CN111410962A - Polypeptide-quantum dot composite probe, preparation method and application - Google Patents
Polypeptide-quantum dot composite probe, preparation method and application Download PDFInfo
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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 dot is a novel biomarker probe formed by connecting polypeptide molecules on the surface of the quantum dot, and 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.
① substitutes the ligand on the surface of the fat-soluble quantum dot by a sulfydryl micromolecule, and then connects with biomolecules such as polypeptide through electrostatic adsorption or a coupling agent, but the electrostatic adsorption is greatly influenced by environment and has poor stability, while the coupling generally needs to introduce a chemical reagent, the reaction efficiency is low, the side reactions are more, the toxicity is higher, ② introduces a connecting group on the biomolecules, the Zn atom on the surface of the quantum dot can form metal affinity coordination with the connecting group, and can directly connect the protein or the polypeptide containing the connecting group to the Zn atom on the surface of the quantum dot CdSe/ZnS, although the stability of the polypeptide quantum dot prepared by the method is better, the method depends on the affinity of the connecting group and the Zn atom on the surface of the quantum dot CdSe/ZnS, and has certain limitation, and the method needs a plurality of steps to realize the modification, compared with the complex preparation method of ①, the preparation of the modification of the polypeptide by a phase modification process, the preparation method of the polypeptide has the defects of complex organic phase modification, the ligand modification of the molecular modification of the quantum dot, the molecular modification of the quantum dots, the molecular modification of the molecular, the molecular modification of the quantum dots, the molecular modification of the.
In order to overcome the defects, the method for synthesizing the quantum dots by using the water phase with the sulfydryl as the surface modification reagent is widely concerned, wherein the sulfydryl mainly comprises α -thioglycerol, thioglycolic acid, mercaptopropionic acid, cysteine, acetylcysteine and the like, Chinese patent CN103897699B discloses a method for preparing a polypeptide-quantum dot nano-composite solution by using the thioglycerol as the surface modification reagent and mixing a cadmium solution, a zinc solution, a tellurium solution and a polypeptide solution, heating at the constant temperature of 90-100 ℃ for 1-48h and then cooling, but the method does not modify a connecting group on the polypeptide, and can cause the unstable combination of a 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 production osazone path, is also easily decomposed intoThe original substrate, and thus the linking group, is cleaved, resulting in poor stability. Chinese patent CN104231035B discloses a method for preparing a quantum dot-polypeptide complex, which comprises initiating ring-opening polymerization of histidine-N-inner carboxylic anhydride by using histidine-N-inner 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 chelating and connecting polyhistidine side chain imidazole group with quantum dot shell layer. 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) -targettingdomain by using a charged amino acid short chain with a Cys at the tail end as a connecting group; and mixing the Te solution and the Cd solution, heating to reflux, slowly dropwise adding the target polypeptide (Cys-X) -targettingdomain 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 a targeted polypeptide (Cys-X) -Targeting domain solution of 1-2m L, 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.
① the polypeptide-quantum dot composite probe provided by the invention uses glutathione as a quantum dot surface modifier, uses a charged amino acid short chain with the end of Cys as a connecting group to modify a polypeptide sequence to obtain a targeted polypeptide (Cys-X) -Targeting domain, wherein the connecting group can be compatible with the glutathione modified on the surface of the quantum dot, ② 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 a few hours, has good stability, ③ the glutathione used by the invention is a peptide naturally synthesized in a human body, has low biocompatibility and toxicity, and can be used for in vivo imaging, and ④ the polypeptide-quantum dot composite probe provided by the invention has the advantages of simple preparation method, mild conditions and can be further synthesized 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 graph of staining of He L a cells with nuclear targeting peptide-quantum dot complex probes;
FIG. 6 staining profile of integrin targeting peptide-quantum dot complex probe for He L a cells;
fig. 7 is a graph of the staining of He L a cells by endoplasmic reticulum-targeting peptide-quantum dot complex 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 solution into 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 cadmium chloride in 30ml of ultrapure water to obtain Cd solution, adjusting the pH value of the obtained solution to 12.0 by 1M of sodium hydroxide, deoxidizing and stirring for standby use, weighing 12.75mg of tellurium powder and 10mg of sodium borohydride, adding 1ml of deoxidized ultrapure water after deoxidization, reacting at 65 ℃ to obtain Te solution, adding 0.55ml of Te solution into the Cd solution, heating and refluxing at 100 ℃, wherein the refluxing time is as follows, 530nm probe refluxing for 15min, 545nm probe refluxing for 30min, 560nm probe refluxing for 45min, 575nm probe refluxing for 60min, 585nm probe refluxing for 70min, 600nm probe refluxing for 90min, 620nm probe refluxing for 120min, 635nm probe refluxing for 150min, 645nm probe refluxing for 180min, 680nm probe refluxing for 240min, refluxing to a specified time, slowly dripping 1ml of targeting peptide CE-Ahx-L NN L H L N-NH-containing 10mg of collagen I2Refluxing the solution for 15min to obtain I type collagen targeting peptide-quantum dot composite probe solutions with different emission wavelengths; polypeptidesAnd cooling the 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 I type collagen targeting peptide-quantum dot composite probe 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 a damaged mouse bladder tissue sample to 4 mu m, cleaning the section, adding confining liquid on the tissue section, placing for 30min, sucking the liquid, taking 0.1mg/ml of the diseased collagen targeting peptide-quantum dot composite probe solution 100 mu L for dyeing, incubating for 6h at 4 ℃, sucking the dyeing liquid, washing for 3 times by using 1x PBS, cleaning the unbound probe by using the washing liquid, dripping the confining liquid, covering a glass slide, and observing and taking a picture by 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, adding confining liquid on the tissue sections after the sections are cleaned, sucking the liquid after the sections are placed for 30min, taking 100 mu L of 0.1mg/ml probe solution to dye, incubating for 6h at 4 ℃, sucking the dyeing liquid, washing for 3 times by using 1x PBS, cleaning unbound probes by using the washing liquid, dripping the confining liquid, covering a glass slide, and observing and taking a picture 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 frozen sections of a rat tail tissue sample to 4 mu m, cleaning the sections, adding a sealing liquid on the tissue sections, standing for 30min, sucking the liquid, taking a probe solution of 0.1mg/ml and 100 mu L for dyeing, incubating for 6h at 4 ℃, sucking the dyeing liquid, washing for 3 times by using 1x PBS, cleaning unbound probes by using the cleaning liquid, dripping a sealing liquid, covering a glass slide, and observing and photographing by 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. Staining He L a cells by using nucleus targeting peptide-quantum dot composite probe
He L a cells were fixed with 3.7% paraformaldehyde for 30min, treated with 0.05% Triton X-100 for 2min, washed with PBS for 1 time, 0.01mg/ml of the nuclear targeting peptide-quantum dot complex probe was stained at room temperature for 2h, DAPI stained for 5min, the cells were washed with PBS for 3 times, and photographed using a fluorescence microscope.
The result of staining He L a cells with the nuclear targeting peptide-quantum dot composite probe with the emission wavelength of 530nm 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.
2. Integrin targeting peptide-quantum dot composite probe for He L a cell staining
He L a cells were stained with 0.01mg/ml integrin targeting peptide-quantum dot composite probe for 6h at room temperature, cells were washed with PBS 3 times, fixed with 3.7% paraformaldehyde for 30min, washed with PBS 1 time, stained with DAPI for 5min, cells were washed with PBS 3 times, and photographed by observation with a fluorescence microscope.
The staining results of He L a cells by the integrin targeting peptide-quantum dot composite probe with the emission wavelength of 620nm are shown in fig. 6, wherein a is the staining graph of the integrin targeting peptide-quantum dot composite probe on the cells, b is the staining graph of DAPI on cell nuclei, and c is the superimposed staining graph of DAPI and the integrin targeting peptide-quantum dot composite probe on the cells.
3. Endoplasmic reticulum targeting peptide-quantum dot composite probe for He L a cell staining
He L a cells were stained with 0.01mg/ml endoplasmic reticulum targeting peptide-quantum dot composite probe for 2h at room temperature, cells were washed with PBS 3 times, fixed with 3.7% paraformaldehyde for 30min, washed with PBS 1 time, stained with DAPI for 5min, cells were washed with PBS 3 times, and photographed by observation with a fluorescence microscope.
The result of staining He L a cells by using the endoplasmic reticulum targeting peptide-quantum dot composite probe with the emission wavelength of 620nm is shown in FIG. 7, wherein a is the staining pattern of the endoplasmic reticulum targeting peptide-quantum dot composite probe on the cells, and b is the staining pattern of the endoplasmic reticulum targeting peptide-quantum dot composite probe and DAPI on the cells at the same time.
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 (16)
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 charged amino acid, the X is connected with the N end and/or the C end of the targettingdomain, and the Cys is combined with the CdTe of the quantum dot.
2. The polypeptide-quantum dot composite probe of claim 1, wherein X is Asp or Glu.
3. The polypeptide-quantum dot composite probe of claim 2, wherein X is Glu.
4. 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.
5. The polypeptide-quantum dot composite probe of claim 4, wherein the Targeting domain is a protein Targeting peptide.
6. The polypeptide-quantum dot composite probe of claim 5, wherein the Targeting domain is a pathological collagen Targeting peptide or a type I collagen Targeting peptide.
7. The polypeptide-quantum dot composite probe of claim 4, wherein the Targeting domain is a cell Targeting peptide.
8. The polypeptide-quantum dot composite probe of claim 7, wherein the Targeting domain is targeted to a cell by Targeting an organelle.
9. The polypeptide-quantum dot composite probe of claim 8, wherein the organelle is endoplasmic reticulum or nucleus.
10. The polypeptide-quantum dot composite probe of claim 7, wherein the Targeting domain targets a cell by Targeting a cellular receptor on the cell.
11. The polypeptide-quantum dot composite probe of claim 10, wherein the cellular receptor is an integrin.
12. The polypeptide-quantum dot composite probe of claim 1, wherein the glutathione is L-glutathione and/or D-glutathione.
13. The method for preparing the polypeptide-quantum dot composite probe as claimed in any one of claims 1 to 12, 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 a targeted polypeptide (Cys-X) -Targeting domain solution of 1-2m L, 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.
14. The method according to claim 13, wherein the concentration of the target polypeptide (Cys-X) -Targeting domain in step (3) is 2.5-10 mg/ml.
15. The method of claim 13, wherein the molar ratio of Cd to Te in step (3) is 1: 0.6-0.3.
16. The use of the polypeptide-quantum dot composite probe as claimed in any one of claims 1-12 in the preparation of protein imaging agent, cell receptor imaging agent or organelle imaging agent.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021190112A1 (en) * | 2020-03-27 | 2021-09-30 | 兰州大学 | Polypeptide-quantum dot composite probe, preparation method, and application |
| CN113512089A (en) * | 2021-06-30 | 2021-10-19 | 兰州大学 | Polypeptide stabilizer of water-soluble quantum dots 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 |
| CN117384246B (en) * | 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） | Polypeptide, hydrogel containing polypeptide, preparation method and application of polypeptide |
Families Citing this family (1)
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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 |
| US9278993B2 (en) * | 2010-09-17 | 2016-03-08 | Agency For Science, Technology And Research | 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 |
|---|
| ISADORA C. CARVALHO ET AL.: "L-cysteine and poly-L-arginine grafted carboxymethyl cellulose/Ag-In-S quantum dot fluorescent nanohybrids for in vitro bioimaging of brain cancer cells", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》 * |
| 王建浩 等: "基于靶向多肽的量子点探针用于结肠癌肿瘤组织识别", 《科学通报》 * |
| 贾静: "功能化多肽量子点的制备及其生物医学应用", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 韩四海: "量子点荧光探针的制备及其在细胞和活体成像中的应用", 《中国博士学位论文全文数据库 基础科学辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021190112A1 (en) * | 2020-03-27 | 2021-09-30 | 兰州大学 | Polypeptide-quantum dot composite probe, preparation method, and application |
| CN113512089A (en) * | 2021-06-30 | 2021-10-19 | 兰州大学 | Polypeptide stabilizer of water-soluble quantum dots and application thereof |
| CN113512089B (en) * | 2021-06-30 | 2023-06-13 | 兰州大学 | Polypeptide stabilizer of water-soluble quantum dot 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 |
| CN117384246B (en) * | 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） | Polypeptide, hydrogel containing polypeptide, preparation method and application of polypeptide |
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