阅读说明：本技术 一种水溶性量子点的多肽稳定剂及应用 (Polypeptide stabilizer of water-soluble quantum dots and application thereof ) 是由 肖建喜 蔡向东 于 2021-06-30 设计创作，主要内容包括：本发明属于半导体量子点材料领域,具体涉及一种水溶性量子点稳定剂,所述水溶性半导体量子点稳定剂为含有Cys-(Xaa)-(n)-Gly的多肽,所述Xaa选自Asp和Glu中的一种或两种组合；所述n为小于等于5的任一整数。所述水溶性半导体量子点稳定剂制备简便,对水溶性半导体量子点具有优越的稳定作用,可用于制备靶向多肽修饰的量子点探针,在生物样品检测和疾病诊断中有广泛的应用前景。(The invention belongs to the field of semiconductor quantum dot materials, and particularly relates to a water-soluble quantum dot stabilizer which contains Cys- (Xaa) n -polypeptide of Gly, said Xaa being selected from one or a combination of two of Asp and Glu; and n is any integer less than or equal to 5. The water-soluble semiconductor quantum dot stabilizer is simple and convenient to prepare,has excellent stabilizing effect on water-soluble semiconductor quantum dots, can be used for preparing targeted polypeptide modified quantum dot probes, and has wide application prospect in biological sample detection and disease diagnosis.)

1. The water-soluble semiconductor quantum dot polypeptide stabilizer is characterized in that the water-soluble semiconductor quantum dot polypeptide stabilizer is polypeptide with a side chain containing carboxyl, and the sequence of the water-soluble semiconductor quantum dot polypeptide stabilizer is Cys- (Xaa)_n-Gly, or Gly- (Xaa)_n-Cys; wherein, the Xaa is selected from one or two combinations of aspartic acid Asp and glutamic acid Glu; and n is any integer less than or equal to 5.

2. The water-soluble semiconductor quantum dot polypeptide stabilizer of claim 1, wherein Xaa is Asp or Glu.

3. The water-soluble semiconductor quantum dot polypeptide stabilizer of claim 2, wherein n is 1, or 2, or 3, or 4, or 5.

4. The water-soluble semiconductor quantum dot polypeptide stabilizer of claim 3, wherein the water-soluble semiconductor quantum dot stabilizer is Cys-Asp-Gly or Cys-Glu-Gly.

5. The use of the water-soluble semiconductor quantum dot polypeptide stabilizer according to any one of claims 1 to 4 for preparing quantum dots or polypeptide-quantum dot composite probes.

6. The use of claim 5, wherein the quantum dots comprise CdTe quantum dots, CdSe quantum dots, ZnS quantum dots, CdS quantum dots, AgS quantum dots.

7. A water-soluble semiconductor quantum dot stabilized by a polypeptide stabilizer, wherein the water-soluble semiconductor quantum dot stabilized by the polypeptide stabilizer is a quantum dot whose surface is coated with the water-soluble semiconductor quantum dot polypeptide stabilizer of any one of claims 1 to 4, and the Cys end in the water-soluble semiconductor quantum dot polypeptide stabilizer is connected with the surface of the quantum dot.

8. The polypeptide stabilizer-stabilized water-soluble semiconductor quantum dot according to claim 7, wherein the quantum dot comprises a CdTe quantum dot, a CdSe quantum dot, a ZnS quantum dot, a CdS quantum dot, an AgS quantum dot.

9. The method for preparing the water-soluble semiconductor quantum dot stabilized by the polypeptide stabilizer according to claim 7 or 8, wherein the method comprises:

(1) solid phase synthesis of the water-soluble semiconductor quantum dot polypeptide stabilizer of any one of claims 1 to 4;

(2) dissolving a water-soluble semiconductor quantum dot polypeptide stabilizer and a metal ion precursor in ultrapure water to obtain a solution A, and adjusting the pH of the solution A to 9-12;

(3) preparing a nonmetal precursor solution B, adding the solution B into the solution A in the step (2), and reacting at a constant temperature of 25-100 ℃ to obtain the water-soluble semiconductor quantum dots with stable polypeptide stabilizers;

wherein the metal ion precursor comprises Cd, Zn and Ag; the non-metal precursor comprises S, Se and Te.

10. A polypeptide-quantum dot composite probe stabilized by a polypeptide stabilizer, which is prepared by connecting a targeting polypeptide to the surface of the water-soluble semiconductor quantum dot stabilized by the polypeptide stabilizer according to claim 7 or 8.

Technical Field

The invention belongs to the field of semiconductor quantum dot materials, and particularly relates to a polypeptide stabilizer of water-soluble quantum dots and application thereof.

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, high fluorescence intensity, wide excitation spectrum range, symmetrical and narrow half-peak width of emission spectrum, 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, and has wide application potential in the aspect of biological imaging.

The fat-soluble quantum dots are widely applied to biological detection. The fat-soluble semiconductor quantum dots are prepared by an organic phase synthesis method. Organic solvents and organometallic reagents are used in large amounts during the preparation; meanwhile, the preparation method has high temperature and high toxicity, and is contrary to the environmental protection concept. Before being applied to biological detection, the fat-soluble quantum dots need to be further modified in a hydrophilic mode to obtain water solubility and reduce biological toxicity. Among them, bifunctional thiol ligands, phospholipid micelles, silica and polymers are the most common hydrophilic modifiers. The further hydrophilic modification causes the fat-soluble quantum dots to have the following defects: the extra modification can cause the particle size of the quantum dot to be enlarged, the fluorescence quantum yield to be reduced, and the biological detection is not facilitated; the modification process is complicated, side reactions are more, the efficiency is low, and the stability of the quantum dots can be influenced.

To overcome the above problems, methods for aqueous phase synthesis of quantum dots are receiving increasing attention. Thiol-containing water-soluble molecules are the most interesting choice. Mercaptoethanol and mercaptopropanol are reported in large quantities as stabilizers for water-soluble CdTe quantum dots; moreover, the quantum dots prepared by using mercaptoethanol and mercaptopropanol as stabilizers have good stability and stable optical properties. However, such organic small molecules have unpleasant odor, poor biocompatibility, high biotoxicity and difficulty in meeting biological detection conditions. Alpha-thioglycerol enters the visual field of people as a stabilizer with better biocompatibility. However, α -thioglycerol itself has the disadvantage of poor stability in alkaline environments. Meanwhile, mercaptoethylamine, cysteine and acetylcysteine with good biocompatibility become important CdTe quantum dot stabilizers, and are used for synthesis of water-soluble CdTe quantum dots. However, quantum dots prepared by the stabilizer have poor stability and are difficult to be used in complex biological systems.

Therefore, the construction of a water-soluble quantum dot stabilizer satisfying the requirement of one-step synthesis of a biologically functionalized quantum dot probe becomes a key problem to be solved urgently. We have unexpectedly found a polypeptide stabilizer that provides a water-soluble quantum dot polypeptide stabilizer having a specific sequence, which has superior stabilizing ability to water-soluble quantum dots; the water-soluble quantum dot polypeptide stabilizer has good biocompatibility and low toxicity, and is suitable for the preparation of different semiconductor quantum dots; the water-soluble quantum dot polypeptide stabilizer can be used for preparing a polypeptide-quantum dot composite probe with good stability by a host-guest peptide system one-step method. The method is mild in condition, environment-friendly and simple in process, and the prepared polypeptide-quantum dot composite probe can be used for biological detection.

Disclosure of Invention

In view of the above technical problems, the present invention aims to provide a polypeptide stabilizer for water-soluble quantum dots and an application of the polypeptide stabilizer for preparing high-stability quantum dots. The method specifically comprises the following steps:

in a first aspect, the invention provides a water-soluble semiconductor quantum dot polypeptide stabilizer, wherein the water-soluble semiconductor quantum dot polypeptide stabilizer is a polypeptide with a side chain containing carboxyl, and the sequence of the water-soluble semiconductor quantum dot polypeptide stabilizer is Cys- (Xaa)_n-Gly, or Gly- (Xaa)_n-Cys; wherein Xaa is selected from one or two of aspartic acid Asp or glutamic acid Glu; and n is any integer less than or equal to 5.

Preferably, Xaa is Asp.

Preferably, Xaa is Glu.

Preferably, the Xaa is a combination of Asp and Glu.

Preferably, the Cys is L-Cys and/or D-Cys.

Preferably, the Asp is L-Asp and/or D-Asp.

Preferably, the Glu is L-Glu and/or D-Glu.

Preferably, n is 1, or 2, or 3, or 4, or 5.

Preferably, the water-soluble semiconductor quantum dot stabilizer is Cys-Asp-Gly or Cys-Glu-Gly.

In a second aspect, the invention provides a use of the water-soluble semiconductor quantum dot polypeptide stabilizer in the first aspect for preparing quantum dots.

Preferably, the quantum dots include CdTe quantum dots, CdSe quantum dots, ZnS quantum dots, CdS quantum dots, AgS quantum dots.

In a third aspect, the invention provides an application of the water-soluble semiconductor quantum dot polypeptide stabilizer in the first aspect in preparation of a polypeptide-quantum dot composite probe.

Preferably, the quantum dots include CdTe quantum dots, CdSe quantum dots, ZnS quantum dots, CdS quantum dots, AgS quantum dots.

In a fourth aspect, the invention provides a water-soluble semiconductor quantum dot stabilized by a polypeptide stabilizer, wherein the water-soluble semiconductor quantum dot stabilized by the polypeptide stabilizer is a quantum dot whose surface is coated with the water-soluble semiconductor quantum dot polypeptide stabilizer of the first aspect, and Cys of the water-soluble semiconductor quantum dot polypeptide stabilizer is connected with the surface of the quantum dot.

Preferably, the quantum dots include CdTe quantum dots, CdSe quantum dots, ZnS quantum dots, CdS quantum dots, AgS quantum dots.

In a fifth aspect, the present invention provides a method for preparing a water-soluble semiconductor quantum dot stabilized by the polypeptide stabilizer of the fourth aspect, the method comprises:

(1) solid-phase synthesis of the water-soluble semiconductor quantum dot polypeptide stabilizer of the first aspect;

(2) dissolving a water-soluble semiconductor quantum dot polypeptide stabilizer and a metal ion precursor in ultrapure water to obtain a solution A, and adjusting the pH of the solution A to 9-12;

(3) preparing a nonmetal precursor solution B, adding the solution B into the solution A in the step (2), and reacting at a constant temperature of 25-100 ℃ to obtain the water-soluble semiconductor quantum dots with stable polypeptide stabilizers;

wherein the metal ion precursor comprises Cd, Zn and Ag; the non-metal precursor comprises S, Se and Te.

Preferably, the method for solid phase synthesis of the water-soluble semiconductor quantum dot polypeptide stabilizer of the first aspect comprises:

(1) rink ammonia resin or chlorine resin is used as resin for solid phase synthesis of polypeptide;

(2) dissolving 4eq of amino acid, HOBt and HBTU in DMF, activating at low temperature for 10-30min, and dropwise adding 4-10eq of DIEA into the solution to obtain a mixed solution;

(3) adding the mixed solution prepared in the step (2) into the polypeptide resin prepared in the step (1), and reacting for 2-4h in a dark place;

(4) treating the resin of step (3) with 20% piperidine DMF solution for 15 min;

(5) circulating the steps (2), (3) and (4) until the synthesis of the target water-soluble semiconductor quantum dot polypeptide stabilizer is finished;

(6) treating the polypeptide resin reacted in the step (5) with cutting fluid for 2-4h, adding ethyl acetate, and obtaining precipitate, namely the polypeptide stabilizer, wherein the volume ratio of the cutting fluid is 95: 2.5: 2.5 trifluoroacetic acid, a radical scavenger and water or from a mixture of water and trifluoroacetic acid in a volume ratio of 90:5:5 trifluoroacetic acid, a radical scavenger and dichloromethane.

In a sixth aspect, the invention provides a polypeptide-quantum dot composite probe stabilized by a polypeptide stabilizer, which is prepared by connecting a targeting polypeptide to the surface of a water-soluble semiconductor quantum dot stabilized by the polypeptide stabilizer in the fourth aspect.

The invention has the beneficial effects that: the water-soluble semiconductor quantum dot polypeptide stabilizer provided by the invention is simple to prepare; the water-soluble semiconductor quantum dot polypeptide stabilizer provided by the invention has good binding capacity with quantum dots, and can remarkably stabilize the quantum dots; the water-soluble semiconductor quantum dot polypeptide stabilizer provided by the invention is widely applicable to different types of semiconductor quantum dots; the water-soluble semiconductor quantum dot polypeptide stabilizer provided by the invention enables the semiconductor quantum dot to have water solubility, biocompatibility, wide pH stability, thermal stability and high ionic strength stability; the water-soluble semiconductor quantum dot polypeptide stabilizer provided by the invention is suitable for a host-guest peptide system, is used for preparing a polypeptide-quantum dot composite probe with good stability and specificity, can be used as a liquid detection reagent for pathogens and disease markers, a biological imaging reagent for germs, cells, tissues and model organisms and an in-vivo imaging reagent for complex organisms, and has wide application prospect.

Drawings

FIG. 1 turbidity analysis of CdTe quantum dots prepared from different types of water-soluble semiconductor quantum dot polypeptide stabilizers;

FIG. 2 stability of CdTe quantum dots prepared from water-soluble semiconductor quantum dot polypeptide stabilizers CGG and CEG at different pH values; where white is CGG and shaded CEG;

FIG. 3 thermal stability results of quantum dots prepared with water-soluble semiconductor quantum dot polypeptide stabilizer CEG;

FIG. 4 shows the result of ionic strength stability of quantum dots prepared by CEG as a water-soluble semiconductor quantum dot polypeptide stabilizer;

FIG. 5 is a fluorescence spectrum of CdTe quantum dots prepared from CEEEG and CEEEEEEEG as water-soluble semiconductor quantum dot polypeptide stabilizers; wherein a is CEEEG and b is CEEEG;

FIG. 6 is a fluorescence spectrum of ZnS, CdS, CdTe, CdSe and AgS quantum dots prepared by water-soluble semiconductor quantum dot polypeptide stabilizer CEG;

FIG. 7 is a spectrum of a host-guest peptide quantum dot-polypeptide probe prepared by a water-soluble semiconductor quantum dot polypeptide stabilizer CEG, wherein the dotted line is a UV-Vis spectrum, and the solid line is a fluorescence spectrum;

FIG. 8 is a fluorescence image of a subject-guest peptide quantum dot-polypeptide probe prepared from a water-soluble semiconductor quantum dot polypeptide stabilizer CEG on rat kidney tissues; wherein a is a host-guest peptide quantum dot-polypeptide probe prepared by a water-soluble semiconductor quantum dot polypeptide stabilizer CEG, and b is a control probe.

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 water-soluble semiconductor quantum dot stabilizer" includes a plurality of such water-soluble semiconductor quantum dot stabilizers, reference to "the highly stable quantum dots" is a reference to one or more highly stable quantum dots 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.

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 the modification of the quantum dot stabilizer described in the present invention on the surface of the CdTe of the quantum dot.

The quantum dot stabilizers CDG (Cys-Asp-Gly), CEG (Cys-Glu-Gly), CEEEG (Cys-Glu-Gly), and CEEEEEG (Cys-Glu-Gly) described in the following examples were all synthesized by solid phase synthesis, but the quantum dot stabilizers described in the present invention are not limited to CDG, CEG, CEEEG, and CEEEEEG.

The polypeptide sequences described in the following examples and comparative examples are shown in table 1 below:

table 1 polypeptide sequences described in the examples and comparative examples

The polypeptide sequence is synthesized by a solid phase synthesis method, and the specific synthesis process comprises the following steps:

weighing 0.3000g of 2-chlorotrityl chloride resin in a polypeptide synthesis tube, and adding 5mL of DCM to suspend the resin; after swelling the resin for 2 hours at room temperature, the polypeptide stabilizer was synthesized by standard Fmoc solid phase polypeptide synthesis method (SPPS): in the sequence synthesis, stepwise coupling of amino acids was performed using Fmoc-protected amino acids (4eq.), HOBt (4eq.), HBTU (4eq.), and DIEA (6 eq.); after each coupling step was completed, washing with DMF (3X 10mL) and DCM (2X 10 mL); the washed resin was freed of the terminal Fmoc protecting group using 20% v/v piperidine DMF solution and washed with DMF (3X 10mL) and DCM (2X 10 mL); after the coupling process and the deprotection process are finished, the ninhydrin color development method is used for checking; when the coupling reaction is finished, detecting the resin to be colorless by using a ninhydrin color development method; when the deprotection reaction is completed, the color is detected to be purple by using a ninhydrin color development method; repeating the coupling and deprotection processes until the synthesis of the polypeptide stabilizer sequence is completed; the resin was washed with DMF (3X 10mL) and DCM (2X 10mL) and 3 times with methanol alternating with DCM; after the resin was air dried, the polypeptide and side chain protecting groups were cleaved using a mixture of TFA, DCM, TIS 90:5:5 at room temperature for 2.5 hours; precipitating the mixed solution after reaction by using ethyl glacial ether with the volume of 10 times, and centrifuging at 6500r/min to obtain a solid; dispersing the solid in glacial ethyl ether, washing, centrifuging at 6500r/min, collecting, and repeating for 3 times to obtain crude product; further purifying the crude product to obtain polypeptide CDG, CEG, CEEEG, CGG, CKG, and CRG.

Example 1 different Water-soluble semiconductor Quantum dot polypeptide stabilizer stabilized CdTe Quantum dots

1.1 Synthesis of Quantum dots

Respectively weighing 90mg of water-soluble semiconductor quantum dot polypeptide stabilizer CDG, CEG, CEEEG, CEEEEEG and 22.7mg of cadmium chloride, dissolving in 30mL of ultrapure water to obtain Cd solution, adjusting the pH of the obtained solution to 11.5 by 1M sodium hydroxide, and deoxidizing and stirring 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 the Cd solution, and heating and refluxing at 100 ℃; after the quantum dot solution is cooled, adding isopropanol with 2 times of volume to precipitate the probe, centrifugally collecting the probe, washing the probe by the isopropanol, and then air-drying the probe to obtain the CdTe quantum dots with different water-soluble semiconductor quantum dot polypeptide stabilizer stabilizations.

1.2 turbidity analysis

Turbidity measurements were recorded by a UV-1750 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Selecting a position of 650nm without ultraviolet and visible absorption as a turbidity measuring wavelength, and measuring the solution turbidity of the CdTe quantum dot reaction solution stabilized by different water-soluble semiconductor quantum dot polypeptide stabilizers after refluxing for 1 hour.

As shown in FIG. 1, the CdTe quantum dots prepared from the water-soluble semiconductor quantum dot polypeptide stabilizers CEG and CDG have good stability and no turbidity. The result shows that the water-soluble semiconductor quantum dot polypeptide stabilizers CEG and CDG show excellent stability.

1.3 pH stability assay

A quantum dot probe solution was prepared at a concentration of 2 μ M. The fluorescence spectra were recorded on an RF-6000 fluorescence spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a xenon lamp as an excitation source. The change of fluorescence intensity of the quantum dots stabilized by the water-soluble semiconductor quantum dot polypeptide stabilizer CEG between pH5-9 was measured.

The results are shown in FIG. 2, where the shaded histogram is CEG. The result shows that the fluorescence intensity of the quantum dots with stable CEG has no obvious change between pH5-9 and shows strong fluorescence; the CEG has more outstanding stability to the quantum dots, and is suitable for detection requirements in a wider pH range.

1.4 thermal stability analysis

The above quantum dot probe solution was prepared at a concentration of 2. mu.M in 10mM phosphate buffer, pH 7.4. The fluorescence spectra were recorded on an RF-6000 fluorescence spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a xenon lamp as an excitation source. The fluorescence intensity change condition of the CdTe quantum dots stabilized by the water-soluble semiconductor quantum dot polypeptide stabilizer CEG at 10-50 ℃ is measured.

The result is shown in figure 3, the fluorescence intensity of the CEG-stabilized CdTe quantum dot probe has no obvious change between 10 ℃ and 50 ℃, and the fluorescence intensity is strong. The result shows that the water-soluble semiconductor quantum dot polypeptide stabilizer has more outstanding stability to quantum dots, and the prepared probe is suitable for detection requirements in a wider temperature range.

1.5 analysis of stability of Ionic Strength

The above quantum dot probe solution was prepared at a concentration of 2. mu.M in 10mM phosphate buffer, pH 7.4. The fluorescence spectra were recorded on an RF-6000 fluorescence spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a xenon lamp as an excitation source. The fluorescence intensity change condition of the CdTe quantum dots stabilized by the water-soluble semiconductor quantum dot polypeptide stabilizer CEG in a 10-125mM NaCl solution is measured.

The result is shown in fig. 4, the fluorescence intensity of the CdTe quantum dot probe stabilized by CEG in NaCl solutions with different concentrations has no obvious change, and the CdTe quantum dot probe is represented as strong fluorescence; the result shows that the water-soluble semiconductor quantum dot polypeptide stabilizer has more outstanding stability performance on quantum dots, and the prepared probe is suitable for the detection requirement in the environment with higher ionic strength.

The turbidity analysis results of the CdTe quantum dots stabilized by the water-soluble semiconductor quantum dot polypeptide stabilizer are shown in table 2 below, and the water-soluble semiconductor quantum dot polypeptide stabilizer provided by the invention has good stability and can stabilize the CdTe quantum dots. The results of pH stability, thermal stability and ionic strength stability show that the CdTe quantum dots stabilized by the water-soluble semiconductor quantum dot polypeptide stabilizer can meet the detection requirement of complex environment.

TABLE 2 stabilization of turbidity and stability of CdTe quantum dots by water-soluble semiconductor quantum dot polypeptide stabilizer

Example 2 preparation of Stable Quantum dots with Water-soluble semiconductor Quantum dot polypeptide stabilizer

2.1 preparation of ZnS Quantum dots

Weighing 90mg of water-soluble semiconductor quantum dot polypeptide stabilizer CEG and 28.7mg of zinc sulfate heptahydrate, dissolving in 30mL of ultrapure water to obtain a Zn solution, adjusting the pH of the obtained solution to 11.0 by 1M sodium hydroxide, and deoxidizing and stirring for later use; weighing 12mg of sodium sulfide, deoxidizing, and adding 1mL of deoxidized ultrapure water to obtain an S solution; adding the S solution into the Cd solution, and heating to react for 2h at 80 ℃. Cooling to room temperature, dialyzing to remove unreacted impurities, and obtaining the ZnS quantum dots.

2.2 preparation of CdS Quantum dots

Weighing 90mg of water-soluble semiconductor quantum dot polypeptide stabilizer CEG and 22.7mg of cadmium chloride, dissolving in 30mL of ultrapure water to obtain a Cd solution, adjusting the pH of the obtained solution to 11.0 by 1M sodium hydroxide, and deoxidizing and stirring for later use; weighing 12mg of sodium sulfide, deoxidizing, and adding 1mL of deoxidized ultrapure water to obtain an S solution; and adding the S solution into the Cd solution, reacting at the constant temperature for 2h at room temperature, and dialyzing to remove unreacted impurities to obtain the CdS quantum dots.

2.3 preparation of CdSe Quantum dots

90mg of water-soluble semiconductor quantum dot polypeptide stabilizer CEG and 22.7mg of cadmium chloride are weighed and dissolved in 30mL of ultrapure water, and the pH of the obtained solution is adjusted to 11.5 by 1M sodium hydroxide to obtain a Cd solution. 31.6mg selenium and 37.8mg NaBH₄Dissolved in 2mL of ultrapure water. Reacting at room temperature until the black color disappears to obtain Se solution. Adding the Se solution into the Cd solution, wherein the molar ratio of Cd to CEG to Se is 1:3:0.25, and heating and refluxing for 3h at 100 ℃. And cooling to room temperature, adding isopropanol with 2 times of volume, centrifuging, collecting precipitate, washing with isopropanol, and air-drying to obtain the CdSe quantum dot.

Preparation of 2.4 AgS Quantum dots

16.9mg AgNO in a three-necked round bottom flask₃And 90mg of water-soluble semiconductor quantum dot polypeptide stabilizer CEG is dissolved in 30mL of deoxidized deionized water to obtain an Ag solution, the pH value is adjusted to 10 by using NaOH, and the Ag solution is deoxidized and stirred for later use. Weighing 12mg of sodium sulfide, deoxidizing, and adding 1mL of deoxidized ultrapure water to obtain an S solution; then the S solution is added into the Ag solution for constant temperature reaction under the condition of vigorous stirring at 50 ℃. And finally, dialyzing to remove unreacted impurities to obtain the AgS quantum dots.

2.5 preparation of CdTe Quantum dots

Respectively weighing 90mg of water-soluble semiconductor quantum dot polypeptide stabilizers CEG, CEEEG and CEEEEEEEG and 22.7mg of cadmium chloride, dissolving 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 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 the Cd solution, and heating and refluxing at 100 ℃; after the quantum dot solution is cooled, adding 2 times of isopropanol by volume to precipitate the probe, centrifugally collecting the probe, washing by the isopropanol, and then air-drying to obtain the CdTe quantum dot.

2.6 Quantum dot optical Property characterization

ZnS quantum dots, CdS quantum dots, CdSe quantum dots, AgS quantum dots, and CdTe quantum dots prepared in the above steps 1 to 5, respectively, were prepared into a quantum dot solution with a concentration of 2. mu.M in 10mM phosphate buffer solution at pH7.4, and the fluorescence spectrum was recorded on an RF-6000 fluorescence spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a xenon lamp as an excitation source.

The fluorescence spectra of the CdTe quantum dots stabilized by the water-soluble semiconductor quantum dot polypeptide stabilizers CEEEG and CEEEEEEEEEEEG are shown in FIG. 5, wherein a is the CdTe quantum dot stabilized by the polypeptide stabilizer CEEEG, and b is the CdTe quantum dot stabilized by the polypeptide stabilizer CEEEEEG, and the quantum dots have maximum emission near 575nm and narrow half-peak width. The result shows that the water-soluble semiconductor quantum dot polypeptide stabilizers CEEEG and CEEEEEEEEEG have excellent stability on CdTe quantum dots, and the prepared quantum dots have outstanding fluorescence properties.

The fluorescence spectra of CdTe quantum dots, ZnS quantum dots, CdS quantum dots, CdSe quantum dots and AgS quantum dots stabilized by water-soluble semiconductor quantum dot polypeptide stabilizer CEG are shown in FIG. 6. The luminous range of the quantum dots stabilized by the water-soluble semiconductor quantum dot polypeptide stabilizer CEG covers blue-violet light to near infrared light. The result shows that the CEG has excellent stability for semiconductor quantum dots with different compositions.

Example 3 preparation and characterization of host-guest peptide Quantum dot-polypeptide probes

Weighing 90mg of water-soluble semiconductor quantum dot polypeptide stabilizer CEG and 22.7mg of cadmium chloride, dissolving 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 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; 0.6mL of Te solution was added to the Cd solution and heated to reflux at 100 ℃. Weighing 10mg of guest targeting polypeptide CE-Ahx-NIDPNAVGN-NH₂Dissolved in 1mL of deoxygenated ultrapure water and slowly added to the quantum dot solution and refluxed for 20 minutes. After the quantum dot solution is cooled, adding 2 times of isopropanol by volume to precipitate the probe, centrifugally collecting the probe, washing by the isopropanol, and then air-drying to obtain the host-guest peptide CdTe quantum dot probe with stable CEG. Meanwhile, quantum dots without the added guest targeting polypeptide are used as control probes.

The ultraviolet-visible absorption spectrum was recorded by a UV-1750 spectrophotometer (Shimadzu Corporation, Kyoto, Japan), and the fluorescence spectrum was recorded on an RF-6000 fluorescence spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a xenon lamp as an excitation source. A solution of the CEG-stabilized host-guest peptide CdTe quantum dot probe prepared in this example at a concentration of 2 μ M in 10mM phosphate buffer, pH7.4, was prepared to accomplish the above characterization. The results are shown in FIG. 7, in which the dotted line is the UV-Vis spectrum and the solid line is the fluorescence spectrum. The result shows that the host-guest peptide CdTe quantum dot probe has strong absorption in near ultraviolet region, so that ultraviolet light can be used as the exciting light of the quantum dot probe for biological imaging. Under the ultraviolet excitation, the maximum emission peak of the quantum dot probe is 635nm, and the quantum dot probe is not influenced by blue self-luminescence generated by the ultraviolet excitation.

Example 4 tissue imaging of host-guest peptide Quantum dot-polypeptide probes

Kidney tissue was taken from 8-10 week old SD rats (200-250 g). Tissues were cryopreserved in Tissue-Tek o.c.t. and the tissues were cut to a thickness of 4 μm and fixed to slides. The tissue sections were air dried at room temperature for use. Tissue sections were fixed with cold methanol at-20 ℃ for 10 min and then washed in 20mM PBS. The sections were then treated with 0.5mL of blocking solution for 30 minutes at room temperature, and the blocking solution was decanted while keeping the sections wet. Host-guest peptide CdTe quantum dot probes and control probe solutions were prepared at a concentration of 0.1mg/mL in 10mM phosphate buffer (pH 7.4). mu.L of each probe solution was used for tissue section staining and the tissue sections were covered with a paraffin film and incubated at 4 ℃ for 6 hours. The parafilm was removed and the solution on the slide was blotted off with absorbent paper. The tissue sections were washed 3 times for 5 minutes each with 10mM phosphate buffer. A drop of anti-quencher was added to the tissue slide and the slide was covered with a coverslip. The Leica DM4000B upright fluorescence microscope collected images.

The results are shown in fig. 8, where a is the imaging result of the host-guest peptide quantum dot-polypeptide probe prepared by CEG, a water-soluble semiconductor quantum dot polypeptide stabilizer, and b is the imaging result of the control probe without guest targeting polypeptide prepared by CEG. The results show that both probes stained tissue under identical conditions, and that the tissue stained with the control probe had no significant fluorescence signal. However, host-guest peptides CdTe quantum dot probe stained tissues showed strong fluorescence. Thus, the guest targeting polypeptide is proved to be a source of host-guest peptide quantum dot probe specificity; quantum dots without guest peptides do not have binding capacity to the target and do not produce non-specific binding. Meanwhile, the water-soluble semiconductor quantum dot polypeptide stabilizer CEG is an ideal host peptide of a host-guest peptide system, and the water solubility and the stability are provided for quantum dots, and the specificity of a guest targeting polypeptide is not influenced.

Comparative example 1 CdTe quantum dot stabilized by polypeptides CGG, CKG and CRG

1.1 Synthesis of Quantum dots

Respectively weighing 90mg of polypeptides CGG, CKG, CRG and 22.7mg of cadmium chloride, dissolving the polypeptides CGG, CKG, CRG and 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 0.6mL of Te solution into the Cd solution, and heating and refluxing at 100 ℃; after the quantum dot solution is cooled, 2 times of isopropanol is added, and precipitates are collected by centrifugation, washed by the isopropanol and dried in the air.

1.2 turbidity analysis

Turbidity measurements were recorded by a UV-1750 spectrophotometer (Shimadzu Corporation, Kyoto, Japan). Selecting a position of 650nm without ultraviolet and visible absorption as a turbidity measuring wavelength, and measuring the solution turbidity of the CdTe quantum dot reaction solution stabilized by different water-soluble semiconductor quantum dot polypeptide stabilizers after refluxing for 1 hour. As a result, as shown in fig. 1, CKG and CRG are not compatible with the alkaline preparation environment of high quality quantum dots, resulting in significant turbidity and poor stability.

1.3 pH stability assay

The concentration of the quantum dot probe solution was 2. mu.M. The fluorescence spectra were recorded on an RF-6000 fluorescence spectrometer (Shimadzu Corporation, Kyoto, Japan) equipped with a xenon lamp as an excitation source. The change of fluorescence intensity of the CGG-stabilized quantum dots between pH5-9 was determined.

The results of the turbidity analysis and the stability of the CdTe quantum dots are shown in the following table 3, and the results show that the polypeptide of the comparative example of the invention has no stability to the quantum dots.

The fluorescence intensity of the CGG-stabilized quantum dots between pH5-9 is shown in FIG. 2, and white is CGG. The result shows that when the CGG is used as a stabilizer and the pH of the prepared quantum dot is changed from 9 to 5, the fluorescence intensity is gradually weakened until the fluorescence disappears, and the stability is poor.

TABLE 3 turbidity and stability of CdTe quantum dots prepared for comparative example polypeptides

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.