Water-phase bio-friendly wide-spectrum up-conversion luminescent mixed-phase CdSe: Yb quantum dot and preparation method thereof

Abstract

The invention relates to an aqueous-phase bio-friendly wide-spectrum up-conversion luminescent mixed-phase CdSe: Yb quantum dot and a preparation method thereof, and belongs to the field of nano luminescent materials. Comprising the following steps: preparing a Se precursor, preparing a Cd + Yb precursor, injecting a Se precursor solution into a Cd + Yb precursor solution according to the molar ratio of Se to Cd + Yb of 1: 3-1: 5, heating from room temperature to 80-90 DEG C in 5-10 minutes, heating from 80-90 DEG C to 100-110 DEG C in 20-30 minutes, continuously heating at the temperature of 100-110 DEG C, condensing, refluxing and reacting for 2-4 hours, and finally generating cubic phase and hexagonal phase coexisting CdSe: Yb quantum dots. The CdSe: Yb quantum dot is low in toxicity and biological friendly, promotes reproduction of macrophages swallowing the quantum dot, and is suitable for tracing cells under weak-intensity multiple full-wave band exciting light matched with a biological fluorescence microscope.

Description

Technical Field

[0001] The present invention belongs to the field of nano-luminescent materials, and particularly relates to a mixed-phase CdSe:Yb quantum dot with aqueous-phase biocompatible broadband up-conversion luminescence and a preparation method thereof. Background Art

[0002] As a kind of zero-dimensional nanocrystal material, CdSe semiconductor quantum dots have important physical properties such as quantum confinement effect, optical properties, and electrical properties. When the size of the quantum dots is smaller than the exciton Bohr radius, a significant quantum confinement effect will occur, resulting in energy level discretization and an increase in the bandgap. [1-3] , this phenomenon enables the emission wavelength of the quantum dots to be precisely regulated by adjusting the particle size. The emission spectrum of CdSe quantum dots can be continuously tuned from the blue light region to the red light region. [4] . The synthesis methods and conditions will significantly affect the properties of the quantum dots. Factors such as reaction temperature, precursor concentration, and solvent type will all affect the size distribution and crystallization quality of the quantum dots. [5] , CdSe quantum dots tend to form a cubic phase at low temperatures, while they are more likely to form a hexagonal phase at high temperatures. [6] . The differences between these two structures are mainly reflected in the atomic arrangement and energy band structure, thereby affecting the optical properties and stability of the quantum dots. Commonly used synthesis methods include thermal injection method, aqueous-phase method, and microwave-assisted method, etc. Among them, the aqueous-phase method has received extensive attention due to its biocompatible characteristics.

[0003] CdSe quantum dots are prepared by chemical methods based on the theory of crystallization kinetics and generally go through two stages: nucleation and growth. According to different synthesis solvents, the colloidal chemical synthesis method can be divided into oil-phase (organic solvent) and aqueous-phase (water solvent) preparations. In 2002, Rogach et al. successfully achieved the aqueous-phase synthesis of CdTe quantum dots through mercapto-carboxylic acid stabilization. [7] , The aqueous-phase synthesis of quantum dots has the advantages of low cost, good biocompatibility, and is suitable for biological labeling. Based on this, the present invention adopts the aqueous-phase preparation of CdSe quantum dots.

[0004] In the field of quantum dot preparation, scientists have been continuously conducting in-depth research. Starting from the early stage, mainly relying on thiol-based compounds as stabilizing media for aqueous-phase synthesis technology, to the later stage, the growth of nanocrystals can be controlled by using liquid-phase coprecipitation processes. The synthesis of CdS quantum dots by combining cadmium chloride with thiosemicarbazide is a typical example. In this reaction, cadmium chloride provides metal ions, and thiosemicarbazide serves as a sulfur donor, successfully obtaining CdS quantum dots with excellent monodispersity characteristics. Focusing on improving the luminescence efficiency and structural stability of materials, the aqueous-phase synthesis process has been further systematically improved. For example, by using polyvinyl alcohol, carboxymethyl cellulose, etc. to replace traditional polyethylene glycol as surface modifiers, the optical properties of quantum dots can be improved. In addition, adopting a one-step synthesis method, such as adjusting with NaOH and passivating with polyethylene glycol, although the quantum yield is low, by introducing a strong reducing agent to activate elemental sulfur as a sulfur source, it provides an innovative path for the preparation of sulfide quantum dots. The preparation route of this method is favored due to its advantages such as simple process flow and strong economy. The obtained quantum dot materials not only exhibit excellent hydrophilic characteristics but also have good biocompatibility. Based on these advantages, they show great potential in application scenarios such as biological detection. However, some challenges faced by this technology cannot be ignored, especially the formation of surface state defects during the crystallization process, which will further lead to problems such as broadening of the emission spectrum and decrease in radiative recombination efficiency. In response to these limitations, developing an efficient aqueous-phase doped quantum dot synthesis method has become one of the important research directions in current materials science.

[0005] Up-conversion Luminescence (UCL), as a typical nonlinear optical effect, its basic principle is to absorb multiple low-energy photons and then emit a high-energy photon, thereby realizing an optical process with anti-Stokes shift. [8] . Up-conversion luminescence is a special fluorescence phenomenon, and its essence belongs to the anti-Stokes emission process. This unique luminescence mechanism can convert incident low-energy near-infrared photons into higher-energy visible-light region photons through nonlinear optical effects. Due to its excellent light penetration ability and low background interference in deep biological tissues, it shows important application value in the biomedical field. The near-infrared excitation characteristics in the first biological window - NIR-I in the 700 - 900 nm band can effectively penetrate high-scattering tissues such as bone and fat, significantly improving the resolution of in vivo imaging (>5 mm) and reducing phototoxicity. Therefore, it has attracted much attention in the fields of tumor-targeted tracing, photodynamic therapy, and real-time monitoring of drug delivery.

[0006] According to the existing research results, the physical mechanisms of up-conversion luminescence can be mainly divided into the following categories [9]: (1) Excited State Absorption (ESA) is one of the most common mechanisms. In this process, electrons in the ground state first absorb a photon and transition to an intermediate energy level, and then absorb another photon to further transition to a higher energy level. Finally, high-energy photons are released through radiative relaxation. This mechanism widely exists in the two-photon absorption process of semiconductor quantum dots such as CdSe / ZnS. (2) Energy Transfer Up-conversion (ETU): Cooperative excitation is achieved through non-radiative energy transfer between adjacent ions, such as the cascade energy transfer between the sensitizer (Yb 3+ / Er 3+ ) and the activator (Er 3+ ) in the rare-earth doped system (NaYF4:Yb 3+ ). This mechanism has been widely used in in vivo deep tissue imaging due to its high efficiency; (3) Phonon-assisted Up-conversion (PAU): The material supplements the transition energy gap by coupling photons with lattice vibrations (phonons). A typical example is the multi-phonon cooperative excitation mode of two-dimensional MoS2 materials; (4) Cooperative Sensitization: Organic molecules generate triplet excitons through singlet fission, and high-energy fluorescence is produced through exciton collision annihilation. This mechanism can enhance the therapeutic effect by regulating the energy transfer of oxygen molecules in photodynamic therapy; (5) Auger Composite Up-conversion (ACU): Energy level transitions are achieved through energy transfer between carriers in a multi-exciton system. For example, the multi-exciton Auger process in WS2 quantum dots can produce an ultrafast fluorescence response. (6) Photon Avalanche (PA): This mechanism is the coupling of ESA and ETU. It was first proposed by Chivian et al. in 1979 based on the Pr 3+ doped system

[10] . Through the directional energy cycle accumulation between the same ions, the population number exponentially increases at a specific energy level, and finally high-efficiency up-conversion luminescence is achieved. Its advantage lies in using the unique spectral characteristics of near-infrared excitation - visible light emission, which can significantly increase the penetration depth of biological tissues (>5 mm), and at the same time avoid the risk of light damage to biological samples by ultraviolet light, providing an innovative technical path for tumor targeted imaging and real-time treatment monitoring.

[0007] Doping rare-earth elements in fluorides is a common strategy for up-conversion luminescent nanoparticles. Such materials are usually doped with Yb 3+ , Er 3+ , Tm 3+ , Ho 3+Rare earth ions such as form an energy level transition system and can emit visible light under near-infrared light excitation. In 2013, the team of Jialiping of Tongji University successfully synthesized high-quality orthorhombic polycrystalline liquid-phase BaMgF4:RE 3+ (RE = Eu, Tb, Sm, Dy, Yb–Er / Tm)

[11] . In 2021, the team of Fuzuoling of Jilin University designed dumbbell-shaped NaYF4:Yb 3+ / RE 3+ @NaYF4:Yb 3+ / RE 3+ @NaNdF4:Yb 3+ (RE 3+ = Ho 3+ or Er 3+ ) nanoparticles improve the upconversion luminescence and sensitivity of nanothermometry

[12] . In 2024, the research group of Jianrong Qiu of Zhejiang University proposed that coupling X-ray excitation in Er 3+ -doped fluoride nanoparticles can achieve persistent luminescence and upconversion, and have various applications

[13] . However, the practical application of upconversion luminescent nanoparticles based on rare earth element-doped fluorides still faces multiple challenges. For example, traditional synthesis routes mostly rely on organic solvent systems, resulting in a lack of hydrophilic groups on the surface of nanoparticles. Their hydrodynamic size generally exceeds 50 nm, which is likely to trigger the capture by the reticuloendothelial system and limit the tumor penetration efficiency. And rare earth ions have a high affinity for oxygen and are prone to form oxide impurity phases, forcing the use of highly reducing fluorides such as NaYF4 as the matrix during the doping process. However, fluorides have poor biodegradability and may release toxic fluoride ions. At the same time, the rare earth doping concentration needs to be precisely regulated within the range of 0.5-5 mol%, as too high a concentration will damage the lattice integrity of the quantum dots. For example, when the lattice distortion of CdSe > 2%, the fluorescence quantum yield decreases by 40%, while too low a concentration is difficult to form an effective sensitizer-activator ion pair. In addition, the thermodynamic self-purification effect during the doping process will cause the lattice to repel foreign ions, and the kinetic aggregation phenomenon further restricts the stability of the upconversion luminescence efficiency.

[0008] To achieve the application of quantum dots excited in the near-infrared band in the fields of biomedical imaging and therapy, the current mainstream strategy for material preparation is to dope rare earth ions such as Yb 3+ , Er 3+ into the quantum dot lattice to construct a composite system with both the quantum dot size effect and rare earth energy level transition characteristics. As early as 2002, Klik et. al. reported the energy transfer mechanism for achieving upconversion luminescence by doping RE 3+ into semiconductors with InP:Yb as an example

[14] . In 2002, Strouse pioneered the incorporation of Eu 3+ into CdSe

[15] But no results were found with Eu 3+ In 2011, Planelles et al. reported the presence of Eu in CdS nanocrystals. 3+ There are two different sites, and no upconversion luminescence properties are proposed.

[16] In 2013, Mukherjee et al. demonstrated that using Tb 3+ and Eu 3+ Doping of ZnS and CdS nanoparticles in which rare earth element ions show a preference for surface sites

[17] , and Tb can be observed in the fluorescence spectrum 3+ and Eu 3+ In 2013, Robin Geitenbeek et al. realized the synthesis of CdSe quantum dots and rare earth ions Yb 3+ Successful coupling

[18] The experimental results show that Yb 3+ The ions were first adsorbed on the CdSe surface and then incorporated into the nanocrystalline semiconductor particles through the growth of the Se shell. However, the coupled quantum dots did not achieve upconversion luminescence.

[0009] The main difficulties are: 1. The mismatch between the crystal structure and the coordination environment. Sulfur quantum dots (such as CdS, PbS) often have cubic (sphalerite) or hexagonal (wurtzite) structures. Generally, a quantum dot has only one lattice structure, and the formation of mixed phases is rare. The cations in the lattice (such as Cd 2+ , Pb 2+ ) is usually 4 (tetrahedral) or 6 (octahedral). And rare earth ions (such as Eu 3+ , Er 3+) tends to be in a high-coordination environment (6-8 coordination), which makes it difficult for it to occupy a stable site in the sulfur-based lattice. This causes rare earth ions to be easily repelled to the surface or form clusters, rather than uniformly doped, which reduces the luminescence efficiency. Therefore, the cubic phase structure is minimized, and hexagonal phases or more asymmetric high-coordination environments are mostly present. 2. Synthetic chemical compatibility issues. The synthesis of sulfur-based quantum dots often requires a high-temperature organic phase (such as oleylamine and octadecene). Organic ligands (such as oleic acid) may preferentially bind to rare earth ions, hindering their entry into the crystal lattice. Moreover, rare earth precursors (such as nitrates and acetylacetonates) are easily decomposed or oxidized in this environment to generate inactive species (such as rare earth oxides). The nucleation and growth rates of rare earth ions and sulfur-based cations are very different, resulting in uneven doping or phase separation. In view of the problems of conflicting synthesis conditions and differences in precursor reaction kinetics, it is a better strategy to choose aqueous phase quantum dots and avoid organic phase quantum dots. 3. The low efficiency of energy transfer is mainly due to the difficulty in matching energy levels and excited state lifetimes and surface state traps. The exciton energy of sulfur-based quantum dots must be precisely matched with the 4f energy level of rare earth ions to achieve effective energy transfer. If the quantum dot band gap is too wide or too narrow, energy transfer is hindered. The quantum dot exciton lifetime (nanosecond level) is much shorter than the rare earth ion excited state lifetime (millisecond level), resulting in reverse energy transfer (rare earth → quantum dot) or excited state quenching. Surface defects in quantum dots may capture carriers, leading to non-radiative recombination. If the doped rare earth ions are located on the surface, their luminescence is easily quenched. 4. Doping with different elements may introduce non-radiative recombination centers, reducing the original luminescence efficiency of quantum dots. The introduction of some rare earth ions may destroy the quantum dot structure, resulting in a decrease in optical and thermal stability. Therefore, when doping quantum dots with rare earth elements, the energy level structure of the two should be considered to effectively improve their optical and thermal stability.

[0010] In response to the above bottlenecks, the present invention focuses on designing a method for synthesizing quantum dots in an aqueous phase that is easily doped with rare earth elements. This method utilizes the biofriendliness of aqueous CdSe quantum dots and achieves efficient near-infrared excitation properties. Combined with the upconversion luminescence of rare earth elements, it can especially promote the proliferation of macrophages that engulf quantum dots, thereby providing new ideas for constructing an efficient and safe upconversion luminescence quantum dot diagnosis and treatment system.

[0011] References

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[18] Martín-Rodríguez R, Geitenbeek R, Meijerink A. Incorporation and luminescence of Yb 3+ in CdSe nanocrystals[J]. Journal of the American Chemical Society, 2013, 135(37): 13668-13671. Summary of the Invention

[0030] The present invention lies in the development and design of a mixed-phase CdSe:Yb quantum dot with aqueous-phase biocompatible broadband upconversion luminescence and a preparation method thereof.

[0031] A preparation method of a mixed-phase CdSe:Yb quantum dot with aqueous-phase biocompatible broadband upconversion luminescence is characterized in that it specifically involves the coordinated preparation of a CdSe:Yb quantum dot with upconversion luminescence by controlling the temperature-rising process in the excessive Cd ratio and nucleation stages, and specifically includes the following:

[0032] Preparation of Se precursor: Se powder and NaBH4 were dissolved in a certain amount of deionized water at a molar ratio of 1:3 - 1:5, and continuously stirred at room temperature for 0.5 - 1 hour under nitrogen protection until the solution became clear, obtaining a Se precursor solution;

[0033] Preparation of Cd + Yb precursor: Yb(NO3)3·5H2O and Cd(NO3)2·4H2O were mixed at a molar ratio of (2 - 5):100, and the molar ratio of the mixture to glutathione (GSH) was controlled in the range of 1:20 - 1:30. The three were dissolved in deionized water together to prepare a co-doped precursor solution, and then NaOH solution was added dropwise to adjust the pH to 8 - 9;

[0034] The Se precursor solution was quickly injected into the Cd + Yb precursor solution according to the molar ratio of Se to Cd + Yb of 1:3 - 1:5. Under the condition of continuous nitrogen flow, it was first heated from room temperature to 80 - 90 °C in 5 - 10 min, and then slowly heated from 80 - 90 °C to 100 - 110 °C in 20 - 30 min by increasing the nitrogen gas flow rate, so as to make the cubic phase and hexagonal phase of CdSe quantum dots coexist, creating favorable conditions for rare earth element doping. Subsequently, it was continuously heated and refluxed at 100 - 110 °C for 2 - 4 hours, and finally CdSe:Yb quantum dots with coexisting cubic phase and hexagonal phase were generated;

[0035] Purification and storage: After the reaction, isopropyl alcohol was added, and centrifuged at 3000 - 5000 rpm for 3 - 10 minutes, and the centrifugation and precipitation were repeated 2 - 5 times to achieve purification. The purified quantum dots were redispersed in deionized water and stored refrigerated.

[0036] Upconversion luminescence: Absorption spectra, emission spectra, and excitation spectra of CdSe and CdSe:Yb quantum dots were obtained using different excitation light sources. The upconversion emission spectrum of CdSe:Yb quantum dots was obtained using a 700 - 900 nm femtosecond laser as the excitation source. For example, attached Figure 4The upconversion fluorescence spectrum of CdSe:Yb quantum dots was obtained by exciting with an 800 nm femtosecond laser. The excitation light range of CdSe:Yb quantum dots can be within the first biological window - NIR-I (700 - 900 nm), and the emission band is in the golden band of human blood (550 - 650 nm). Compared with other rare earth element-doped quantum dots, this quantum dot has the advantages of high luminescence efficiency, wide-spectrum tunability, large penetration depth, and low biological toxicity, making it more suitable for biological applications. After observation and analysis, it was found that the fluorescence signal intensity of the CdSe:Yb quantum dots synthesized in the water-based solvent in this invention increased significantly and upconversion luminescence could be achieved. At the same time, it also has the effect of promoting the proliferation of macrophages that phagocytize quantum dots. Because of its biocompatibility and the effect of promoting the proliferation of macrophages that phagocytize quantum dots, and tracing cells under the weak-intensity and multi-wavelength excitation light of a biological fluorescence microscope, cell biological tracing can be better carried out.

[0037] Tracing of macrophages RAW264.7 with upconversion luminescent CdSe:Yb quantum dots: The RAW264.7 cells in the logarithmic growth phase were seeded in a 24-well plate. The next day, when the cell confluence reached 70% - 80%, the next experiment was carried out; the above-mentioned CdSe:Yb quantum dots with different volumes were added to the 24-well plate to prepare various different concentrations from 1.0×10 -3 mol / L to 1.0×10 -4 mol / L. After 24 - 72 h, the culture medium was aspirated and discarded, washed 1 - 3 times with PBS, and 0.5 - 2 mL of complete culture medium was added. The tracing of CdSe:Yb quantum dots by RAW264.7 cells was observed under different wavelength fluorescence channels with a fluorescence microscope. The biological fluorescence microscope was equipped with a weak LED light source with a wavelength range of 340 - 680 nm to excite the CdSe:Yb quantum dots with a concentration of 2.0×10 -4 -1.0×10 -4 mol / L to achieve the best cell tracing effect. Among them, 340 nm, 470 nm, and 560 nm are for fluorescence tracing, and 680 nm is for upconversion tracing. Description of the Drawings

[0038] Figure 1 Schematic diagram of the preparation and cell tracing process of CdSe:Yb quantum dots and schematic diagram of the formation of chemical bonds in the mixed crystal phase. Figure 2 Comparison chart and peak statistical chart of the XRD peaks of CdSe, CdSe:Er, CdSe:Eu, and CdSe:Yb mixed crystal phase quantum dots with standard single crystal crystals. a. XRD pattern b. Peak statistics.

[0039] Figure 3 Morphology characterization of CdSe and CdSe:Yb quantum dots a. TEM of CdSe quantum dots b. TEM of CdSe:Yb quantum dots c. EDS of CdSe:Yb quantum dots.

[0040] Figure 4 Optical property characterization of CdSe and CdSe:Yb, CdSe:Er, CdSe:Eu quantum dots. a. Upconversion fluorescence spectrum under 355 nm xenon lamp excitation light. b. Excitation spectrum of the strongest fluorescence peak of the quantum dots. c. Upconversion fluorescence spectrum under 800 nm femtosecond excitation light. d. Dependence of the maximum fluorescence intensity on the laser power.

[0041] Figure 5 Transient absorption diagram of CdSe:Yb quantum dots. a. and b. Evolution of the pump-probe signal with time under the conditions of 710 nm and 11.5 mw. c. Evolution of the signal with wavelength. d. Evolution of the signal with time.

[0042] Figure 6 Under the condition of the same cell density (concentration of 8.5*10 -5 ) during the same plate seeding, state diagram of macrophages RAW264.7 co-cultured with quantum dots for 48 h. a. Control group without phagocytized quantum dots, blue represents the bioluminescence-stained cell nuclei and also represents the number of live cells; b. Phagocytized CdTe quantum dots, red represents the quantum dots entering the cells, and the diffuse red indicates that the more toxic quantum dots will trigger cell apoptosis; c. Phagocytized CdTe core-shell quantum dots coated with CdSe on the outside, with the same toxicity as CdSe quantum dots, showing a significant increase in the number of cells and promoting cell division and proliferation.

[0043] Figure 7 After macrophages RAW264.7 were co-cultured with quantum dots for 48 h, fluorescence microscopy imaging diagram of CdSe:Yb quantum dot cell tracing. Before taking pictures with the fluorescence microscope, the solution was replaced with a quantum dot-free culture medium. The pseudo-color bright spots shown are the quantum dots inside the cells. Among them, the concentration of 1*10 -4 The corresponding cell concentration and number are similar to Figure 6 (c) above, and CdSe:Yb quantum dots also promote cell division and proliferation. Detailed implementation manners

[0044] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited to the following embodiments.

[0045] Embodiment 1

[0046] Step 1, prepare CdSe quantum dots

[0047] Preparation of Se precursor: Dissolve 1 mmol of selenium powder and 4 mmol of NaBH4 in 4 mL of deionized water, and continuously stir at room temperature for 45 min under nitrogen protection until the solution becomes clear to obtain the Se precursor solution.

[0048] Cd Precursor Preparation: Dissolve 0.01 mmol of Cd(NO3)2·4H2O and 0.25 mmol of GSH in 10 mL of deionized water to prepare a Cd precursor solution, and then add dropwise 1 mmol·mL -1 NaOH solution to adjust the pH to 8.5.

[0049] Quickly inject 120 μL of the Se precursor solution into the Cd precursor solution (the molar ratio of Cd 2+ to Se 2- is 4:1). Under the condition of continuously introducing nitrogen, heat up to 85 °C in 5 min, and then increase the gas flow rate and slowly heat up from 85 °C to 100 °C in 25 min. Subsequently, continuously heat and carry out a condensation reflux reaction for 3 hours, and finally generate CdSe quantum dots with a coexistence of cubic and hexagonal phases at a concentration of 0.003 mmol·mL -1 . The doping of rare earth element Yb significantly affects the relative content of the characteristic crystal planes of the hexagonal phase. Figure 2 .

[0050] Purification and Storage: After the reaction, add isopropanol and centrifuge at 4000 rpm for 5 minutes, and repeat centrifugation and precipitation 3 times to achieve purification. The purified quantum dots are redispersed in deionized water and stored refrigerated at 4 °C.

[0051] Step 2, Preparation of CdSe:Yb Quantum Dots

[0052] Se Precursor Preparation: Dissolve 1 mmol of selenium powder and 4 mmol of NaBH4 in 4 mL of deionized water, and continuously stir at room temperature for 45 min under nitrogen protection until the solution becomes clear to obtain a Se precursor solution.

[0053] Cd+Yb Precursor Preparation: Dissolve 0.098 mmol of Cd(NO3)2·4H2O, 0.002 mmol of Yb(NO3)3·5H2O and 0.25 mmol of GSH in 10 mL of deionized water to prepare a co-doped precursor solution, and then add dropwise 1 mmol·mL -1 NaOH solution to adjust the pH to 8.5.

[0054] Quickly inject 120 μL of the selenium precursor solution into the doped precursor solution (the molar ratio of Cd+Yb to Se is 4:1). Under the condition of continuously introducing nitrogen, heat up to 85 °C in 5 min, and then increase the gas flow rate and slowly heat up from 85 °C to 100 °C in 25 min. Subsequently, maintain a reflux reaction at 100 °C for 3 hours, and finally generate CdSe:Yb quantum dots that can achieve upconversion. Their optical properties and upconversion effects are shown in Figures 3 - 5

[0055] Figure 3The grain diameter of CdSe:Yb quantum dots is 5.65 nm, and that of CdSe quantum dots is 7.54 nm. Image comparison shows that doping reduces the size of quantum dots, but the lattice diffraction fringe spacing remains obvious, indicating that Yb doping does not change the original crystal structure. The presence of Yb in the EDS elemental spectrum of CdSe:Yb quantum dots fully proves the success of doping. The upconversion luminescence properties of CdSe:Yb quantum dots are shown in Figure 4 . The downconversion emission spectrum obtained with 355 nm as the excitation light is shown in Figure 4 a in Figure 4 . The emission spectrum signal of CdSe quantum dots synthesized in aqueous solvent excited by an 800 nm fs Ti:Sapphire laser is very weak, but the signal intensity in the quantum dots increases significantly after Yb doping (see Figure 4 b in Figure 4 ). Moreover, Figure 5 in c of

[0056] the emission peak in the upconversion spectrum changes from a narrow peak to a wide peak containing multiple peaks, which is in line with the characteristics of upconversion luminescence. CdSe:Yb quantum dots are excited by an 800 nm fs Ti:Sapphire laser with different powers, and the specific values of the maximum fluorescence intensity and laser power are statistically analyzed. There is an approximate linear relationship between the logarithm of the laser power and the fluorescence intensity (see Figure 1 d in

[0057] ), which is consistent with the two-photon absorption characteristics, that is, upconversion luminescence is achieved. Figure 6 Figure 6 Step 3: Seed RAW264.7 cells in the logarithmic growth phase in a 24-well plate. The next day, when the cell confluence reaches 75%, proceed with the next experiment. Treat RAW264.7 cells with quantum dots at concentrations ranging from 0 to 300 μM for 48 hours. After 48 hours, aspirate the culture medium, wash twice with PBS, add 1 mL of complete culture medium, and then analyze with CCK8. n = 3 biological replicates. The cell status is shown in Figure 6Blue represents the fluorescence display of nuclear organism staining, and red represents the fluorescence display of quantum dot staining. The quantum dots are effectively phagocytosed into the interior of macrophages and adhere between the cell wall and the nucleus. Compared with the control group, for pure CdTe quantum dots, the number of cells significantly decreases, and the quantum dots are diffusely distributed, with cell rupture, while for CdSe-coated quantum dots, the number of cells increases, promoting cell division and growth. Combining Figure 7 with the corresponding concentration of 1*10 -4 , and the number of cells is similar, indicating that CdSe:Yb has the effect of promoting cell division and growth.

[0058] Step 4, RAW264.7 cells phagocytose CdSe:Yb QDs

[0059] Cell fluorescence imaging was performed using a Discover Echo RVL-100-M microscope. RAW264.7 cells in the logarithmic growth phase were seeded in 24-well plates. The next day, when the cell confluence reached 75%, the next experiment was carried out; 500 μL, 250 μL, 100 μL, and 50 μL of CdSe:Yb quantum dots with an initial concentration of 3.0×10 -3 mol / L were added to the 24-well plates to make the final concentrations 1.0×10 -3 mol / L, 5.0×10 -4 mol / L, 2.0×10 -4 mol / L, and 1.0×10 -4 mol / L respectively. After 48 h, the culture medium was aspirated and discarded, washed twice with PBS, 1 mL of complete medium was added, and the quantum dot cell tracing was observed using the forward and reverse integrated fluorescence microscopy technique. The process schematic diagram is shown in the appendix Figure 1 . The cell tracing images under bright field, ultraviolet (340 nm), blue light (470 nm), red light (560 nm), infrared (680 nm) conditions and after mixing are shown in Figure 7 . The fluorescence brightness is the most obvious under 470 nm excitation, and at the same time, the human eye is most sensitive to this wavelength range, resulting in the best tracking effect. In addition, upconversion fluorescence tracing was achieved under 680 nm excitation. Although the labeling abilities of quantum dots with different concentrations are different, the fluorescence labeling ability shown by CdSe:Yb quantum dots after being excited by near-infrared light is of great significance for future biological research, especially very promising in the field of deep biological tracking.

[0060] A hybrid-phase CdSe:Yb quantum dot with aqueous bio-friendly broadband upconversion luminescence was successfully designed and prepared. Its advantage lies in that the doping preparation process occurs in an aqueous solvent, improving the bio-friendliness and being more suitable for cell tracing. The Yb element provides intermediate states, allowing the quantum dots to undergo multi-photon broadband absorption and upconversion absorption. The signal fluctuates around 0, and the lifetime of the intermediate energy state is long enough to ensure the stability of the double conversion luminescence under continuous excitation, as shown in Figure 5 . The upconversion luminescence enables near-infrared excitation, which has the advantages of significantly enhancing the penetration depth of biological tissues, while avoiding the risk of photodamage to biological samples by ultraviolet light and promoting cell division and growth, as shown in Figure 6 .

Claims

1. A preparation method of hybrid-phase CdSe:Yb quantum dots with aqueous-phase biocompatible broadband upconversion luminescence, characterized in that, Specifically related to the co-preparation of upconversion luminescent CdSe:Yb quantum dots by excessive Cd ratio and controlling the heating process during the nucleation stage, specifically including the following: Preparation of Se precursor: Dissolve Se powder and NaBH4 in a certain amount of deionized water at a molar ratio of 1:3 - 1:5, and continuously stir at room temperature for 0.5 - 1 hour under nitrogen protection until the solution becomes clear to obtain the Se precursor solution; Preparation of Cd + Yb precursor: Mix Yb(NO3)3·5H2O and Cd(NO3)2·4H2O at a molar ratio of (2 - 5):100, control the molar ratio of the mixture to glutathione (GSH) in the range of 1:20 - 1:30, dissolve the three in deionized water to prepare a co-doped precursor solution, and then add NaOH solution to adjust the pH to 8 - 9; Rapidly inject the Se precursor solution into the Cd + Yb precursor solution according to the molar ratio of Se to Cd + Yb of 1:3 - 1:

5. Under the condition of continuously introducing nitrogen, heat it. First, raise the temperature from room temperature to 80 - 90°C in 5 - 10 minutes, and then slowly raise the temperature from 80 - 90°C to 100 - 110°C in 20 - 30 minutes by increasing the nitrogen gas flow rate to make the cubic and hexagonal phases of CdSe quantum dots coexist, creating favorable conditions for rare earth element doping. Subsequently, continuously heat and carry out condensation reflux reaction at 100 - 110°C for 2 - 4 hours to finally generate CdSe:Yb quantum dots with coexisting cubic and hexagonal phases; Purification and storage: After the reaction, add isopropanol, centrifuge at a speed of 3000 - 5000 rpm for 3 - 10 minutes, and repeat centrifugation and precipitation 2 - 5 times to achieve purification. The purified quantum dots are redispersed in deionized water and stored refrigerated.

2. The CdSe:Yb quantum dots prepared by the method according to claim 1. The CdSe:Yb quantum dots prepared by the method according to claim 1 are characterized in that, Quantum dots with coexisting cubic and hexagonal phases. The CdSe:Yb quantum dots prepared by the method according to claim 1 are characterized in that, Having upconversion luminescent properties. The CdSe:Yb quantum dots prepared by the method according to claim 1 are characterized in that, The excitation light range of the CdSe:Yb quantum dots can be within the first biological window - NIR-I (700 - 900 nm), and the emission band is in the golden band of human blood (550 - 650 nm). At the same time, it also has the effect of promoting the reproduction of macrophages that phagocytize quantum dots.

6. Use of the CdSe:Yb quantum dots prepared by the method according to claim 1, characterized in that, Use upconversion luminescent CdSe:Yb quantum dots for cell tracing.

7. Use of the CdSe:Yb quantum dots prepared by the method according to claim 6, characterized in that, Trace macrophages RAW264.

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8. Use of the CdSe:Yb quantum dots prepared by the method according to claim 7, characterized in that, The biofluorescence microscope is configured with a weak LED light source in the wavelength range of 340 - 680 nm to excite CdSe:Yb quantum dots with a concentration of 2.0×10 -4 -1.0×10 -4 mol / L to achieve the best effect of cell tracing. Among them, 340 nm, 470 nm, and 560 nm are for fluorescence tracing, and 680 nm is for upconversion tracing.

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