot; secondly, the polypeptide-quantum dot composite probe provided by the invention can complete dyeing without being damaged in a cell culture medium with complex components for several hours, and has good stability; the glutathione used by the invention is a peptide naturally synthesized in human body, has low biocompatibility and toxicity, and can be used for in vivo imaging; the polypeptide-quantum dot composite probe provided by the invention has the advantages of simple preparation method, mild conditions and one-step synthesis without further quantum dot modification.
Drawings
FIG. 1 emission spectra of polypeptide-quantum dot probes of different emission wavelengths;
FIG. 2 is a staining graph of pathological collagen targeting peptide-quantum dot composite probes with different emission wavelengths on pathological bladder tissues of a mouse;
FIG. 3 is a staining diagram of the specificity of a pathological collagen targeting peptide-quantum dot composite probe;
FIG. 4 is a staining graph of a mouse tail tissue with a collagen type I targeting peptide-quantum dot composite probe;
FIG. 5 is a staining graph of HeLa cells with the nuclear targeting peptide-quantum dot composite probe;
FIG. 6 is a staining graph of a HeLa cell with an integrin targeting peptide-quantum dot composite probe;
FIG. 7 is a staining diagram of HeLa cells by endoplasmic reticulum targeting peptide-quantum dot composite probes.
Detailed Description
In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments. The scope of the invention is not limited to the examples described below.
Definition of
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Representative exemplary methods and compositions are now described, although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a polypeptide-quantum dot composite probe" includes a plurality of such polypeptide-quantum dot composite probes, reference to "the polypeptide-quantum dot composite probe" is a reference to one or more polypeptide-quantum dot composite probes and equivalents thereof known to those skilled in the art, and so forth.
"comprising" means "including but not limited to," and is not intended to exclude, for example, other components, integers, and the like. In particular, where the statement "includes a metal ion targeting peptide," it is expressly intended to include the listed (unless a limiting term such as "consisting of") which is meant to not be intended to exclude other components not listed in the laser, for example.
Where a range of values is provided, it is understood that each intervening value, to the extent that there is no such stated or intervening value in the stated range, to the upper and lower limits of that range, and any other stated or intervening value in that stated range, is encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also included in the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.
It is appreciated that certain features are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination.
It will be apparent to those skilled in the art upon reading this disclosure that each of the individual embodiments described and illustrated herein has independent components and features that can be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method may be performed in the order of events recited or in any other order that is logically possible.
The term "quantum dot" may also be referred to as a semiconductor nanocrystal or artificial atom, which is a semiconductor crystal containing any number of electrons between 100 and 1,000 and having a size of about 2nm to 10nm, and is composed mainly of elements of groups II-VI (e.g., CdTe, CdS, CdSe, etc.) or III-V (InP, InAs) and IV-VI (e.g., PbS, PbSe) in the periodic Table of elements.
The term "coating" refers to that the surface of the CdTe of the quantum dot is modified with glutathione, and a connecting group Cys-X in the targeting polypeptide combines the targeting polypeptide and the quantum dot.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to two or more amino acid residues linked 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 mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers that contain modified residues and non-naturally occurring amino acid polymers.
Example 1 preparation of diseased collagen targeting peptide-Quantum dot composite Probe
1. Preparation of pathological change collagen targeted peptide-quantum dot composite probe
Weighing 90mg of glutathione and 22.7mg of cadmium chloride, dissolving the glutathione and the 22.7mg of cadmium chloride in 30ml of ultrapure water to obtain a Cd solution, adjusting the pH of the obtained solution to 11.5 by 1M sodium hydroxide, and deoxidizing and stirring the solution for later use; weighing 12.75mg of tellurium powder and 10mg of sodium borohydride, deoxidizing, adding 1ml of deoxidized ultrapure water, and reacting at 65 ℃ to obtain a Te solution; adding 0.5ml of Te solutionPutting the probe into a Cd solution, heating and refluxing at 100 ℃, wherein the refluxing time of the composite probes with different generation wavelengths is as follows: refluxing with a 530nm probe for 15min, refluxing with a 545nm probe for 30min, refluxing with a 560nm probe for 45min, refluxing with a 575nm probe for 60min, refluxing with a 585nm probe for 70min, refluxing with a 600nm probe for 90min, refluxing with a 620nm probe for 120min, refluxing with a 635nm probe for 150min, refluxing with a 645nm probe for 180min, and refluxing with a 680nm probe for 240 min. After refluxing for a prescribed time, 1ml of CE- (GPO) containing 10mg of pathological collagen targeting peptide is slowly added dropwise8-NH2Refluxing the solution for 15min to respectively obtain polypeptide-quantum dot composite probe solutions with different emission wavelengths; and after cooling the polypeptide-quantum dot composite probe solution, adding isopropanol with the volume being 2 times that of the polypeptide-quantum dot composite probe solution to precipitate the probe, centrifugally collecting the probe, washing the probe by the isopropanol, and then air-drying the probe to obtain the pathological change collagen targeting peptide-quantum dot composite probes with different emission wavelengths.
Example 2 preparation of type I collagen targeting peptide-Quantum dot composite Probe
Weighing 90mg of glutathione and 22.7mg of cadmium chloride, dissolving the glutathione and the 22.7mg of cadmium chloride in 30ml of ultrapure water to obtain a Cd solution, adjusting the pH of the obtained solution to 12.0 by 1M of sodium hydroxide, and deoxidizing and stirring the solution for later use; weighing 12.75mg of tellurium powder and 10mg of sodium borohydride, deoxidizing, adding 1ml of deoxidized ultrapure water, and reacting at 65 ℃ to obtain a Te solution; 0.55ml of Te solution is added into the Cd solution, and the mixture is heated and refluxed at 100 ℃ for the following time: refluxing with a 530nm probe for 15min, refluxing with a 545nm probe for 30min, refluxing with a 560nm probe for 45min, refluxing with a 575nm probe for 60min, refluxing with a 585nm probe for 70min, refluxing with a 600nm probe for 90min, refluxing with a 620nm probe for 120min, refluxing with a 635nm probe for 150min, refluxing with a 645nm probe for 180min, and refluxing with a 680nm probe for 240 min. After refluxing for a given time, 1ml of CE-Ahx-LRELHLNNN-NH containing 10mg of type I collagen targeting peptide is slowly added dropwise2Refluxing the solution for 15min to obtain I type collagen targeting peptide-quantum dot composite probe solutions with different emission wavelengths; and after cooling the polypeptide-quantum dot composite probe solution, adding isopropanol with the volume being 2 times that of the polypeptide-quantum dot composite probe solution to precipitate the probe, centrifugally collecting the probe, washing the probe by the isopropanol, and then air-drying the probe to obtain the type I collagen targeted peptide-quantum dot composite probes with different emission wavelengths.
Example 3 preparation of Nuclear targeting peptide-Quantum dot composite Probe
Weighing 90mg of glutathione and 22.7mg of cadmium chloride, dissolving the glutathione and the 22.7mg of cadmium chloride in 30ml of ultrapure water to obtain a Cd solution, adjusting the pH of the obtained solution to 12.0 by 1M of sodium hydroxide, and deoxidizing and stirring the solution for later use; weighing 12.75mg of tellurium powder and 10mg of sodium borohydride, deoxidizing, adding 1ml of deoxidized ultrapure water, and reacting at 65 ℃ to obtain a Te solution; adding 0.5ml of Te solution into Cd solution, heating and refluxing at 100 ℃, wherein the refluxing time is as follows: refluxing with a 530nm probe for 15min, refluxing with a 545nm probe for 30min, refluxing with a 560nm probe for 45min, refluxing with a 575nm probe for 60min, refluxing with a 585nm probe for 70min, refluxing with a 600nm probe for 90min, refluxing with a 620nm probe for 120min, and refluxing with a 635nm probe for 150 min. After refluxing for a specified time, slowly dripping 1.5ml of solution containing 7mgTAT targeting peptide, and refluxing for 15min to obtain cell nucleus targeting peptide-quantum dot composite probe solutions with different emission wavelengths; and cooling the cell nucleus targeting peptide-quantum dot composite probe solution, adding 2 times of isopropanol by volume to precipitate the probe, centrifugally collecting the probe, washing by the isopropanol, and air-drying to obtain the cell nucleus targeting peptide-quantum dot composite probes with different emission wavelengths.
Example 4 preparation of integrin targeting peptide-Quantum dot composite Probe
Weighing 90mg of glutathione and 22.7mg of cadmium chloride, dissolving the glutathione and the 22.7mg of cadmium chloride in 30ml of ultrapure water to obtain a Cd solution, adjusting the pH of the obtained solution to 12.5 by 1M of sodium hydroxide, and deoxidizing and stirring the solution for later use; weighing 12.75mg of tellurium powder and 10mg of sodium borohydride, deoxidizing, adding 1ml of deoxidized ultrapure water, and reacting at 65 ℃ to obtain a Te solution; adding 0.6ml of Te solution into Cd solution, heating and refluxing at 100 ℃, wherein the refluxing time is as follows: refluxing with a 530nm probe for 15min, refluxing with a 545nm probe for 30min, refluxing with a 560nm probe for 45min, refluxing with a 575nm probe for 60min, refluxing with a 585nm probe for 70min, refluxing with a 600nm probe for 90min, refluxing with a 620nm probe for 120min, and refluxing with a 635nm probe for 150 min. After refluxing for a specified time, slowly dripping 1ml of solution containing 5mg of RGD targeting peptide, and refluxing for 15min to obtain integrin targeting peptide-quantum dot composite probe solutions with different emission wavelengths; and after cooling the integrin targeting peptide-quantum dot composite probe solution, adding isopropanol with the volume being 2 times that of the cooled integrin targeting peptide-quantum dot composite probe solution to precipitate the probe, centrifugally collecting the probe, washing the probe by the isopropanol, and then air-drying the probe to obtain the integrin targeting peptide-quantum dot composite probes with different emission wavelengths.
EXAMPLE 5 preparation of endoplasmic reticulum-targeting peptide-Quantum dot composite Probe
Weighing 90mg of glutathione and 22.7mg of cadmium chloride, dissolving the glutathione and the 22.7mg of cadmium chloride in 30ml of ultrapure water to obtain a Cd solution, adjusting the pH of the obtained solution to 12.5 by 1M of sodium hydroxide, and deoxidizing and stirring the solution for later use; weighing 12.75mg of tellurium powder and 10mg of sodium borohydride, deoxidizing, adding 1ml of deoxidized ultrapure water, and reacting at 65 ℃ to obtain a Te solution; adding 0.6ml of Te solution into Cd solution, heating and refluxing at 100 ℃, wherein the refluxing time is as follows: refluxing with a 530nm probe for 15min, refluxing with a 545nm probe for 30min, refluxing with a 560nm probe for 45min, refluxing with a 575nm probe for 60min, refluxing with a 585nm probe for 70min, refluxing with a 600nm probe for 90min, refluxing with a 620nm probe for 120min, and refluxing with a 635nm probe for 150 min. After refluxing for a specified time, slowly dripping 1ml of solution containing 5mgER targeting peptide, and refluxing for 15min to obtain endoplasmic reticulum targeting peptide-quantum dot composite probe solutions with different emission wavelengths; and after cooling the polypeptide-quantum dot composite probe solution, adding isopropanol with the volume being 2 times that of the polypeptide-quantum dot composite probe solution to precipitate the probe, centrifugally collecting the probe, washing the probe by the isopropanol, and then air-drying the probe to obtain endoplasmic reticulum targeted peptide-quantum dot composite probes with different emission wavelengths.
EXAMPLE 6 polypeptide-Quantum dot Probe emission Spectroscopy at different emission wavelengths
Preparing a polypeptide-quantum dot probe solution with the concentration of 1 mu g/ml, and taking 2ml of the solution to determine the fluorescence emission spectrum. The excitation wavelength is 365nm, and the emission wavelength scanning range is 450 nm and 700 nm. The results are shown in FIG. 1, in which curve 1 is the type I collagen targeting peptide-quantum dot composite probe with an emission wavelength of 530nm, and curve 3 is the type I collagen targeting peptide-quantum dot composite probe with an emission wavelength of 560 nm; the curve 10 is a type I collagen targeting peptide-quantum dot composite probe with the emission wavelength of 680 nm; curve 2 is a diseased collagen targeting peptide-quantum dot composite probe with an emission wavelength of 545 nm; curve 4 is a diseased collagen targeting peptide-quantum dot composite probe with an emission wavelength of 575 nm; curve 6 is a diseased collagen targeting peptide-quantum dot composite probe with emission wavelength of 600 nm; curve 7 is a diseased collagen targeting peptide-quantum dot composite probe with an emission wavelength of 620 nm; curve 9 is a diseased collagen targeting peptide-quantum dot composite probe with an emission wavelength of 645 nm; the curve 8 is a cell nucleus targeting peptide-quantum dot composite probe with the emission wavelength of 635 nm; curve 5 is an integrin targeting peptide-quantum dot composite probe with emission wavelength 585 nm. The results show that the emission wavelength of the polypeptide-quantum dot composite probe prepared by the method can cover the wavelength range from green light to near infrared.
Example 7 tissue staining
1. Staining of lesion collagen targeting peptide-quantum dot composite probe with different emission wavelengths on injured mouse bladder tissue
Preparing a frozen section of the injured mouse bladder tissue sample to 4 mu m; after the section is cleaned, adding confining liquid on the tissue section, placing for 30min, sucking away the liquid, taking 100 mu L of the pathological collagen targeting peptide-quantum dot composite probe solution with the concentration of 0.1mg/ml for dyeing, and incubating for 6h at 4 ℃; after the staining solution is absorbed; wash 3 times with 1x PBS and wash to wash unbound probes; the mounting solution was dropped, and the plate was covered with a glass slide and photographed by observation using a fluorescence microscope.
The staining results of the different emission wavelength pathological change collagen targeting peptide-quantum dot composite probe on the injured mouse bladder tissue are shown in fig. 2, wherein a and e are the staining results of the 530nm emission wavelength pathological change collagen targeting peptide-quantum dot composite probe, b and f are the staining results of the 545nm emission wavelength pathological change collagen targeting peptide-quantum dot composite probe, c and g are the staining results of the 585nm emission wavelength pathological change collagen targeting peptide-quantum dot composite probe, and d and h are the staining results of the 620nm emission wavelength pathological change collagen targeting peptide-quantum dot composite probe.
2. Specific staining of pathological change collagen targeting peptide-quantum dot composite probe on pathological change tissue
Taking tissue samples of different parts of a mouse to prepare frozen sections to 4 mu m; after the section is cleaned, adding confining liquid on the tissue section, placing for 30min, sucking the liquid, taking 100 mu L of 0.1mg/ml probe solution for dyeing, and incubating for 6h at 4 ℃; after the staining solution is absorbed; wash 3 times with 1x PBS and wash to wash unbound probes; dropping the sealing liquid, covering a glass slide, and observing and photographing by using a fluorescence microscope;
the staining results of the diseased collagen targeting peptide-quantum dot composite probe with the emission wavelength of 530nm on various tissues are shown in fig. 3, wherein a is the staining result of the diseased collagen targeting peptide-quantum dot composite probe on mouse diseased tail tissues, b is the staining result of the diseased collagen targeting peptide-quantum dot composite probe on mouse normal tail tissues, c is the staining result of quantum dots which are not marked by the targeting peptide on mouse diseased tail tissues, d is the staining result of the diseased collagen targeting peptide-quantum dot composite probe on mouse diseased ear tissues, e is the staining result of the diseased collagen targeting peptide-quantum dot composite probe on mouse normal ear tissues, and f is the staining result of quantum dots which are not marked by the targeting peptide on mouse diseased ear tissues. The staining result shows that the pathological tissue can be stained by the pathological collagen targeting peptide-quantum dot composite probe but cannot be stained by the unmarked quantum dot; meanwhile, the pathological collagen targeting peptide-quantum dot composite probe does not stain normal tissues. The results show that the polypeptide-quantum dot composite probe prepared by the method provides the inherent excellent luminescent property of the quantum dot, and simultaneously endows the probe with excellent target recognition capability.
Staining mouse tail tissue by type I collagen targeting peptide-quantum dot composite probe
Preparing a rat tail tissue sample into frozen sections of 4 mu m; after the section is cleaned, adding confining liquid on the tissue section, placing for 30min, sucking the liquid, taking 100 mu L of 0.1mg/ml probe solution for dyeing, and incubating for 6h at 4 ℃; after the staining solution is absorbed; wash 3 times with 1x PBS and wash to wash unbound probes; the mounting solution was dropped, and the plate was covered with a glass slide and photographed by observation using a fluorescence microscope.
The staining results of the type I collagen targeting peptide-quantum dot composite probe on the mouse tail tissue are shown in fig. 4, wherein a is a staining pattern of the type I collagen targeting peptide-quantum dot composite probe with an emission wavelength of 585nm on the mouse tail tissue, and b is a staining pattern of the type I collagen targeting peptide-quantum dot composite probe with an emission wavelength of 600nm on the mouse tail tissue, and the staining patterns show that the type I collagen targeting peptide-quantum dot composite probes with different emission wavelengths can stain the mouse tail tissue.
Example 8 cell staining
1. Dyeing HeLa cells by using cell nucleus targeting peptide-quantum dot composite probe
HeLa cells were fixed with 3.7% paraformaldehyde for 30min and treated with 0.05% Triton X-100 for 2 min. Washed 1 time with PBS. And (3) staining 0.01mg/ml of the cell nucleus targeting peptide-quantum dot composite probe for 2h at room temperature and staining the cell nucleus targeting peptide-quantum dot composite probe for 5min by DAPI. Cells were washed 3 times with PBS. The photographs were observed using a fluorescence microscope.
The staining result of the nuclear targeting peptide-quantum dot composite probe with the emission wavelength of 530nm on the HeLa cell is shown in FIG. 5, wherein a is the staining pattern of DAPI on the cell nucleus, b is the staining pattern of the nuclear targeting peptide-quantum dot composite probe on the cell nucleus, and c is the superimposed staining pattern of DAPI and the nuclear targeting peptide-quantum dot composite probe on the cell nucleus. The above staining results indicate that the probe targets the stained cell nucleus.
2. Dyeing HeLa cells by integrin targeting peptide-quantum dot composite probe
HeLa cells were stained with 0.01mg/ml integrin targeting peptide-quantum dot composite probe at room temperature for 6h, and cells were washed 3 times with PBS. 3.7% paraformaldehyde fixed for 30min and washed 1 time with PBS. DAPI stained for 5min, cells were washed 3 times with PBS. The photographs were observed using a fluorescence microscope.
The staining results of HeLa cells by the integrin targeting peptide-quantum dot composite probe with the emission wavelength of 620nm are shown in fig. 6, wherein a is a staining graph of the cell by the integrin targeting peptide-quantum dot composite probe, b is a staining graph of cell nucleus by DAPI, and c is a superimposed staining graph of the cell by the DAPI and the integrin targeting peptide-quantum dot composite probe. The dyeing result shows that the integrin targeting peptide-quantum dot composite probe targets the integrin in the cell membrane.
3. Endoplasmic reticulum targeted peptide-quantum dot composite probe for dyeing HeLa cells
HeLa cells were stained with 0.01mg/ml endoplasmic reticulum targeting peptide-quantum dot composite probe at room temperature for 2h, and cells were washed 3 times with PBS. 3.7% paraformaldehyde fixed for 30min and washed 1 time with PBS. DAPI stained for 5min, cells were washed 3 times with PBS. The photographs were observed using a fluorescence microscope.
The dyeing result of the endoplasmic reticulum targeting peptide-quantum dot composite probe with the emission wavelength of 620nm on the HeLa cells is shown in figure 7, wherein a is the dyeing graph of the endoplasmic reticulum targeting peptide-quantum dot composite probe on the cells, and b is the dyeing graph of the endoplasmic reticulum targeting peptide-quantum dot composite probe and DAPI on the cells simultaneously. The staining results indicated that the probe stained endoplasmic reticulum in a targeted manner.
The above description is only for details of a specific exemplary embodiment of the present invention, and it is obvious to those skilled in the art that various modifications and changes may be made in the present invention in the practical application process according to specific preparation conditions, and the present invention is not limited thereto. All that comes within the spirit and principle of the invention is to be understood as being within the scope of the invention.
Claims (14)
1. The polypeptide-quantum dot composite probe is characterized by comprising a quantum dot CdTe with the surface coated with glutathione and a Targeting polypeptide (Cys-X) -Targeting domain; the Targeting domain is a Targeting polypeptide sequence; the Cys-X is a connecting group, the X is Glu, the X is connected with the N end and/or the C end of the Targeting domain, and the Cys is combined with the CdTe of the quantum dot.
2. The polypeptide-quantum dot composite probe of claim 1, wherein the Targeting domain comprises one or more of a protein Targeting peptide, a cell Targeting peptide, and a metal ion Targeting peptide.
3. The polypeptide-quantum dot composite probe of claim 2, wherein the Targeting domain is a protein Targeting peptide.
4. The polypeptide-quantum dot composite probe of claim 3, wherein the Targeting domain is a pathological collagen Targeting peptide or a type I collagen Targeting peptide.
5. The polypeptide-quantum dot composite probe of claim 2, wherein the Targeting domain is a cell Targeting peptide.
6. The polypeptide-quantum dot composite probe of claim 5, wherein the Targeting domain is targeted to a cell by Targeting an organelle.
7. The polypeptide-quantum dot composite probe of claim 6, wherein the organelle is endoplasmic reticulum or nucleus.
8. The polypeptide-quantum dot composite probe of claim 5, wherein the Targeting domain targets a cell by Targeting a cellular receptor on the cell.
9. The polypeptide-quantum dot composite probe of claim 8, wherein the cellular receptor is an integrin.
10. The polypeptide-quantum dot composite probe of claim 1, wherein the glutathione is L-glutathione and/or D-glutathione.
11. The method for preparing the polypeptide-quantum dot composite probe as claimed in any one of claims 1 to 10, wherein the method comprises the following steps:
(1) dissolving glutathione and cadmium chloride in ultrapure water to obtain a Cd solution, adjusting the pH of the obtained solution to 10-13 by sodium hydroxide, and deoxidizing and stirring for later use;
(2) weighing tellurium powder and sodium borohydride, deoxidizing, adding deoxidized ultrapure water, and reacting at 50-70 ℃ to obtain a Te solution;
(3) adding the Te solution into the Cd solution, heating to reflux at 100 ℃, slowly dropwise adding 1-2mL of targeted polypeptide (Cys-X) -Targeting domain solution, and refluxing for 15-300min to obtain a polypeptide-quantum dot composite probe solution;
(4) and (3) cooling the polypeptide-quantum dot composite probe solution in the step (3), adding 1-2 times of isopropanol in volume to precipitate the probe, centrifugally collecting the probe, washing the probe by the isopropanol, and air-drying to obtain the polypeptide-quantum dot composite probe.
12. The method according to claim 11, wherein the concentration of the target polypeptide (Cys-X) -Targeting domain in the step (3) is 2.5 to 10 mg/ml.
13. The production method according to claim 11, wherein the molar ratio of Cd to Te in the step (3) is 1: 0.6-0.3.
14. The use of the polypeptide-quantum dot composite probe as claimed in any one of claims 1-10 in the preparation of protein imaging agent, cell receptor imaging agent or organelle imaging agent.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010226767.1A CN111410962B (en) | 2020-03-27 | 2020-03-27 | Polypeptide-quantum dot composite probe, preparation method and application |
| PCT/CN2021/072873 WO2021190112A1 (en) | 2020-03-27 | 2021-01-20 | Polypeptide-quantum dot composite probe, preparation method, and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010226767.1A CN111410962B (en) | 2020-03-27 | 2020-03-27 | Polypeptide-quantum dot composite probe, preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111410962A CN111410962A (en) | 2020-07-14 |
| CN111410962B true CN111410962B (en) | 2021-02-02 |
Family
ID=71487771
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010226767.1A Active CN111410962B (en) | 2020-03-27 | 2020-03-27 | Polypeptide-quantum dot composite probe, preparation method and application |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN111410962B (en) |
| WO (1) | WO2021190112A1 (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111410962B (en) * | 2020-03-27 | 2021-02-02 | 兰州大学 | Polypeptide-quantum dot composite probe, preparation method and application |
| CN113512089B (en) * | 2021-06-30 | 2023-06-13 | 兰州大学 | Polypeptide stabilizer of water-soluble quantum dot and application thereof |
| CN114920798A (en) * | 2022-05-09 | 2022-08-19 | 国家纳米科学中心 | Self-assembly material constructed in situ at wound part and preparation method and application thereof |
| CN117384246A (en) * | 2023-12-11 | 2024-01-12 | 北京大学第三医院（北京大学第三临床医学院） | Polypeptide, hydrogel containing polypeptide, preparation method and application of polypeptide |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103830745A (en) * | 2014-03-11 | 2014-06-04 | 南通大学 | Preparation and application of quantum dot-based multifunctional nano siRNA (Small Interfering Ribonucleic Acid) carrier system |
| CN106568757A (en) * | 2016-11-10 | 2017-04-19 | 常州大学 | Quantum dot targeting probe kit for detecting tumor of colon cancer |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1793283A (en) * | 2005-12-08 | 2006-06-28 | 上海交通大学 | Process for auxiliary synthesizing high lumineseent cadmium telluride quantum point by cysteine-containing polypeptide |
| SG179378A1 (en) * | 2010-09-17 | 2012-04-27 | Agency Science Tech & Res | Cell-targeting nanoparticles and uses thereof |
| CN110231327A (en) * | 2019-03-11 | 2019-09-13 | 兰州大学 | A kind of polypeptide Raman microprobe of targets identification collagen and its preparation and imaging method |
| CN111410962B (en) * | 2020-03-27 | 2021-02-02 | 兰州大学 | Polypeptide-quantum dot composite probe, preparation method and application |
-
2020
- 2020-03-27 CN CN202010226767.1A patent/CN111410962B/en active Active
-
2021
- 2021-01-20 WO PCT/CN2021/072873 patent/WO2021190112A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103830745A (en) * | 2014-03-11 | 2014-06-04 | 南通大学 | Preparation and application of quantum dot-based multifunctional nano siRNA (Small Interfering Ribonucleic Acid) carrier system |
| CN106568757A (en) * | 2016-11-10 | 2017-04-19 | 常州大学 | Quantum dot targeting probe kit for detecting tumor of colon cancer |
Non-Patent Citations (4)
| Title |
|---|
| L-cysteine and poly-L-arginine grafted carboxymethyl cellulose/Ag-In-S quantum dot fluorescent nanohybrids for in vitro bioimaging of brain cancer cells;Isadora C. Carvalho et al.;《International Journal of Biological Macromolecules》;20190422;第133卷;739-753 * |
| 功能化多肽量子点的制备及其生物医学应用;贾静;《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》;20140315;B020-206 * |
| 基于靶向多肽的量子点探针用于结肠癌肿瘤组织识别;王建浩 等;《科学通报》;20130310;第58卷(第7期);556-560 * |
| 量子点荧光探针的制备及其在细胞和活体成像中的应用;韩四海;《中国博士学位论文全文数据库 基础科学辑》;20130815(第8期);第A006-225页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2021190112A1 (en) | 2021-09-30 |
| CN111410962A (en) | 2020-07-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111410962B (en) | Polypeptide-quantum dot composite probe, preparation method and application | |
| KR100943839B1 (en) | Method for the production of bio-imaging nanoparticles with high yield by early introduction of irregular structure | |
| Ali et al. | Red fluorescent carbon nanoparticle-based cell imaging probe | |
| KR101088147B1 (en) | Novel water-soluble nanocrystals and methods of preparing the same | |
| US9683992B2 (en) | Ligand passivated gold nanoparticles | |
| AU2017238024B2 (en) | Ratiometric sensing compound and device made from the compound | |
| Lan et al. | Highly stable organic fluorescent nanorods for living-cell imaging | |
| US20190212335A1 (en) | AIE Nanoparticle Conjugates And Methods Therefor | |
| US20140255696A1 (en) | Biotin-Decorated Fluorescent Silica Nanoparticles With Aggregation-Induced Emission for Tumor Cell Targeting and Long-Term Tumor Cell Tracking | |
| Lin et al. | CuInS 2 quantum dots@ silica near-infrared fluorescent nanoprobe for cell imaging | |
| KR20080017149A (en) | Single nanoparticle containing organic-inorganic composite nanoparticle and method for preparing the same | |
| US11116853B2 (en) | Monolayer protected nanoclusters and methods of making and using thereof | |
| Feng et al. | Red-emission hydrophobic porphyrin structure carbon dots linked with transferrin for cell imaging | |
| Song et al. | Synthesis of fluorescent silica nanoparticles and their applications as fluorescence probes | |
| Sun et al. | Polycation-functionalized gold nanodots with tunable near-infrared fluorescence for simultaneous gene delivery and cell imaging | |
| Wang et al. | Synthesis and characterization of self-assembled CdHgTe/gelatin nanospheres as stable near infrared fluorescent probes in vivo | |
| Zhang et al. | Highly efficient near-infrared organic dots based on novel AEE fluorogen for specific cancer cell imaging | |
| CN108310397B (en) | Diagnosis and treatment reagent with SERS/fluorescence bimodal targeting tumor cell imaging function and preparation method thereof | |
| Wang et al. | Dual-emission carbon dots achieved by luminescence center modulation within one-pot synthesis for a fluorescent ratiometric probe of pH, Hg 2+, and glutathione | |
| 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 | |
| Adegoke et al. | Conjugation of mono-substituted phthalocyanine derivatives to CdSe@ ZnS quantum dots and their applications as fluorescent-based sensors | |
| US10927221B2 (en) | Dendrimeric metallacrowns | |
| Li et al. | RGDS-conjugated CdSeTe/CdS quantum dots as near-infrared fluorescent probe: preparation, characterization and bioapplication | |
| KR101334779B1 (en) | Highly monodisperse silica nanoparticles as reversible internal light switch for living biological systems | |
| CN113512089B (en) | Polypeptide stabilizer of water-soluble quantum dot and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |