Glycan-based water-soluble CuInS2 ternary quantum dot as well as preparation method and application thereof

Abstract

The invention discloses a glycan-based water-soluble CuInS ternary quantum dot as well as a preparation method and application thereof. Hydrothermal synthesis is regulated and controlled through a sulfhydrylated glycan ligand (Glu / Gal-PGEMA-SH), the CuInS quantum dot with the particle size being 3-4 nm and the surface coated with glycan is prepared, and the CuInS quantum dot has visible / near-infrared fluorescence emission, high quantum yield (larger than or equal to 16%) and excellent biocompatibility. Based on good biological penetration performance and stable fluorescence characteristics, the quantum dot not only can be used for high-precision tumor microenvironment dynamic analysis, but also can be used as an excellent fluorescent nanoprobe for tumor monitoring analysis and fluorescence imaging application in living organisms.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of nanomaterials and biomedical technology, and particularly relates to a water-soluble CuInS2 ternary quantum dot (QD) with a polysaccharide molecule as a surface ligand, a preparation method of the quantum dot, and application of the quantum dot in the fields of biological imaging and tumor diagnosis. BACKGROUND

[0002] Nanobiosensors play a key role in rapid diagnosis of cancer and clinical medicine, and have attracted extensive attention in the field of biological science. In recent years, glyconanoparticles (GNPs) have attracted great interest of scientists, which effectively combine the unique chemical-physical and optical properties of various nanoparticles and the properties of carbohydrate coating. Based on the modification of sugar molecules to endow them with unique biological activity functions, the carbohydrate structure grafted on the surface of GNPs enhances the water solubility and colloidal stability of the nanoparticles, effectively reduces the biological toxicity of the material, and improves the biocompatibility. More importantly, the inherent biological recognition properties of carbohydrates enable the nanoparticles to have specific recognition ability. Therefore, GNPs show great potential in biomedical applications, especially in cell labeling and imaging. Therefore, research on the biological effects related to carbohydrates and development of new GNPs have become a hot topic of researchers in recent years.

[0003] Since the first synthesis of carbohydrate-functionalized gold nanoparticles in 2001, various nanoparticles (NPs), including gold, iron oxide and semiconductor quantum dots (QDs), have been used as carriers of carbohydrates to prepare GNPs. These materials have a small size, a large surface area, and may have better sensitivity and specificity. Among them, QDs have become an extremely attractive biomedical resource because of their wide absorption, high quantum yield and excellent light stability, and have been widely used in multicolor imaging and targeted drug delivery in vitro and in vivo.

[0004] Quantum dots (QDs) as a typical semiconductor nanomaterial, due to its unique optical properties (such as size-dependent fluorescence emission, wide excitation spectrum, narrow emission spectrum and light stability) in biological imaging, optoelectronic devices, energy conversion and sensing field show broad application prospects. Although traditional QDs containing cadmium (such as CdSe, CdTe) have excellent fluorescence performance, but its biological toxicity and environmental hazards limit its application in the field of biomedicine. Therefore, the development of low toxicity, environmentally friendly new QDs has become a research hotspot in recent years. Among them, I-III-VI ternary semiconductor CuInS2 QDs due to its low toxicity of constituent elements, band gap adjustable (1.5-2.0 eV), visible-near infrared luminescence characteristics and good water solubility, biocompatibility, etc., these are the basic elements of biomedical applications. In addition, many studies have shown that CuInS2 QDs have great potential in cancer detection and treatment, and are considered as an ideal candidate material to replace traditional binary cadmium, lead-based QDs.

[0005] However, the practical application of CuInS2 QDs still faces two major challenges: first, the traditional synthesis method depends on high-temperature organic phase system (such as octadecene, oleylamine), which leads to the hydrophobicity of quantum dots, and the water solubility and biocompatibility need to be realized through complex ligand exchange or coating process; the second, its fluorescence quantum yield (QY) is generally lower than that of traditional binary cadmium, lead-based QDs, which limits the detection sensitivity of practical application. In view of the above problems, the present application adopts aqueous synthesis strategy, through taking polysaccharide molecules as stabilizer and surface functionalization ligand, the water solubility and biocompatibility of quantum dots are improved while the synthesis steps are simplified. Polysaccharide compounds are widely used in green synthesis and functionalization of nanomaterials due to their specific recognition function, rich functional groups (such as amino, hydroxyl), excellent biodegradability and easy chemical modification characteristics. However, the regulation mechanism of different polysaccharide molecular structures on the nucleation kinetics, surface defect state and optical properties of CuInS2 QDs is still unclear, and the structure-activity relationship research of polysaccharide-based QDs is still relatively scarce. Therefore, it is of research prospect and significance to develop a new type of polysaccharide-based QDs material with water solubility and environmental protection by using carbohydrate as ligand molecule capping, and to apply it as a fluorescent probe in biology and various fields. At present, there are few reports on the synthesis and application of CuInS2 QDs based on polysaccharide molecules as surface ligand modification.

[0006] Based on the excellent biological functionality of the prepared glycan molecular ligand, the application studies a simple and green synthesis method for preparing multifunctional water-soluble CuInS2 QDs optical material, that is, two multifunctional green and environmentally friendly glycan-based QDs, namely Glu / Gal-Pglyco-CuInS2 QDs, are constructed in aqueous solution by a hydrothermal method. First, based on glucose and galactose, glycan molecules containing thiol-SH (Glu-PGEMA-SH and Gal-PGEMA-SH) are synthesized. Then, with Glu-PGEMA-SH and Gal-PGEMA-SH as ligands, copper chloride (CuCl2·2H2O) and indium chloride (InCl3·4H2O) as metal sources, and thiourea (CH4N2S) as a sulfur precursor, two water-soluble glycan-based quantum dots, glucose-modified type (Glu-Pglyco-CuInS2 QDs) and galactose-modified type (Gal-Pglyco-CuInS2 QDs), are successfully prepared by a one-step hydrothermal method. The research results show that the two prepared water-soluble Pglyco-CuInS2 QDs have good dispersity and morphology stability, and both emit bright deep red / near-infrared fluorescence in the visible / near-infrared I region (650 nm and 750 nm), exhibit excitation wavelength-dependent fluorescence tuning characteristics, and have excellent light stability. They have great application potential in the field of life science.

[0007] In addition, the biocompatibility and in vitro imaging potential of the two glycan-based QDs are further explored to provide a theoretical basis for their application in the biomedical field. The application not only provides a new idea for the controllable synthesis of high-performance biologically functional CuInS2 QDs, but also lays an experimental foundation for the rational design of glycan-semiconductor nanocomposites. SUMMARY

[0008] The purpose of the application is to provide a glycan-based water-soluble CuInS2 ternary quantum dot and a preparation method and application thereof.

[0009] I. Preparation of glycan-based water-soluble CuInS2 ternary quantum dots 1. Synthesis of thiolated glycan molecules: (1) Glycosylation reaction of hydroxyethyl methacrylate (HEMA) and 2', 3', 4', 6'-tetra-O-acetyl-α-D-glucopyranose / galactopyranosyl bromide under argon protection, acetylation, and column chromatography purification to obtain Glu / Gal-ACGEMA; The glycosidation reaction conditions are: reaction temperature -35~ -45℃, reaction time 45~50 hours, catalyst AgOTf, solvent dry dichloromethane, and 3Å molecular sieve is added to remove water; the molar ratio of hydroxyethyl methacrylate (HEMA) to 2', 3', 4', 6'-tetra-O-acetyl-α-D-glucosyl / galactosyl bromide is 3:1~4:1.

[0010] (2) The monomer Glu / Gal-GEMA is obtained by catalyzing the deacetylation reaction of Glu / Gal-ACGEMA with sodium methoxide and purifying by silica gel column chromatography; The deacetylation reaction of Glu / Gal-ACGEMA is stirred in MeONa MeOH solution at 20~30℃ for 10~20 minutes; in MeONa MeOH solution, the mass fraction of MeONa is 1%.

[0011] (3) The monomer Glu / Gal-GEMA is polymerized in a water / ethanol mixed system (the volume ratio of water to ethanol is 9:1) to obtain Glu / Gal-PGEMA, with 4-cyano-4-(thiobenzoylthio) pentanoic acid (CTP) as a chain transfer agent and 4,4'-azobis(4-cyanopentanoic acid) (V-501) as an initiator; The polymerization reaction of Glu / Gal-GEMA is carried out at 60~80℃ under argon protection for 3~4 hours, the molar ratio of Glu / Gal-GEMA monomer to chain transfer agent CTP 4-cyano-4-(thiobenzoylthio) pentanoic acid is 90:1~100:1; the molar ratio of Glu / Gal-GEMA monomer to initiator V-501 4,4'-azobis(4-cyanopentanoic acid) is 190:1~200:1.

[0012] (4) The thiolation reaction of Glu / Gal-PGEMA with 3-mercaptopropionic acid is catalyzed by EDC·HCl and DMAP to prepare thiolated glycan ligand Glu / Gal-PGEMA-SH; The thiolation reaction is carried out in DMSO solvent at room temperature for 20~25 h; the molar ratio of Glu / Gal-PGEMA to 3-mercaptopropionic acid is 1:1~1:1.5.

[0013] 2. Preparation of quantum dots: Glu / Gal-PGlyco-CuInS2 quantum dots are prepared by dissolving Glu / Gal-PGEMA-SH in DMSO / H2O mixed solvent, adding CuCl2·2H2O and InCl3·4H2O, adjusting pH to 10~12, then adding thiourea, and hydrothermal reaction at 140~160℃ for 20~25h. The synthesis route of Glu / Gal-PGlyco-CuInS2 QDs Figure 1shown.

[0014] Among them, Cu 2+ 、In 3+ 、S 2- The mass ratio of Glu / Gal-PGEMA-SH is 1:1:2:6 to 1:1:2:12. In the DMSO / H₂O mixed solvent, the volume ratio of DMSO to H₂O is 1:1. After the hydrothermal reaction, the product is purified by centrifugation at 20,000 rpm for 10 minutes, and then dispersed in ultrapure water.

[0015] 2. Characterization and Properties of Glu / Gal-Pglyco-CuInS2QDs 1. Effect of experimental variables on the fluorescence properties of Glu / Gal-Pglyco-CuInS2QDs In order to explore the effect of experimental variables on the fluorescence properties of Glu / Gal-Pglyco-CuInS2QDs, the mass ratio between Cu, In, S and polysaccharide ligands, the pH and temperature of the reaction system were changed to explore the effects of different experimental variables on the photophysical properties of Glu / Gal-Pglyco-CuInS2QDs. Figures 2-4 Figures 2 and 3 show the fluorescence intensity and emission wavelength of Glu / Gal-Pglyco-CuInS2QDs synthesized under different experimental conditions. The results show that when the mass ratios of Cu, In, S, and polysaccharide ligands are 1:1:2:12 and 1:1:2:6, respectively, the Glu-Pglyco-CuInS2QDs and Gal-Pglyco-CuInS2QDs achieve optimal emission wavelengths at pH 12, 13, and 150°C. When the mass ratios of Cu, In, S, and polysaccharide ligands are 1:1:2:12 and 1:1:2:6, respectively, the fluorescence intensities of the Glu-Pglyco-CuInS2QDs and Gal-Pglyco-CuInS2QDs are maximized at pH 11, 12, and 150°C. In summary, under these preparation conditions, the Glu / Gal-Pglyco-CuInS2QDs achieve excellent photophysical properties. Therefore, two structurally well-defined, water-soluble, and biocompatible Glu / Gal-Pglyco-CuInS2QDs with polysaccharide molecules as surface ligands were synthesized by a hydrothermal method.

[0016] 2. Structural Characterization of Glu / Gal-Pglyco-CuInS2 QDs The chemical composition of the prepared Glu / Gal-Pglyco-CuInS2QDs was systematically characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) to verify the successful preparation of CuInS2QDs with thiolated polysaccharide molecules as surface ligands. First, crystallographic analysis was used to explore the regulatory effect of polysaccharide molecules on the crystal structure and morphology of quantum dots. Figure 5 As shown, the XRD spectrum of Glu / Gal-Pglyco-CuInS2 QDs shows three characteristic diffraction peaks, whose Bragg diffraction angles (2θ) are located at 27.9°, 46.3° and 55.1° respectively. After comparing with the standard card (CuInS2, JCPDS47-1372), these diffraction peaks can be attributed to the (112), (024) and (132) crystal planes of tetragonal CuInS2. According to the Debye-Scherrer equation (Equation 2-1): Where θ is the Bragg angle, λ is the X-ray wavelength (0.154 nm), B is the half-width (FWHM) of the diffraction peak, and K is the Scherrer constant (0.89). Calculated average crystallite sizes of the Glu / Gal-Pglyco-CuInS2 QDs are 3.53 nm and 3.89 nm, respectively. The diffuse peaks indicate their small size, while the absence of diffraction peaks associated with other impurity phases in the spectrum demonstrates the excellent phase purity of the prepared samples.

[0017] The Glu / Gal-Pglyco-CuInS2QDs stabilized by thiolated polysaccharide molecules were characterized by X-ray photoelectron spectroscopy (XPS). Figure 6 ), systematically analyzing its elemental composition, chemical state and surface coordination mechanism. Full spectrum analysis shows that quantum dots exhibit characteristic signal peaks at binding energies of 285 eV (C 1s), 531 eV (O 1s), 952 / 932 eV (Cu 2p), 452 / 445 eV (In 3d) and 163.3 eV (S 2p). In order to obtain deeper structural information about CuInS2QDs, high-resolution XPS spectra of each element were further analyzed. The C 1s spectrum can be decomposed into four chemical states at 284.1 eV (CH), 284.6 eV (CC), 285.5 eV (CO) and 287.8 eV (C=O), which are highly consistent with the functional group information characterized by FTIR. The O 1s spectrum shows a characteristic peak of carboxyl oxygen at 531 eV, proving the stable coating of the surface polysaccharide molecular ligand. The Cu 2p fine spectrum only has a peak at 932 eV (2p 3 / 2 ) and 951 eV (2p 1 / 2) characteristic double peaks, while the Cu²⁺ peak at 944 eV disappears, proving that in the process of preparing Glu / Gal-Pglyco-CuInS2QDs, Cu 2+ The oxidized state of Cu is reduced to Cu by the thiolated polysaccharide molecules. + In addition, 3+ Valence state (In 3d 5 / 2 , ~445 eV and In 3d 3 / 2 , ~452 eV) was also confirmed by XPS spectroscopy. Finally, analysis of the high-resolution XPS spectrum of S 2p confirmed the valence state of the S element to be -2. These XPS test results demonstrate the successful preparation of Glu / Gal-Pglyco-CuInS2QDs.

[0018] 3. Morphological Characterization of Glu / Gal-Pglyco-CuInS2QDs In order to study the nanostructure characteristics of Glu / Gal-Pglyco-CuInS2QDs, their microstructure and size distribution were systematically characterized by thermal field emission transmission electron microscopy (TEM) and dynamic light scattering (DLS) techniques. TEM analysis ( Figure 7 a1-a2) show that the Glu / Gal-Pglyco-CuInS2QDs functionalized with polysaccharide ligands exhibit highly uniform spherical morphology, with a particle size distribution concentrated in the range of 3-4 nm ( Figure 7 b1-b2), which is in good agreement with the grain size calculated from the XRD diffraction pattern. Typical lattice fringes can be seen in the inset of the high-resolution TEM image, and the measured interplanar spacings are 3.19 Å and 2.87 Å, respectively, corresponding to the (112) crystal plane of the CuInS2 crystal, confirming the integrity of its single crystal structure. In addition, the lattice continuity of the QDs edge region is not impaired by the modification of the surface polysaccharide molecule ligands, indicating that the glycosylation process does not affect the crystal structure of CuInS2QDs.

[0019] In order to further analyze the elemental composition characteristics of QDs, the elemental composition of Glu / Gal-Pglyco-CuInS2QDs was characterized by scanning electron microscopy-energy dispersive spectroscopy (SEM-EDS). Energy dispersive spectroscopy analysis confirmed that both QDs contained characteristic element peaks of carbon (C), oxygen (O), copper (Cu), indium (In) and sulfur (S), among which the C and O signals were contributed by the surface polysaccharide ligands. Figure 8The spatial distribution of each element was visualized, and the results showed that C, N, O, Cu, In, and S were evenly distributed on the surface of the Glu / Gal-Pglyco-CuInS2 QDs. The high-intensity signal areas of Cu and In elements reflected the enrichment characteristics of the corresponding elements. The elemental analysis results further proved the successful preparation of Glu / Gal-Pglyco-CuInS2 QDs.

[0020] 4. Optical properties of Glu / Gal-Pglyco-CuInS2 QDs On the basis of the morphological structure characterization of Glu / Gal-Pglyco-CuInS2 QDs, we systematically explored their optical physical properties. As shown in Figure 9 UV-Vis analysis revealed the light absorption behavior of the two kinds of glyco-CuInS2 QDs. As can be seen from the figure, Glu / Gal-Pglyco-CuInS2 QDs showed strong absorption near 300 nm, which was mainly due to the absorption of thiolated glycan molecules. At the same time, Glu / Gal-Pglyco-CuInS2 QDs showed strong UV-Vis absorption in the range of 300-500 nm, which was consistent with the characteristic absorption of CuInS2 QDs reported in the literature. However, it is worth noting that Gal-Pglyco-CuInS2 QDs showed certain light absorption ability in the near-infrared region (> 700 nm), indicating that they had the potential for near-infrared light emission. In summary, Glu / Gal-Pglyco-CuInS2 QDs showed good light absorption ability in the visible and near-infrared regions, which would be beneficial to their application in biological imaging.

[0021] In the process of exploring the optimal synthesis conditions of two kinds of Pglyco-CuInS2 QDs, based on the respective needs for long-wavelength emission and high fluorescence intensity, the optimal synthesis conditions were explored. As shown in Figure 10Glu-Pglyco-CuInS2 QDs with the longest emission wavelength of 630 nm were prepared when the mass ratio of Cu, In, S and Glu-Pglyco polysaccharide molecular ligand was 1:1:2:12, pH = 12 and the synthesis condition was 150℃. The Glu-Pglyco-CuInS2 QDs with the highest fluorescence intensity were prepared when the mass ratio of Cu, In, S and Glu-Pglyco was 1:1:2:12, pH = 11 and the synthesis condition was 150℃, but the emission wavelength was blue-shifted to 610 nm. Similarly to the Glu-Pglyco-CuInS2 QDs, Gal-Pglyco-CuInS2 QDs with the longest emission wavelength of 750 nm were prepared when the mass ratio of Cu, In, S and Gal-Pglyco polysaccharide molecular ligand was 1:1:2:6, pH = 13 and the synthesis condition was 150℃. The Gal-Pglyco-CuInS2 QDs with the highest fluorescence intensity were prepared when the mass ratio of Cu, In, S and Gal-Pglyco polysaccharide molecular ligand was 1:1:2:6, pH = 12 and the synthesis condition was 150℃, but the emission wavelength was blue-shifted to 580 nm. The above test results show that the emission wavelength of the quantum dots can be controlled by adjusting the synthesis conditions, that is, the QDs can be effectively controlled from green light to red light to near-infrared light according to the requirements.

[0022] Then, the fluorescence quantum yield (PLQY) and fluorescence lifetime (τ) values of the two polysaccharide-based quantum dot materials were tested, as shown in Table 1. The PLQY of Glu / Gal-Pglyco-CuInS2 QDs in aqueous solution was more than 16 %, which showed a relatively high PLQY, of which Φ f (Glu-Pglyco-CuInS2 QDs) = 16.53 %, Φ f (Gal-Pglyco-CuInS2 QDs) = 18.19 %. In addition, the average fluorescence lifetime of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs in water was 489 ns and 575 ns, respectively, which was much higher than that of traditional organic fluorescent dyes, and met the characteristics of high fluorescence lifetime of quantum dots. Therefore, the high quantum yield and long fluorescence lifetime of Glu / Gal-Pglyco-CuInS2 QDs in water environment are conducive to their application in the field of biological imaging.

[0023] wherein, λ ex - excitation wavelength; λ em - emission wavelength; τ - fluorescence lifetime; χ2 - Confidence factor; Φ f - Quantum yield.

[0024] 5. Stability of Glu / Gal-Pglyco-CuInS2 QDs 5.1 Fluorescence stability As a key tool in the field of bio-fluorescent labeling and cell imaging, the photostability of QDs probes is a particularly important parameter for fluorescence imaging, especially for long-term dynamic tracking of living samples. Among them, the study of the luminescence stability of Glu / Gal-Pglyco-CuInS2 QDs is particularly crucial. First, through fluorescence spectrum analysis, the fluorescence intensity decay law of two kinds of Glu / Gal-Pglyco-CuInS2 QDs aqueous solution was systematically evaluated within 1 day, 7 days and 14 days of storage period. As shown in Figure 5.1, both Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs aqueous solution showed good light stability after 14 days of placement. The emission wavelength hardly changed, and the PL intensity also changed only slightly. The test results show that Glu / Gal-Pglyco-CuInS2 QDs have good luminescence stability. Figure 11

[0025] 5.2 pH sensitivity Because cancer cells produce excess lactic acid and hydrogen ions and other acidic substances during special metabolic processes, which cannot be returned to the blood system in time and accumulate outside the cells, causing the pH of the microenvironment around the tumor tissue to gradually change from the original normal weak alkaline (pH 7.4±0.05) to weak acid (pH 6.0~6.9), therefore, the bio-imaging fluorescent probe must have the luminescence characteristics of acidic environment responsiveness. Therefore, the relationship between pH and the fluorescence intensity of Glu / Gal-Pglyco-CuInS2 QDs was studied. Figure 12 The fluorescence spectra of Glu / Gal-Pglyco-CuInS2 QDs aqueous solution at pH values of 2-13 are shown in Figure 5.2. As the pH of the system increases from 2 to 13, the fluorescence intensity of the two QDs first increases and then decreases, and emits the strongest fluorescence in the weak acid environment of pH = 6~7, which will be conducive to its application in cancer cell tracking and bio-imaging.

[0026] ​In summary, the present application provides an effective method for synthesizing green and environmentally friendly polysaccharide-based CuInS2 QDs in one step in an aqueous phase. First, based on glucose and galactose, two polysaccharide molecular ligands (Glu-PGEMA and Gal-PGEMA) are synthesized by thiol functionalization modification. Then, two water-soluble polysaccharide-based ternary QDs, namely, Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs, are prepared under the regulation of the polysaccharide molecular ligands by using copper chloride and indium chloride as metal precursors and thiourea as a sulfur source in one-step hydrothermal synthesis. The molecular structure of the ligands is analyzed by nuclear magnetic resonance hydrogen spectrum (1H NMR), mass spectrometry (MS), and Fourier transform infrared spectroscopy (FTIR); the crystal structure and surface chemical state of the two QDs are confirmed by X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). Transmission electron microscopy (TEM) combined with dynamic light scattering (DLS) analysis shows that the average particle size of the two quantum dots is distributed in the range of 3-4 nm, and they have monodisperse spherical morphology and excellent colloidal stability. Notably, the water solubility of the Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs modified by the polysaccharide ligands is excellent, which lays an important foundation for their application in the biomedical field. The optical research results show that the Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs emit bright deep red / near-infrared fluorescence in the visible / near-infrared I region (650 nm and 750 nm), respectively, and exhibit obvious excitation dependence, excellent optical stability, high fluorescence quantum yield (16.53% and 18.19%), and long fluorescence lifetime (489 ns and 575 ns). More importantly, the Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs both exhibit the strongest fluorescence emission performance in the physiological range of pH = 6-7.

[0027] The two polysaccharide-based quantum dots prepared exhibit good water dispersibility, excellent visible / near-infrared luminescence performance, and excellent stability under light, high temperature, different pH environments, and long-term storage conditions, which is conducive to their application in the fields of biological imaging and in vitro detection research.

[0028] III. Use of Glu / Gal-Pglyco-CuInS2 QDs 1. Cell fluorescence imaging of Glu / Gal-Pglyco-CuInS2 QDs We evaluated the cytotoxicity of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs by MTT assay. Hela cells, MKN-45 cells, Hep-G2 cells were cultured for 24 h at different concentration gradients (0, 20, 40, 60, 80 and 100 μg / mL), respectively. As shown in Figure 13 When the concentration of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs solution was up to 100.0 μg / mL, the survival rate of Hela cells, MKN-45 cells, Hep-G2 cells was up to 95% or more, which indicated that the cytotoxicity of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs was extremely low, and had good biocompatibility. Therefore, Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs material was suitable for intracellular detection and imaging.

[0029] Subsequently, we studied the imaging ability of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs on Hela cells, MKN-45 cells, Hep-G2 cells at different incubation times. Hela cells, MKN-45 cells, Hep-G2 cells were incubated in the culture medium containing 100 μg / mL of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs for 0.5 h, 2 h, 4 h and 12 h, respectively, Figure 14(a1-b3) is its fluorescence imaging figure. It can be seen from the figure that Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs can be highly aggregated on the cell membranes of Hela, MKN-45, and Hep-G2 tumor cells, and the fluorescence intensity continuously increases over time. After co-incubation for 0.5 h, a certain amount of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs are enriched on the cell membranes, and red fluorescence signals can be observed on the cell membranes of HeLa, MKN-45, and Hep-G2. With the extension of incubation time, more Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs samples are observed to be enriched on the cell membranes at 2 h, and the fluorescence signals obtained are stronger than those at 0.5 h. After continuing to incubate for 4 h, the fluorescence brightness on the cell membrane is significantly enhanced, and when continuing to incubate for 12 h, the fluorescence signal intensity on the cell membrane does not significantly increase, indicating that after 4 h of staining, the two QDs have reached the maximum enrichment on the cell membrane. The above study shows that Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs can enter the tumor cell membrane, and are enriched in the tumor cells within a certain time, making the fluorescence signal intensity gradually increase.

[0030] 2. Cell membrane targeting fluorescence imaging of Glu / Gal-Pglyco-CuInS2 QDs It can be observed from the above cell imaging pictures that Glu / Gal-Pglyco-CuInS2 QDs are mainly enriched on the cell membranes of the three tumor cells for imaging. This is mainly because Glu / Gal-Pglyco-CuInS2 QDs can specifically stain the cell membranes of HeLa, MKN-45, Hep-G2, and other tumor cells by recognizing the sugar cell membrane receptor protein. In order to further verify the membrane localization function of QDs in living cells, a co-staining experiment was performed. The red fluorescence signal of Glu / Gal-Pglyco-CuInS2 QDs in HeLa and MKN-45 cells is co-localized with the blue fluorescence signal of DAPI (a commercial cell nucleus positioning fluorescent dye). It should be noted that since Glu / Gal-Pglyco-CuInS2 QDs samples emit red fluorescence signals, they coincide with the red fluorescence signals of Dil (a commercial cell membrane positioning fluorescent dye), and therefore cannot be used. Therefore, the cell nucleus dye DAPI is selected for co-localization to explore the membrane positioning staining of QDs. It can be seen from the figure that the red fluorescence signals of Glu / Gal-Pglyco-CuInS2 QDs in HeLa and MKN-45 cells are co-localized with the blue fluorescence signals of DAPI, which is a commercial cell nucleus positioning fluorescent dye. This result further verifies that Glu / Gal-Pglyco-CuInS2 QDs can specifically stain the cell membranes of HeLa and MKN-45 cells. Figure 15It can be clearly observed that the red fluorescence signal emitted by QDs in the red channel and the blue signal emitted by DAPI in the blue channel have no obvious overlap, and the blue imaging area is wrapped by red fluorescence, which proves that Glu / Gal-Pglyco-CuInS2 QDs have good targeting imaging function for cell membranes. In addition, the previous research of the application has proved that Glu / Gal-Pglyco-CuInS2 QDs have excellent optical stability and good anti-photobleaching ability. Therefore, Glu / Gal-Pglyco-CuInS2 QDs can be used as an ideal cell membrane imaging agent, and have very important tumor positioning application value.

[0031] 3. Multicellular 3D tumor microsphere imaging of Glu / Gal-Pglyco-CuInS2 QDs At present, the challenges faced by QDs include poor penetration depth of tumor tissue, which makes them only gather in the periphery of the tumor in vivo, limiting their aggregation and imaging inside the tumor. Multicellular tumor spheroids (MCTS) are widely recognized in evaluating the penetration and retention of molecular probes in the tumor microenvironment. However, there are still few reports on the spatiotemporal distribution dynamics of QDs-based nanoprobes in three-dimensional spheroid models. Therefore, the application takes MCTS constructed by HeLa and MKN-45 cells as research carriers, and systematically explores the tumor penetration behavior and imaging efficiency of Glu / Gal-Pglyco-CuInS2 QDs. Figure 16 (a, b) Confocal microscopic images clearly reveal that at the initial incubation stage (3 h), the red fluorescence signal of Glu / Gal-Pglyco-CuInS2 QDs is mainly enriched in the peripheral region of the spheroid, indicating that the probe has not yet broken through the dense outer cell barrier; as the co-culture time is prolonged (6-12 h), the fluorescence signal gradually diffuses to the middle layer of the spheroid, and the signal intensity is significantly improved; by 24 h, the sample has penetrated to the core region of the spheroid, and is uniformly dispersed in the entire three-dimensional space. Experimental data show that the fluorescence intensity of Glu / Gal-Pglyco-CuInS2 QDs is time-dependent and increases, and is uniformly dispersed in the entire tumor cell, and Glu / Gal-Pglyco-CuInS2 QDs have excellent tumor penetration ability.

[0032] Based on the excellent optical properties, water-solubility and luminescence stability of the prepared glycan-based quantum dots Glu / Gal-Pglyco-CuInS2 QDs, the bio-imaging ability of the two kinds of QDs was systematically studied. Firstly, the two kinds of glycan-based quantum dots exhibit excellent water-solubility, excellent biocompatibility and no significant cytotoxicity. Based on this characteristic, it has important application prospects in the field of bio-fluorescence imaging. The experimental results show that by staining three kinds of tumor cells (Hela, MKN-45 and Hep-G2), Glu / Gal-Pglyco-CuInS2 QDs all present bright red fluorescence signals on the cell membrane, showing excellent cell membrane targeting imaging ability. More importantly, in the 3D tumor microsphere (MCTS) imaging study, Glu / Gal-Pglyco-CuInS2 QDs exhibit excellent penetration in MCTS, and its fluorescence signal can penetrate the dense cell matrix layer by layer and enrich in the core necrotic area of the microsphere. Therefore, Glu / Gal-Pglyco-CuInS2 QDs are expected to be used as excellent fluorescent nanoprobes for tumor monitoring and analysis.

[0033] The beneficial effects of the present application are: (1) The present application is based on glucose and galactose, and two kinds of glycan molecules containing thiol-SH are synthesized: Glu-PGEMA-SH and Gal-PGEMA-SH. Then, two kinds of water-soluble glycan-based QDs: Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs are synthesized by using them as ligands and adopting hydrothermal method. The experimental results show that the two kinds of QDs all have the advantages of small size, strong stability, good biocompatibility and the like. Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs respectively emit bright deep red / near-infrared fluorescence in the visible / near-infrared I region (650 nm and 750 nm), and show obvious excitation-dependent adjustable fluorescence emission and excellent optical stability. In addition, Glu / Gal-Pglyco-CuInS2 QDs exhibit excellent bio-fluorescence characteristics, which provides an important material basis for bio-imaging application.

[0034] (2) Based on the prepared two kinds of glycan-based quantum dots (Pglyco-CuInS2 QDs) exhibit excellent optical response characteristics, good water dispersibility and excellent luminescence performance, the application of biological imaging was systematically studied. The research results show that Glu / Gal-Pglyco-CuInS2 QDs have excellent fluorescence imaging ability in the red channel, and show good specific fluorescence imaging targeting function to tumor cell membrane. More importantly, Glu / Gal-Pglyco-CuInS2 QDs exhibit excellent three-dimensional space penetration ability in the study of multi-cell tumor spheroids (MCTS), successfully constructing a three-dimensional visualization model of complex tumor microenvironment, significantly improving the limitations of traditional fluorescent probes in tumor microsphere penetration. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a schematic diagram of the synthesis of Glu / Gal-Pglyco-CuInS2 QDs.

[0036] Figure 2 (a, b) Fluorescence emission spectra of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs synthesized under different ligand content ratios.

[0037] Figure 3 (a, b) Fluorescence emission spectra of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs synthesized under different pH values.

[0038] Figure 4 (a, b) Fluorescence emission spectra of Glu-Pglyco-CuInS2 QDs and Gal-Pglyco-CuInS2 QDs synthesized at different temperatures.

[0039] Figure 5 is an XRD diagram of Glu / Gal-Pglyco-CuInS2 QDs synthesized with glycan molecules as ligands.

[0040] Figure 6 (a) X-ray photoelectron spectroscopy of Glu-Pglyco-CuInS2 QDs, (b) Gal-Pglyco-CuInS2 QDs.

[0041] Figure 7(a1, a2) TEM images of Glu-Pglyco-CuInS2QDs, Gal-Pglyco-CuInS2QDs, scale bar is 10 nm and 20 nm (inset: HR-TEM images of Glu / Gal-Pglyco-CuInS2QDs and their lattice spacing, scale bar is 1 nm); (b1, b2) Particle size distribution of Glu-Pglyco-CuInS2QDs, Gal-Pglyco-CuInS2QDs. (inset: Visualized photographs of Glu / Gal-Pglyco-CuInS2QDs under excitation wavelength light irradiation and their path maps).

[0042] Figure 8 EDS elemental mapping of Glu-Pglyco-CuInS2QDs and Gal-Pglyco-CuInS2QDs.

[0043] Figure 9 (a) UV-Vis absorption spectra of Glu-Pglyco-CuInS2QDs, (b) Gal-Pglyco-CuInS2QDs.

[0044] Figure 10 (a1, b1) UV-Vis absorption spectra and optimal emission wavelength fluorescence spectra of Glu / Gal-Pglyco-CuInS2QDs; (a2, b2) UV-Vis absorption spectra and strongest fluorescence intensity fluorescence spectra of Glu / Gal-Pglyco-CuInS2QDs.

[0045] Figure 11 (a1, a2) Fluorescence stability of Glu-Pglyco-CuInS2QDs in solution (b1, b2) Gal-Pglyco-CuInS2QDs. The curves in the figure respectively refer to the fluorescence spectra of Glu / Gal-Pglyco-CuInS2QDs after 1, 7 and 14 days.

[0046] Figure 12 (a) pH-responsive fluorescence spectra of Glu-Pglyco-CuInS2QDs (b) Gal-Pglyco-CuInS2QDs. (inset: Relationship between pH and luminescence intensity of Glu / Gal-Pglyco-CuInS2QDs).

[0047] Figure 13 (a, b) Cell viability changes after incubation with different concentration gradients (20, 40, 60, 80, 100 μg / mL) of Glu-Pglyco-CuInS2QDs, Gal-Pglyco-CuInS2QDs in three kinds of cancer cells, respectively.

[0048] Figure 14 (a1, a2, a3) HeLa, MKN-45, Hep-G2 cell imaging images after incubation with 100 μg / mL of Glu-Pglyco-CuInS2 QDs for 0.5 h, 2 h, 4 h and 12 h; (b1, b2, b3) HeLa, MKN-45, Hep-G2 cell imaging images after incubation with 100 μg / mL of Gal-Pglyco-CuInS2 QDs for 0.5 h, 2 h, 4 h and 12 h. (Scale bar: 20 μm).

[0049] Figure 15 Confocal study of Glu / Gal-Pglyco-CuInS2 QDs with DAPI (commercial nuclear dye). (a1, a2) Fluorescence images of HeLa cells co-cultured with Glu-Pglyco-CuInS2 QDs, Gal-Pglyco-CuInS2 QDs for 3 hours; (b1, b2) Fluorescence images of MKN-45 cells co-cultured with Glu-Pglyco-CuInS2 QDs, Gal-Pglyco-CuInS2 QDs for 3 hours. (Scale bar: 20 μm) Figure 16 (a) Fluorescence expression of Glu / Gal-Pglyco-CuInS2 QDs at different times in 3D Hela cell microspheres (scale: 500 μm); (b) Fluorescence expression of Glu / Gal-Pglyco-CuInS2 QDs at different times in 3D MKN-45 cell microspheres (scale: 500 μm).

[0050] Figure 17 NMR hydrogen spectrum of Glu-ACGEMA (400 MHz, CDCl3).

[0051] Figure 18 NMR hydrogen spectrum of Glu-GEMA (400 MHz, CD3OD).

[0052] Figure 19 NMR hydrogen spectrum of Glu-PGEMA (400 MHz, D2O).

[0053] Figure 20 Gel permeation chromatogram of Glu-PGEMA.

[0054] Figure 21 NMR hydrogen spectrum of Glu-PGEMA-SH (400 MHz, DMSO-d6).

[0055] Figure 22 H NMR of Gal-ACGEMA (400 MHz, CDCl3).

[0056] Figure 23 H NMR of Gal-GEMA (400 MHz, CD3OD).

[0057] Figure 24 H NMR of Gal-PGEMA (400 MHz, D2O).

[0058] Figure 25 Gel permeation chromatogram of Gal-PGEMA.

[0059] Figure 26 H NMR of Gal-PGEMA-SH (400 MHz, DMSO-d6). DETAILED DESCRIPTION

[0060] Example One 1. Synthesis of thiolated glycan molecule Glu-PGEMA-SH (1) Synthesis of Glu-ACGEMA Hydroxyethyl methacrylate (HEMA) (1.899 g, 14.30 mmol) and 2', 3', 4', 6'-tetra-O-acetyl-a-D-glucopyranosyl bromide (2.000 g, 4.760 mmol) were dissolved in dry dichloromethane (DCM). 3A powdered molecular sieves (2.400 g) were added, stirred for 30 min under argon protection, then the mixture was transferred to a -40 °C reaction bath, and excess silver trifluoromethanesulfonate (AgOTf) (1.515 g, 5.720 mmol) was added to the mixture and reacted for 48 h. After the reaction was completed, the solvent was removed by a rotary evaporator, and the residue was dissolved in a mixture of pyridine (50 mL) and acetic anhydride (20 mL) to fully acetylate the unreacted HEMA, and reacted overnight under argon protection. Then, the solvent was removed, and the residue was dissolved in DCM (50 mL). Water-soluble impurities were washed away with hydrochloric acid (0.25 M, 30 mL), saturated brine solution, saturated NaHC03solution, and evaporated under vacuum to remove the solvent. Finally, the product was separated by silica gel column chromatography (ethyl acetate: hexane, 3:7) to obtain 1.55 g of light yellow solid product Glu-ACGEMA with an approximate yield of 40%. The molecular structure of Glu-ACGEMA was confirmed by1H NMR (as shown in Figure 1). 1 H NMR of Gal-ACGEMA (400 MHz, CDCl3). Figure 17

[0061] 1 ​H NMR (400 MHz, Chloroform- d ) δ 6.12 (dd, J = 2.3, 1.3 Hz, 1H), 5.59(h, J = 1.7 Hz, 1H), 5.19 (t, J = 9.5 Hz, 1H), 5.08 (t, J = 9.7 Hz, 1H), 5.00 (dd, J = 9.6, 8.0 Hz, 1H), 4.56 (d, J = 7.9 Hz, 1H), 4.32 – 4.23 (m, 3H), 4.13 (dd, J =12.3, 2.4 Hz, 1H), 4.05 (ddd, J = 11.3, 5.1, 3.5 Hz, 1H), 3.82 (ddd, J = 11.2,7.0, 3.7 Hz, 1H), 3.69 (ddd, J = 10.0, 4.7, 2.4 Hz, 1H), 2.09 (s, 3H), 2.02 (s,3H), 2.00 (d, J = 2.5 Hz, 6H), 1.94 (t, J = 1.3 Hz, 3H). (2) Synthesis of monomer Glu-GEMA To a solution of 2-O-((2',3',4',6'-tetra-O-acetyl)-a-D-glucopyranosyl)methyl acrylate Glu-ACGEMA (1.2 g, 2.6 mmol) in MeOH (5 mL) was added to a solution of 1% MeONa in MeOH (0.1 mL) and the mixture was stirred at 25 °C for 15 min. After the reaction was completed, the reaction mixture was neutralized with AcOH and the solvent was removed by distillation under reduced pressure. Finally, the product was purified by silica gel column chromatography (20% MeOH in chloroform) to give 0.42 g of yellow oil of Glu-GEMA with a yield of about 35%. The molecular structure of Glu-GEMA was confirmed by1H NMR (as shown in Figure 2). 1 Figure 18 ).

[0062] 1 H NMR (400 MHz, Methanol- d ​4) δ 6.11 (dd, 1H, J = 1.0 Hz, J = 1.5 Hz),5.62 (t, 1H, J = 1.5 Hz), 4.83 (d, 1H, J =1.5 Hz), 4.35 (ddd, 1H, J = 3.0 Hz,J = 6.0 Hz, J = 12.0 Hz), 4.32 (ddd, 1H, J = 3.0 Hz, J = 6.0 Hz, J = 12.0Hz), 3.96 (ddd, 1H, J = 3.0 Hz, J = 6.0 Hz, J =11.5 Hz), 3.81−3.84 (m, 2H),3.74 (ddd, 1H, J = 2.5 Hz, J = 6.0 Hz, J = 11.5 Hz), 3.73 (t, 1H, J =5.5 Hz),3.69 (dd, 1H, J = 3.5 Hz, J = 10.0 Hz), 3.64 (t, 1H, J = 9.5 Hz), 3.55−3.59(m, 1H), 1.97 (s, 3H). (3) Synthesis of polymer Glu-PGEMA Into a 10 mL reaction vial, Glu-GEMA (200 mg, 0.685 mmol) and CTP (1.96 mg, 7 umol) were dissolved in 5 mL of water and ethanol (9:1 by volume) mixture, the reaction vial was sealed with a rubber septum, purged with argon for 30 min, into another reaction vial, V-501 (0.98 mg, 3.5 umol) and 1 ml of deionized water were added, purged with argon, and then added into the above reaction mixture by syringe, and reacted at 70 °C for 3-4 h. After the reaction was completed, it was precipitated with a large amount of methanol, centrifuged, washed, and freeze-dried to obtain 50 mg of pink solid product Glu-PGEMA. The yield was about 25%. The molecular structure of Glu-PGEMA was confirmed by 1 H NMR, GPC (as Figure 19 , Figure 20 ).

[0063] 1 H NMR (400 MHz, Deuterium Oxide) δ 4.47 (d, J = 7.3 Hz, 7H), 4.16 (d, J = 43.0 Hz, 27H), 3.93 (s, 11H), 3.76 (d, J= 41.5 Hz, 19H), 3.44 (dd, J = 29.9,14.1 Hz, 24H), 2.06 (d, J = 71.9 Hz, 15H), 0.98 (d, J = 72.5 Hz, 24H). The molecular weight of polymer Glu-PGEMA was determined by gel permeation chromatography (GPC) with water as mobile phase, as shown in Figure 1. Figure 20 The GPC elution curve of polymer Glu-PGEMA is a single peak, the number average molecular weight (M n ) is 2.5 x 10 5 , the weight average molecular weight (M w ) is 2.8 x 10 5 , and the PDI calculated from (M w / M n ) is 1.12, the molecular weight distribution is very narrow, the degree of polymerization (DP) is about 500, which is consistent with the characteristics of RAFT active polymerization.

[0064] GPC (unit g / mol): Mn = 2.5 x 10 5 , Mw = 2.8 x 10 5 , PDI (M w / M n ) = 1.12 (4) Synthesis of Glu-PGEMA-SH 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl, 0.157 g, 0.816 mmol), 3-mercaptopropionic acid (3-MPA, 0.070 g, 0.620 mmol) and 4-dimethylaminopyridine DMAP (0.006 g, 0.051 mmol) were dissolved in a round-bottom flask with 5 mL of DMSO. Then Glu-PGEMA (0.150 g, 0.510 mmol) was added to the reaction system and reacted at room temperature for 24 h. After the reaction was completed, the reaction solution was precipitated with a large amount of methanol to obtain a white flocculent precipitate, which was centrifuged, washed and dried to obtain 70 mg of the final product Glu-PGEMA-SH, with a yield of about 45 %. The molecular structure of Glu-PGEMA-SH was confirmed by 1 H NMR. Figure 21

[0065] 1 H NMR (400 MHz, DMSO- d 6) δ 4.88 (d, J ​= 42.8 Hz, 13H), 4.48 (s, -7H),4.11 (q, J = 5.2 Hz, 176H), 3.24 – 2.84 (m, 423H), 1.91 (s, -12H), 1.23(s, 1H),1.20 – 0.62 (m, 35H). 2. Preparation of aqueous glycan-based quantum dots Glu-Pglyco-CuInS2 QDs The prepared glycan ligand Glu-PGEMA-SH (0.040 g, 0.105 mmol) was dissolved in 10 mL of a mixed solvent of dimethyl sulfoxide (DMSO) and distilled water (volume ratio DMSO:H2O = 1:1), and the Glu-PGEMA-SH was fully dissolved by magnetic stirring for 30 min. Then, copper chloride dihydrate (CuCl2·2H2O, 0.026 g, 0.15 mmol) and indium chloride tetrahydrate (InCl3·4H2O, 0.044 g, 0.15 mmol) were added to the above mixture in a stoichiometric ratio, and argon was continuously passed at 25°C for 30 min. After the reaction system changed from light yellow to light green, the pH was adjusted to 11 by dropwise addition of freshly prepared sodium hydroxide solution (2.50 M). When the color of the system was further changed to brown yellow, stirring was maintained for 10 min, and then thiourea (CS(NH2)2, 0.0228 g, 0.30 mmol) was added to complete the precursor assembly, and the magnetic stirring was continued at 25°C for 40 min. Then, the reaction system was transferred to a reaction kettle (15 mL), and after hydrothermal treatment at 150°C for 21 h, the brown Glu-Pglyco-CuInS2 QDs liquid was obtained by filtration. Finally, the product was collected by centrifugation at 20000 r / min for 10 min, and the obtained product was dispersed in ultrapure water for standby.

[0066] Example Two 1. Synthesis of thiolated glycan molecule Gal-PGEMA-SH (1) Synthesis of Gal-ACGEMA HEMA (1.899 g, 14.30 mmol) and 2',3',4',6'-tetra-O-acetyl-a-D-galactopyranosyl bromide (2.000 g, 4.760 mmol) were dissolved in dry DCM solvent. 3A powdered molecular sieves (2.400 g) were added and after stirring for 30 minutes under argon the mixture was transferred to a -40 °C reaction bath. To the mixture was added an excess of AgOTf (1.515 g, 5.720 mmol) and the reaction was allowed to proceed for 48 hours. The solvent was removed by rotary evaporator and the residue was dissolved in a mixture of pyridine (50 mL) and acetic anhydride (20 mL). The unreacted HEMA was allowed to acetylate fully. The mixture was allowed to react overnight under argon. The solvent was removed and the residue was dissolved in DCM (50 mL). The water soluble impurities were washed away with hydrochloric acid (0.25 M, aliquot 30 mL), saturated brine solution, saturated NaHC03solution. The solvent was evaporated under vacuum to remove the solvent. Finally, the product was isolated by silica gel column chromatography (ethyl acetate: hexane, 3:7) to give 1.75 g of Gal-ACGEMA as a light yellow oily liquid with a yield of about 45 %. The molecular structure of Gal-ACGEMA was confirmed by1H NMR. 1 H NMR (400 MHz, Chloroform- Figure 22 ).

[0067] 1 H NMR (400 MHz, Chloroform- d ) δ 6.12 (dd, J = 2.3, 1.3 Hz, 1H), 5.59(h, J = 1.7 Hz, 1H), 5.19 (t, J = 9.5 Hz, 1H), 5.08 (t, J = 9.7 Hz, 1H), 5.00 (dd, J = 9.6, 8.0 Hz, 1H), 4.56 (d, J = 7.9 Hz, 1H), 4.32 – 4.23 (m, 3H), 4.13 (dd, J =12.3, 2.4 Hz, 1H), 4.05 (ddd, J = 11.3, 5.1, 3.5 Hz, 1H), 3.82 (ddd, J = 11.2,7.0, 3.7 Hz, 1H), 3.69 (ddd, J= 10.0, 4.7, 2.4 Hz, 1H), 2.09 (s, 3H), 2.02 (s,3H), 2.00 (d, J = 2.5 Hz, 6H), 1.94 (t, J = 1.3 Hz, 3H). (2) Synthesis of monomer Gal-GEMA To a solution of 2-0-((2',3',4',6'-tetra-O-acetyl)-a-D-galactopyranosyl) methyl acrylate Gal-ACGEMA (1.2 g, 2.6 mmol) in MeOH (5 mL) was added to a solution of 1% MeONa in MeOH (0.1 mL). The mixture was stirred at 25 °C for 15 min. The reaction mixture was neutralized with AcOH. The solvent was evaporated under reduced pressure to give the desired product. Then purified by silica gel column chromatography (20% MeOH in chloroform) to give 0.36 g of the product as yellow oil. The yield was 30%. The molecular structure of Gal-GEMA was confirmed by1H NMR. 1 H NMR (400 MHz, Methanol- Figure 23 ).

[0068] 1 H NMR (400 MHz, Methanol- d 4) δ 6.15 (dd, 1H, J = 1.0 Hz, J = 1.5 Hz), 5.65 (t, 1H, J = 1.5 Hz), 4.83 (d, 1H, J = 1.5 Hz), 4.35 (ddd, 1H, J = 3.0 Hz, J = 6.0 Hz, J = 12.0 Hz), 4.32 (ddd, 1H, J = 3.0 Hz, J = 6.0 Hz, J = 12.0 Hz), 3.96 (ddd, 1H, J = 3.0 Hz, J = 6.0 Hz, J = 11.5 Hz), 3.81−3.84 (m, 2H), 3.74 (ddd, 1H, J = 2.5 Hz, J = 6.0 Hz, J = 11.5 Hz), 3.73 (t, 1H, J = 5.5 Hz), 3.69 (dd, 1H, J = 3.5 Hz, J = 10.0 Hz), 3.64 (t, 1H, J = 9.5 Hz), 3.55−3.59 (m, 1H), 1.96 (s, 3H). (3) Synthesis of polymer Gal-PGEMA To a 10 mL reaction vial was added Gal-GEMA (200 mg, 0.685 mmol) and CTP (1.96 mg, 7 umol) dissolved in a mixture of water and ethanol (9:1 by volume). The reaction vial was sealed with a rubber septum and immersed in a preheated oil bath at 70 °C, then purged with argon for 30 min. To another reaction vial was added V-501 (0.98 mg, 3.5 umol) and 1 mL of deionized water, which was purged with argon, then added to the monomer vial by syringe. The reaction was allowed to proceed for 3-4 h, then precipitated with a large volume of methanol, centrifuged, washed, and lyophilized to give 56 mg of pink solid product Gal-PGEMA. The yield was 28 %. The molecular structure of Gal-PGEMA was confirmed by 1 H NMR, GPC Figure 24 , Figure 25 ).

[0069] 1 H NMR (400 MHz, Deuterium Oxide) δ 4.95 (s, 2H), 4.48 (s, 133H), 4.16 (d, J = 40.9 Hz, 460H), 3.92 (d, J = 12.0 Hz, 287H), 3.82 (s, 99H), 3.77 - 3.54 (m, 256H), 3.54 - 3.33 (m, 419H), 2.21 - 1.70 (m, 329H), 0.98 (d, J = 74.1 Hz, 515H). The molecular weight of polymer Gal-PGEMA was determined by gel permeation chromatography (GPC) with water as the mobile phase. As shown in Figure 25 , the GPC elution curve of polymer Gal-PGEMA was a single peak, with a number average molecular weight (M n ) of 1.4 x 10 5 , a weight average molecular weight (M w ) of 1.9 x 10 5 , and a PDI of 1.36 calculated from (M w / M n ), with a narrow molecular weight distribution and a degree of polymerization (DP) of about 500, consistent with the characteristics of RAFT living polymerization.

[0070] GPC (unit g / mol): Mn = 1.4 x 10 5 , Mw = 1.9 x 10 5 , PDI (Mw / Mn) = 1.36 (4) Synthesis of Gal-PGEMA-SH Under magnetic stirring, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC-HCl, 0.157 g, 0.816 mmol), 3-mercaptopropionic acid (3-MPA, 0.070 g, 0.620 mmol) and 4-dimethylaminopyridine (DMAP, 0.006 g, 0.051 mmol) were accurately weighed into a 50 mL three-necked flask, which was preloaded with 5 mL of anhydrous dimethyl sulfoxide (DMSO). Then, Gal-PGEMA (0.150 g, 0.510 mmol) was added to the reaction system. After magnetic stirring at 25 °C for 24 h, a large amount of methanol was added to obtain a white flocculent precipitate. After centrifugation, washing and drying, 0.068 g of the final product Gal-PGEMA-SH was obtained. The yield was 45 %. The molecular structure of Glu-PGEMA-SH was confirmed by 1 H NMR (400 MHz, DMSO- Figure 26 ).

[0071] 1 H NMR (400 MHz, DMSO- d 6) δ 4.88 (d, J = 42.8 Hz, 12H), 4.48 (s, -3H),4.11 (q, J = 5.2 Hz, 137H), 3.26 – 2.79 (m, 352H), 1.91 (s, -12H), 1.23(s, -1H), 1.20 – 0.59 (m, 25H). 2. Preparation of Gal-Pglyco-CuInS2 QDs The preparation method of Gal-Pglyco-CuInS2 QDs was the same as that of Glu-Pglyco-CuInS2 QDs in Example One, except that the ligand Glu-PGEMA-SH was replaced by the ligand Gal-PGEMA-SH.

Claims

1. A method for preparing polysaccharide-based water-soluble CuInS2 ternary quantum dots, comprising the following steps: (1) Synthesis of thiolated polysaccharide molecules: Hydroxyethyl methacrylate was glycosylated with 2',3',4',6'-tetra-O-acetyl-α-D-glucopyranose / galactose bromide under argon protection, and Glu / Gal-ACGEMA was obtained after acetylation and column chromatography purification. Glu / Gal-ACGEMA was deacetylated by sodium methoxide and purified by silica gel column chromatography to obtain monomeric Glu / Gal-GEMA; Glu / Gal-PGEMA was synthesized by polymerization of Glu / Gal-GEMA monomers in a water / ethanol mixture using 4-cyano-4-(thiobenzoylthio)valeric acid as a chain transfer agent and 4,4'-azobis(4-cyanovaleric acid) as an initiator. Glu / Gal-PGEMA was thiolated with 3-mercaptopropionic acid using EDC·HCl and DMAP as catalysts to produce the thiolated polysaccharide molecule Glu / Gal-PGEMA-SH. (2) Preparation of quantum dots: Glu / Gal-PGEMA-SH was dissolved in a DMSO / H2O mixed solvent, and CuCl2·2H2O and InCl3·4H2O were added. After adjusting the pH to 10-13, thiourea was added and the reaction was hydrothermally reacted at 140-160℃ for 20-25h to obtain polysaccharide-based CuInS2 quantum dots.

2. The preparation method according to claim 1, characterized in that The glycosidation reaction conditions in step (1) are as follows: reaction temperature of -35 to -45°C, reaction time of 45 to 50 hours, catalyst of AgOTf, solvent of dry dichloromethane, and addition of 3Å molecular sieves to remove water; the molar ratio of hydroxyethyl methacrylate to 2',3',4',6'-tetra-O-acetyl-α-D-pyranose glucose / galactosyl bromide is 3:1 to 4:

1.

3. The preparation method according to claim 1, characterized in that The deacetylation reaction of Glu / Gal-ACGEMA in step (1) is carried out in a methanol solution of sodium methoxide with stirring at 20-30° C. for 10-20 minutes; the mass fraction of sodium methoxide in the methanol solution of sodium methoxide is 1%.

4. The preparation method according to claim 1, characterized in that The polymerization reaction of the Glu / Gal-GEMA monomer in step (1) is carried out at 60-80° C. under argon protection for 3-4 hours, the molar ratio of the Glu / Gal-GEMA monomer to the chain transfer agent CTP is 90:1-100:1; the molar ratio of the Glu / Gal-GEMA monomer to the initiator V-501 is 190:1-200:1; and in the water / ethanol mixed system, the volume ratio of water to ethanol is 9:

1.

5. The preparation method according to claim 1, characterized in that In step (1), the thiolation reaction is carried out in DMSO solvent at room temperature for 20 to 25 hours; the molar ratio of Glu / Gal-PGEMA to 3-mercaptopropionic acid is 1:1 to 1:1.

5.

6. The preparation method according to claim 1, characterized in that In step (2), Cu 2+ 、In 3+ 、S 2- , the mass ratio of Glu / Gal-PGEMA-SH is 1:1:2:6~1:1:2:

12.

7. The preparation method according to claim 1, characterized in that In step (2), the volume ratio of DMSO to H2O in the DMSO / H2O mixed solvent is 1:1; after the hydrothermal reaction, the product needs to be purified by centrifugation at 20,000 r / min for 10 min, and the product is dispersed in ultrapure water.

8. A polysaccharide-based water-soluble CuInS2 ternary quantum dot prepared by the method of any one of claims 1 to 7, characterized in that: The surface of quantum dots is coated with polysaccharide ligands, with a particle size of 3-4 nm. They emit fluorescence in the visible light or near-infrared I region, with a fluorescence quantum yield of ≥16%.

9. Use of the polysaccharide-based CuInS2 quantum dots according to claim 8 in the preparation of bioluminescent imaging reagents.

10. Use of the polysaccharide-based CuInS2 quantum dots according to claim 8 as fluorescent probes in the preparation of tumor monitoring reagents.

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