Copper pre-organized quantum dots / hydrogen-bonded organic framework composite system and its application in glutathione ratio fluorescence detection

Abstract

This invention provides a copper pre-organized quantum dot / hydrogen-bonded organic framework composite system and its application in glutathione ratio fluorescence detection, relating to the field of glutathione detection technology. It includes quantum dots, hydrogen-bonded organic frameworks, and copper species. The quantum dots are composited with the hydrogen-bonded organic framework, and the copper species are pre-organized and anchored at the composite interface between the quantum dots and the hydrogen-bonded organic framework. Therefore, by confining and coordinating the copper species in a QDs / HOF composite system with a well-defined pore structure and interfacial microenvironment, when GSH interacts strongly with the pre-organized copper sites, not only does complexation occur, but a systematic reconstruction of the local coordination environment, valence state distribution, and interfacial electronic coupling relationship of the copper sites is triggered. This activates new low-energy emission channels, achieves redistribution of emission channels, and generates a clear ratio fluorescence signal, successfully overcoming the technical problems of existing GSH fluorescence detection technologies, such as single response dimension, poor stability in complex environments, and ambiguous structure-signal correlation.

Description

Technical Field

[0001] This invention belongs to the field of glutathione detection technology, specifically relating to a copper pre-organized quantum dot / hydrogen bond organic framework composite system and its application in glutathione ratio fluorescence detection. Background Technology

[0002] Glutathione (GSH) is an endogenous tripeptide composed of glutamic acid, cysteine, and glycine. It is also one of the most abundant non-protein thiols in cells, playing a crucial role in maintaining cellular redox homeostasis, scavenging reactive oxygen species, participating in detoxification metabolism, and regulating protein function and cell signal transduction. Because GSH levels are closely related to cellular oxidative stress, metabolic activity, and disease development, it is widely considered an important biomarker for assessing cellular health and pathological changes. Studies have shown that abnormal GSH levels are closely associated with various diseases, including diabetes, neurodegenerative diseases, cardiovascular diseases, liver damage, and immune system disorders. Therefore, developing sensitive, rapid, and highly selective GSH detection methods is of great significance for early disease diagnosis, monitoring of biological processes, and research on related mechanisms.

[0003] Currently, GSH detection methods mainly include high-performance liquid chromatography (HPLC), electrochemical analysis, colorimetric analysis, mass spectrometry, and fluorescence analysis. Among these, fluorescence analysis exhibits unique advantages in GSH recognition due to its high sensitivity, fast response speed, ease of operation, and ease of visualization. In particular, the strong interaction between the thiol group in the GSH molecule and metal ions provides an important foundation for constructing fluorescence sensing systems based on metal-mediated recognition. However, most existing GSH fluorescent probes still primarily rely on a "metal ion quenching—GSH competitive coordination to recover fluorescence" response mode, and their signal output is usually limited to a single intensity on / off change. While these systems are simple to design, they generally suffer from limited anti-interference capabilities, insufficient signal stability in complex environments, and unclear intrinsic correlation between structural changes and optical responses.

[0004] Among numerous metal recognition media, Cu 2+ Due to its strong coordination ability, significant fluorescence quenching effect, and high affinity for thiol molecules, Cu is widely used in GSH sensing systems. 2+ In mediating fluorescent probes, copper ions typically exist in solution either through free diffusion or simple complexation. In this state, Cu... 2+ It is susceptible to non-specific competition or interference from other coexisting substances in the system (such as various amino acids, metal ions and thiols), resulting in poor recognition selectivity.

[0005] Therefore, existing GSH fluorescence detection technologies generally suffer from problems such as single response dimension, insufficient stability in complex environments, and unclear structure-signal correlation, making it difficult to meet the needs for high reliability and high selectivity detection of GSH in complex biological samples. Summary of the Invention

[0006] In view of this, the present invention provides a copper pre-organized quantum dot / hydrogen bond organic framework composite system that can meet the requirements of high reliability and high selectivity detection of GSH in complex biological samples.

[0007] It is also necessary to provide an application of a copper pre-organized quantum dot / hydrogen-bonded organic framework composite system in the detection of glutathione ratio fluorescence.

[0008] The technical solution adopted by this invention to solve its technical problem is:

[0009] A copper pre-organized quantum dot / hydrogen bonded organic framework composite system, the composite system comprising quantum dots as luminescent cores, a confined matrix and interface-controlled hydrogen bonded organic framework as quantum dots, and copper species, wherein the quantum dots are composited with the hydrogen bonded organic framework, and the copper species are pre-organized and anchored at the composite interface between the quantum dots and the hydrogen bonded organic framework.

[0010] The copper species, together with quantum dots and hydrogen-bonded organic frameworks, constitute a reconfigurable local coordination environment. In the presence of glutathione, this environment can induce a redistribution of the luminescent channels in the composite system, resulting in a ratiometric fluorescence response.

[0011] Preferably, the quantum dot is a ZnSe / ZnS core-shell quantum dot, the hydrogen-bonded organic framework is formed by the self-assembly of trimesic acid and melamine, and the copper species is copper ions.

[0012] A method for preparing a copper pre-organized quantum dot / hydrogen-bonded organic framework composite system includes the following steps:

[0013] S1: Preparation of core-shell quantum dots with surface rich in carboxyl functional groups;

[0014] S2: Core-shell quantum dots are co-assembled with hydrogen-bonded organic framework precursors, and quantum dot / hydrogen-bonded organic framework composite materials are constructed through in-situ encapsulation or chemical bonding.

[0015] S3: The quantum dot / hydrogen bond organic framework composite material is reacted with a copper salt solution. After the reaction, the mixture is centrifuged and washed. Copper ions are pre-organized and anchored in the interface and pores of the composite material through coordination, thus obtaining a copper pre-organized quantum dot / hydrogen bond organic framework composite system.

[0016] Preferably, in step S2, "the co-assembly of core-shell quantum dots and hydrogen-bonded organic framework precursors, and the construction of quantum dot / hydrogen-bonded organic framework composite materials through in-situ encapsulation or chemical bonding" specifically includes the following steps:

[0017] S21: Disperse core-shell quantum dots in water, then add EDC and NHS aqueous solution, stir at room temperature to activate the core-shell quantum dots, and obtain an activated quantum dot solution;

[0018] S22: Trimethylbenzene and melamine are dissolved in DMF to obtain trimethylbenzene DMF solution and melamine DMF solution, respectively. Trimethylbenzene DMF solution and melamine DMF solution are added to the activated quantum dot solution in sequence, mixed and reacted to obtain quantum dot / hydrogen bond organic framework composite material.

[0019] Preferably, the reaction time of the activated quantum dot solution with the pyromellitic acid DMF solution and the melamine DMF solution is 20 h-28 h, and the reaction temperature is 55℃-65℃.

[0020] Preferably, the mass ratio of pyromellitic acid to melamine is 3:1-2.

[0021] Preferably, the mass ratio of the quantum dot / hydrogen bond organic framework composite material to the copper salt solution is 1:1-1.5.

[0022] Preferably, in step S3, the reaction temperature is room temperature and the reaction time is 3.5-4.5 h.

[0023] A ratiometric fluorescence detection method for glutathione includes the following steps:

[0024] S1: The sample to be tested is mixed with and incubated with a copper pre-organized quantum dot / hydrogen bonded organic framework composite system to obtain the detection system;

[0025] S2: Excite the detection system with excitation light and simultaneously detect the fluorescence intensity of the detection system at the short-wavelength emission peak and the long-wavelength emission peak;

[0026] S3: Calculate the fluorescence intensity ratio of the two emission peaks, and determine the concentration of glutathione in the sample to be tested according to the preset standard curve.

[0027] Preferably, the short-wave emission peak and the long-wave emission peak are 465 nm and 610 nm, respectively.

[0028] As described above, the copper pre-organized quantum dot / hydrogen-bonded organic framework composite system was used in the preparation of a detection kit for glutathione-related diseases.

[0029] Preferably, the glutathione-related diseases include one of diabetes, neurodegenerative diseases, cardiovascular diseases, liver damage, and immune system diseases.

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0031] The copper pre-organized quantum dot / hydrogen-bonded organic framework composite system provided by this invention includes quantum dots as the luminescent core, a confined matrix and interface-controlled hydrogen-bonded organic framework as the quantum dots, and copper species. The quantum dots are composited with the hydrogen-bonded organic framework, and the copper species are pre-organized and anchored at the composite interface between the quantum dots and the hydrogen-bonded organic framework. The copper species, quantum dots, and hydrogen-bonded organic framework together constitute a reconfigurable local coordination environment. Therefore, by confining and coordinating the copper species in a QDs@HOF composite system with a well-defined pore structure and interface microenvironment, the function of the copper sites is enhanced. Elevating GSH from a passively competing "quencher" to an "interfacial luminescence regulator" that can be specifically induced and reconstructed by GSH, GSH not only complexes with pre-organized copper sites when they interact strongly, but also triggers a systematic reconstruction of the local coordination environment, valence state distribution, and interfacial electronic coupling relationship of the copper sites. This activates new low-energy luminescence channels, achieves the redistribution of luminescence channels, and generates a clear ratiometric fluorescence signal (I long wavelength / I short wavelength). This successfully overcomes the technical problems of existing GSH fluorescence detection technologies, such as single response dimension, poor stability in complex environments, and fuzzy structure-signal correlation. Attached Figure Description

[0032] Figure 1 For (a) HOF, (b) QDs, (c) QDs@HOF, (d) Cu 2+ - SEM image of QDs@HOF.

[0033] Figure 2 XRD patterns of (a) MA-BTA-HOF, MA-BTA, BTA, and MA; (b) XRD patterns of MA-BTA-HOF, QDs@HOF, and Cu. 2+ XRD pattern of QDs@HOF composite material.

[0034] Figure 3 Cu 2+ -FT-IR spectra of QDs@HOF, QDs@HOF, QDs, MA-BTA-HOF, BTA, MA.

[0035] Figure 4 Cu 2+ - Selectivity plot of QDs@HOF sensor.

[0036] Figure 5 Cu 2+ -QDs@HOF fluorescence comparison detection diagram with each component.

[0037] Figure 6 The image shows the results of the anti-interference test.

[0038] Figure 7 This is a standard curve graph. Detailed Implementation

[0039] The technical solutions and effects of the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0040] This invention provides a copper pre-organized quantum dot / hydrogen bonded organic framework composite system. The composite system includes a quantum dot as a luminescent core, a confined matrix and interface-controlled hydrogen bonded organic framework as a quantum dot, and a copper species. The quantum dot is composited with the hydrogen bonded organic framework, and the copper species is pre-organized and anchored at the composite interface between the quantum dot and the hydrogen bonded organic framework.

[0041] In this system, the copper species, together with quantum dots and hydrogen-bonded organic frameworks, constitute a reconfigurable local coordination environment. In the presence of glutathione, this environment can induce a redistribution of the luminescent channels in the composite system, generating a ratiometric fluorescence response. Therefore, by confining and coordinating the copper species in the QDs@HOF composite system with a well-defined pore structure and interfacial microenvironment, the function of the copper sites is elevated from a passively competing "quencher" to an "interfacial luminescence regulator" that can be specifically induced and reconfigured by GSH. When GSH interacts strongly with the pre-organized copper sites, it not only complexes but also triggers a systematic reconstruction of the local coordination environment, valence state distribution, and interfacial electronic coupling relationship of the copper sites. This activates new low-energy luminescent channels, achieves the redistribution of luminescent channels, and generates a clear ratiometric fluorescence signal (I long wavelength / I short wavelength).

[0042] Furthermore, the quantum dots are ZnSe / ZnS core-shell quantum dots, the hydrogen-bonded organic framework is formed by the self-assembly of trimesic acid and melamine, and the copper species is copper ions.

[0043] A method for preparing a copper pre-organized quantum dot / hydrogen-bonded organic framework composite system includes the following steps:

[0044] S1: Preparation of core-shell quantum dots with surface rich in carboxyl functional groups;

[0045] S2: Core-shell quantum dots are co-assembled with hydrogen-bonded organic framework precursors, and quantum dot / hydrogen-bonded organic framework (QDs@HOF) composite materials are constructed through in-situ encapsulation or chemical bonding.

[0046] S3: The quantum dot / hydrogen-bonded organic framework composite material is reacted with a copper salt solution. After the reaction, the mixture is centrifuged and washed. Copper ions are pre-organized and anchored in the interface and channels of the composite material through coordination, resulting in a copper-pre-organized quantum dot / hydrogen-bonded organic framework (Cu). 2+ -QDs@HOF) composite system.

[0047] Specifically, the quantum dot / hydrogen-bonded organic framework composite material prepared by S2 was dispersed in deionized water under light-protected conditions to form a homogeneous suspension. An aqueous solution of Cu(NO3)2·3H2O was slowly added dropwise to the suspension under continuous stirring. The reaction was carried out at room temperature with stirring. After the reaction was completed, the mixture was centrifuged and washed to remove unbound copper ions, finally yielding Cu. 2+ -QDs@HOF composite materials.

[0048] Utilizing the abundant carboxyl and amino functional groups at the interface of the QDs@HOF composite material and the confined space provided by the HOF channels, Cu 2+ The copper sites are pre-organized and stably anchored near the quantum dot luminescent center through coordination interactions. This "pre-organization" allows for precise control of the spatial distribution and local coordination environment of the copper sites, laying the structural foundation for their subsequent specific interaction with GSH and triggering the reconstruction of the interface electronic structure. This differs from the traditional method of pre-organizing Cu... 2+ There is a fundamental difference between simply and haphazardly adding it to the testing system.

[0049] Furthermore, in step S1, "preparing core-shell quantum dots with surface rich in carboxyl functional groups" specifically includes the following steps:

[0050] Preparation of S11 ZnSe cores: Zn(ClO4)2·6H2O and N-acetyl-L-cysteine ​​(NAC) were dissolved in deionized water, and the pH was adjusted to 8.5. NAC acts as a stabilizer and surface ligand, controlling quantum dot growth and endowing their surfaces with abundant carboxyl functional groups. Subsequently, NaHSe solution was added, and the pH of the system was precisely adjusted to 6.0 to ensure precursor stability. The mixed solution was transferred to a high-pressure reactor for reaction, yielding a ZnSe core precursor solution. Quantum dot cores with high crystallinity and excellent luminescence properties were obtained through high-temperature and high-pressure reaction.

[0051] The S12 ZnS shell coating: NAC and Zn(ClO4)2·6H2O were dissolved in deionized water, the pH was adjusted to 4.0, and then Na2S·9H2O was added. The pH was adjusted to 6.0 again to obtain a ZnS shell precursor solution; this was used to passivate ZnSe core surface defects and improve quantum yield and stability.

[0052] S13 Microwave-Assisted Core-Shell Growth: A ZnSe core precursor solution and a ZnS shell precursor solution are mixed and transferred to a microwave reactor for reaction, which facilitates the formation of a uniform and dense ZnS shell to generate a ZnSe / ZnS core-shell quantum dot solution.

[0053] Therefore, by combining hydrothermal synthesis with microwave-assisted synthesis, a ZnSe / ZnS core-shell quantum dot solution with a surface rich in carboxyl groups, stable luminescence, and high quantum yield was generated.

[0054] Furthermore, in step S2, "the co-assembly of core-shell quantum dots and hydrogen-bonded organic framework precursors, and the construction of quantum dot / hydrogen-bonded organic framework composite materials through in-situ encapsulation or chemical bonding" specifically includes the following steps:

[0055] S21: Disperse core-shell quantum dots in water, then add aqueous solutions of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and stir at room temperature to activate the core-shell quantum dots, obtaining an activated quantum dot solution; activate the carboxyl groups on the surface of the quantum dots with EDC / NHS chemical coupling agent to enable them to undergo amidation reaction with HOF precursors (containing amino groups), thereby enhancing the interfacial bonding force between the quantum dots and the hydrogen-bonded organic framework, ensuring that the quantum dots are stably anchored in the HOF matrix and preventing leakage during subsequent use.

[0056] S22: Trimethylbenzene and melamine were dissolved in DMF to obtain trimethylbenzene DMF solution and melamine DMF solution, respectively. Trimethylbenzene DMF solution and melamine DMF solution were then added sequentially to the activated quantum dot solution, mixed thoroughly, and reacted to obtain a quantum dot / hydrogen-bonded organic framework composite material. Trimethylbenzene acts as a hydrogen bond donor and melamine as a hydrogen bond acceptor. During this process, trimethylbenzene and melamine self-assemble through intermolecular hydrogen bonds to form a hydrogen-bonded organic framework (HOF) with a periodic porous structure. Simultaneously, the carboxyl groups on the surface of the activated quantum dots can react with the amino groups of melamine, or be in-situ encapsulated in the pores or framework of the HOF through hydrogen bonding interactions, thus forming the QDs@HOF composite material. This composite material combines the high luminescence efficiency of quantum dots with the porosity, structural designability, and interface tunability of HOF.

[0057] Furthermore, the reaction time of the activated quantum dot solution with the pyromellitic acid DMF solution and the melamine DMF solution is 20 h-28 h, and the reaction temperature is 55℃-65℃.

[0058] Furthermore, the mass ratio of the pyromellitic acid to melamine is 3:1-2.

[0059] Furthermore, the mass ratio of the quantum dot / hydrogen bond organic framework composite material to the copper salt solution is 1:1-1.5.

[0060] Furthermore, in S3, the reaction temperature is room temperature and the reaction time is 3.5-4.5 h.

[0061] A ratiometric fluorescence detection method for glutathione includes the following steps:

[0062] S1: The sample to be tested is mixed with and incubated with a copper pre-organized quantum dot / hydrogen bonded organic framework composite system to obtain the detection system;

[0063] S2: Excite the detection system with excitation light and simultaneously detect the fluorescence intensity of the detection system at the short-wavelength emission peak and the long-wavelength emission peak; specifically, use 350nm as the wavelength of the excitation light and simultaneously detect the fluorescence intensity of the detection system at the short-wavelength emission peak and the long-wavelength emission peak.

[0064] S3: Calculate the fluorescence intensity ratio of the two emission peaks, and determine the concentration of glutathione in the sample to be tested according to the preset standard curve.

[0065] Furthermore, the short-wave emission peak and the long-wave emission peak are 465 nm and 610 nm, respectively.

[0066] In the initial state, pre-organized Cu 2+ The intrinsic fluorescence of the quantum dots (short-wavelength emission at approximately 465 nm) was effectively quenched via electron or energy transfer mechanisms. When GSH was added to the system, the thiol groups in the GSH molecule underwent strong affinity complexation with the pre-organized copper sites. This interaction is not limited to simple coordination competition; more importantly, it induces the reconstruction of the local coordination environment of the copper sites, accompanied by partial Cu... 2+ To Cu + The transformation of the valence-coordination state of the copper sites triggers a reconfiguration of the interfacial electronic coupling, leading to a reorganization of the hole / electron distribution and surface state distribution on the quantum dot surface. The final macroscopic manifestation is that while short-wavelength emission is partially or completely recovered, a new and significantly enhanced long-wavelength emission band appears at approximately 610 nm. Thus, with increasing GSH concentration, the system exhibits a clear redistribution of luminescence channels, i.e., the intensity ratio of short-wavelength emission to long-wavelength emission (Ig) is significantly higher. 610 / I 465 ) undergoes regular changes. By establishing I 610 / I 465 A standard curve between the ratio and GSH concentration can be used to achieve highly sensitive and selective ratio fluorescence detection of GSH.

[0067] As described above, the copper pre-organized quantum dot / hydrogen-bonded organic framework composite system was used in the preparation of a detection kit for glutathione-related diseases.

[0068] Furthermore, the glutathione-related diseases include one of the following: diabetes, neurodegenerative diseases, cardiovascular diseases, liver damage, and immune system diseases.

[0069] The following experiment further illustrates this point:

[0070] I. Copper Prestructure QDs@HOF (Cu) 2+ Preparation of QDs@HOF composite materials:

[0071] Step 1: Preparation of core-shell quantum dots (QDs)

[0072] 1. Preparation of ZnSe nuclei: 1.0 mmol Zn(ClO4)2·6H2O and 2.4 mmol NAC were dissolved in 50 mL of deionized water, and the pH was adjusted to 8.5. Then, freshly prepared 0.2 mmol NaHSe solution was added, and the pH of the system was precisely adjusted to 6.0. The mixed solution was transferred to a high-pressure reactor and reacted at 200 °C for 60 min to obtain the ZnSe nuclei precursor solution.

[0073] 2. Coating with ZnS shell: Dissolve 7.2 mmol NAC and 3.0 mmol Zn(ClO4)2·6H2O in 50 mL of deionized water, adjust the pH to 4.0, add 0.6 mmol Na2S·9H2O, and adjust the pH to 6.0 again to obtain the ZnS shell precursor solution.

[0074] 3. Microwave-assisted core-shell growth: The ZnSe core precursor solution and the ZnS shell precursor solution were mixed and transferred to a microwave reactor. The temperature was raised to 140℃ within 30 min and held for 30 min. A ZnSe / ZnS core-shell quantum dot solution was generated, denoted as (QDs).

[0075] Step 2: Construction of Quantum Dot / Hydrogen-Bonded Organic Framework (QDs@HOF) Composites

[0076] 1. Quantum dot surface activation: Take 2 mL of purified ZnSe / ZnS core-shell quantum dot aqueous dispersion, add 20 μL of 500 mM EDC and 20 μL of 1 M NHS aqueous solution, and react with gentle stirring at room temperature for 30 min to obtain the activated quantum dot solution.

[0077] 2. Assembly and composite of HOF framework: 5.0 mg of pyromellitic acid and 4.0 mg of melamine were weighed and dissolved in 5 mL of DMF. 3 mL of pyromellitic acid solution and 2 mL of melamine solution were added to the activated quantum dot solution in sequence. After vortex mixing, the mixture was placed in a 60℃ oven and allowed to stand for 24 h to generate QDs@HOF composite material.

[0078] Step 3: Copper pre-structured QDs@HOF (Cu 2+ Preparation of QDs@HOF composite materials

[0079] 20 mg of the QDs@HOF composite material prepared in step two was dispersed in 5 mL of deionized water to form a homogeneous suspension. Under continuous stirring, 5 mL of an aqueous solution containing 24 mg of Cu(NO3)2·3H2O was slowly added dropwise to the suspension. The entire reaction was carried out at room temperature for 4 h under light-protected conditions. After the reaction was completed, the mixture was centrifuged and washed to remove unbound copper ions, finally yielding Cu. 2+ -QDs@HOF composite material, denoted as Cu 2+ -QDs@HOF.

[0080] II. Preparation of Related Systems

[0081] Cu 2+ It is a 5 mL aqueous solution containing 24 mg Cu(NO3)2·3H2O;

[0082] The QDs were prepared using the same method described in step one above for preparing core-shell quantum dots (QDs).

[0083] HOF (MA-BTA-HOF) was prepared as follows: 5.0 mg of pyromellitic acid and 4.0 mg of melamine were weighed and dissolved in 5 mL of DMF. After vortexing and mixing, the mixture was placed in an oven at 60 °C and allowed to stand for 24 h to generate HOF.

[0084] MA-BTA is obtained by simply mixing 5.0 mg of pyromellitic acid and 4.0 mg of melamine;

[0085] The QDs@HOF system was prepared using the quantum dot / hydrogen bond organic framework (QDs@HOF) composite material preparation method described in step two above.

[0086] Cu 2+ - The QDs system is prepared by simply mixing 5 mL of an aqueous solution containing 24 mg Cu(NO3)2·3H2O with the core-shell quantum dots (QDs) prepared in step one.

[0087] Cu 2+The QDs@HOF system employs a copper prestructure QDs@HOF (Cu) 2+ The composite material was prepared by a method for preparing QDs@HOF.

[0088] Cu 2+ - The HOF system is obtained by simply mixing 5 mL of an aqueous solution containing 24 mg Cu(NO3)2·3H3O with the HOF prepared in step two without adding quantum dots (i.e., the blank control prepared in the same way);

[0089] III. Characterization

[0090] 1. The effects of scanning electron microscopy (SEM) on HOF, quantum dots (QDs), QDs@HOF, and the Cu prepared above were investigated. 2+ The microstructure of QDs@HOF was characterized, such as... Figure 1 As shown.

[0091] like Figure 1 As shown in (a), pure HOF exhibits a relatively regular cubic morphology with a smooth and dense surface and no obvious defects; while Figure 1 (b) shows QDs that are uniformly dispersed nanoparticles with sizes ranging from tens of nanometers, exhibiting a typical quantum dot morphology. From Figure 1 (c) It can be seen that when QDs are loaded onto HOF, the QDs@HOF composite material still maintains a blocky structure, but the surface becomes rough and a large number of fine granular protrusions appear, indicating the successful composite of QDs and HOF. Further introduction of Cu... 2+ Afterwards, as Figure 1 As shown in (d), Cu 2+ -QDs@HOF retain the HOF framework morphology, and the surface quantum dot particles are uniformly distributed without aggregation, detachment, or framework collapse, indicating that copper ion coordination modification has not damaged the main structure of the composite material, and the QDs remain stably dispersed on the HOF surface. In summary, SEM results show that QDs were successfully anchored on the HOF surface, and Cu... 2+ The modified composite material has a complete skeleton and stable structure, providing a reliable microstructure basis for subsequent GSH fluorescence sensing.

[0092] 2. To investigate the crystal structure and chemical composition of the materials, HOF, QDs, and a series of composite materials were characterized by X-ray diffraction (XRD) and Fourier transform infrared spectroscopy (FT-IR), such as... Figure 2 , 3 As shown.

[0093] like Figure 2(a) shows the XRD patterns of MA-BTA-HOF, MA-BTA, BTA, and MA. MA (melamine) exhibits multiple sharp diffraction peaks in the 2θ = 10°–30° range, while BTA (trimethylammonium phthalate) also shows obvious crystal characteristic peaks. When MA and BTA self-assemble through hydrogen bonding to form the MA-BTA complex, the diffraction peak positions are significantly different from those of the two monomers, and new characteristic diffraction peaks appear, indicating that effective hydrogen bonding interactions have occurred between MA and BTA, forming an ordered supramolecular structure. After further assembly to form MA-BTA-HOF, the spectrum shows even richer and stronger diffraction peaks, especially a series of new peaks in the low-angle region of 2θ = 5°–10°. This is a typical feature of HOF materials with periodic pore structures, confirming the successful construction of a three-dimensional hydrogen-bonded organic framework. Figure 2 (b) Comparison of MA-BTA-HOF, QDs@HOF and Cu 2+ XRD patterns of QDs@HOF. The diffraction patterns of QDs@HOF are basically consistent with those of MA-BTA-HOF. The positions of the characteristic peaks did not shift significantly, and the peak shapes remained sharp, indicating that the loading of QDs did not destroy the intrinsic crystal framework of HOF. The QDs are uniformly dispersed in the channels or surface of HOF in the form of amorphous or nanoclusters. The introduction of Cu... 2+ After that, Cu 2+ The XRD patterns of -QDs@HOF show a significant decrease in the intensity of HOF characteristic diffraction peaks, while some peaks exhibit broadening. This is attributed to Cu. 2+ Coordination interactions with functional groups such as carboxyl and amino groups in the HOF skeleton cause some perturbation to the hydrogen bond network, resulting in a slight decrease in long-range order. The absence of characteristic peaks for copper compounds such as CuO or Cu₂O in the spectrum indicates that Cu… 2+ They are uniformly dispersed in the composite system in the form of single atoms or amorphous nanoclusters.

[0094] The FT-IR spectroscopy system verified the construction of HOF, the loading of QDs, and Cu. 2+ Interface pre-organization, such as Figure 3 As shown. First, the stretching vibration peaks of -NH2 in free MA are located at 3467 and 3419 cm⁻¹. -1 After self-assembly with BTA, these two peaks broadened and redshifted to 3372 cm⁻¹. -1 Meanwhile, the triazine ring skeletal vibration peak decreased from 812 cm⁻¹. -1 Moved to 783 cm -1 The C=O stretching peak decreased from 1717 cm⁻¹. -1 Reduced to 1686 cm -1These changes are attributed to the formation of -OH…N, -NH…N, and -NH…O hydrogen bonds, which lowered the force constants of the corresponding chemical bonds, confirming the successful construction of the MA-BTA hydrogen bond framework.

[0095] Secondly, after introducing QDs into the HOF system, the spectrum of the QDs@HOF composite material retains the broad hydrogen bond absorption of HOF (~3300 cm⁻¹). -1 ) and triazine ring vibration (~773 cm) -1 In addition, a new amide II band (~1554 cm⁻¹) belonging to the NAC ligands on the surface of QDs also appeared. -1 This demonstrates that QDs have been successfully loaded onto HOF. Finally, Cu is introduced. 2+ After that, Cu 2+ -QDs@HOF spectra exhibit clear coordination characteristics: pristine ~1686 cm⁻¹ -1 The C=O peaks are still present, indicating that the main structure of the HOF has not been destroyed; at the same time, new peaks at 1671 and 1640 cm⁻¹ are added. -1 The two peaks are attributed to Cu, respectively. 2+ Coordinated carboxyl and amide carbonyl groups. Furthermore, ~1554 cm⁻¹ -1 The amide II band shifted to ~1560 cm⁻¹ -1 ~729 cm⁻¹ appears in the low wavenumber region. -1 The new peak is attributed to the Cu-O / Cu-N vibration. The retention of the original carbonyl peak, the appearance of the new coordinated carbonyl peak, and the metal-ligand bond peak clearly confirm that Cu 2+ It is pre-organized in a coordination manner at the QDs@HOF interface without disrupting the overall framework structure.

[0096] IV. Selective Detection Test

[0097] Experimental steps:

[0098] The copper prestructured QDs@HOF composite material (Cu) was prepared using the method described in Example 1 of this invention. 2+ -QDs@HOF system), fluorescence detection was performed on the following different substances: Blank control, citric acid (CA), glutamic acid (Glu), ammonium chloride (NH4Cl), thiodiacetic acid (TDGA), glycine (Gly), creatinine (Cre), serine (Ser), uric acid (UA), aspartic acid (Asp), sodium chloride (NaCl), cysteine ​​(Cys), urea (Urea), homocysteine ​​(Hcy), ascorbic acid (AA), Mg 2+ K + Ca 2+ Glutathione (GSH).

[0099] Specifically:

[0100] 1. Place Cu 2+ - The QDs@HOF system was prepared into a stock solution of 1 mg / mL with deionized water.

[0101] 2. Prepare 0.5 mmol / L solutions of the different substances mentioned above with deionized water; the blank control is: take 1 mL of Cu 2+ -QDs@HOF stock solution + 1 mL deionized water.

[0102] 3. Take 19 centrifuge tubes and take 1 mL of Cu. 2+ The stock solution of the -QDs@HOF system was placed in different centrifuge tubes, and then 1 mL of solutions of different substances were placed in different centrifuge tubes. The solutions were mixed evenly and reacted at 25℃±1℃ in the dark for 10 min.

[0103] 4. After the reaction is complete, transfer the reaction solution from each centrifuge tube to a 1 cm quartz cuvette and detect it using a fluorescence spectrophotometer.

[0104] The recommended instrument parameter settings are as follows:

[0105] Emission spectral scanning range: 400-900 nm

[0106] Excitation and emission slit width: 5 nm

[0107] Scan speed: 600 nm / min

[0108] Photomultiplier tube voltage: 400 V

[0109] Record the fluorescence intensity of each sample at the maximum emission wavelength. Each sample was measured three times, and the average value was taken. The results are as follows: Figure 4 As shown.

[0110] Experimental results:

[0111] To systematically investigate Cu 2+ The selective recognition ability and response mechanism of the QDs@HOF fluorescence sensor for glutathione (GSH) were studied in this study based on the "Cu 2+ Based on the principle of "induced fluorescence quenching—GSH coordination-triggered fluorescence recovery," selectivity, component comparison, anti-interference, and concentration linearity response experiments were conducted, and the results are as follows: Figure 4As shown. Equivalent amounts of material were added to each analyte at a uniform concentration (blank control, citric acid (CA), glutamic acid (Glu), ammonium chloride (NH4Cl), thiodiacetic acid (TDGA), glycine (Gly), creatinine (Cre), serine (Ser), uric acid (UA), aspartic acid (Asp), sodium chloride (NaCl), cysteine ​​(Cys), urea (Urea), homocysteine ​​(Hcy), ascorbic acid (AA), Mg). 2+ K + Ca 2+ Glutathione (GSH), selective results as follows Figure 4 As shown, the fluorescence intensity at 610 nm of the system is significantly enhanced only when GSH is added, exhibiting a clear "on" response. The fluorescence curves of the other interfering substances almost overlap with the blank group, and the fluorescence emission of the quantum dot at 465 nm shows a recovery trend to some extent, proving that the sensor has excellent selectivity for GSH. Therefore, ratiometric fluorescence (Ig) can be constructed... 610 / I 465 The probe enables specific detection of GSH.

[0112] V. Fluorescence Contrast Test

[0113] Experimental steps:

[0114] Choose Cu 2+ QDs, HOF, Cu 2+ -QDs, QDs@HOF, Cu 2+ -HOF and Cu 2+ The QDs@HOF system was used to detect the fluorescence of glutathione.

[0115] Testing of each system:

[0116] 1.2.1. Place Cu 2+ QDs, HOF, Cu 2+ -QDs, QDs@HOF, Cu 2+ -HOF and Cu 2+ The QDs@HOF system was prepared into a 1 mg / mL stock solution with deionized water.

[0117] 1.2.2. Prepare a 0.1 mmol / L solution of GSH using deionized water;

[0118] 1.2.3. Take 7 centrifuge tubes, add 1 mL of the stock solution of each system to different centrifuge tubes, add 1 mL of GSH solution to different centrifuge tubes, mix well, and react in the dark at 25℃±1℃ for 10 min.

[0119] 1.2.4 The detection process is the same as step 4 in section IV, Selective Detection Test. The results are as follows: Figure 5 As shown.

[0120] Experimental results: From the above Figure 5 It can be seen that QDs, HOF, and QDs@HOF alone do not have a significant ratio response to GSH.

[0121] Cu 2+ Simple mixing with QDs (Cu 2+ -QDs) only showed the traditional "fluorescence recovery" phenomenon, with no new peak at 610 nm.

[0122] Cu 2+ -HOF also cannot produce 610 nm characteristic emission.

[0123] Only in Cu 2+ In systems pre-organized at the QDs@HOF interface, GSH can simultaneously retain 465 nm emission (from QDs) and induce a new 610 nm emission band (from Cu). 2+ (Coordination environment reconstruction or changes in interfacial electronic coupling).

[0124] These comparative experiments strongly demonstrate that the technical effect of the present invention does not originate from simple component addition or known quenching-recovery mechanisms, but rather depends on the specific reconstruction of the "copper pre-structured QDs@HOF composite interface" under the action of GSH.

[0125] Meanwhile, this invention constructs Cu 2+ In the QDs@HOF composite system, copper species are pre-organized at the QDs@HOF composite interface. The introduction of GSH not only leads to coordination competition but also induces local coordination environment and valence state distribution of copper sites (Cu). 2+ / Cu + The systematic reconstruction of the conversion and interfacial electronic coupling relationship led to a significant redistribution of emission channels under the action of GSH: while retaining short-wavelength emission (approximately 465 nm), a new long-wavelength emission band appeared at approximately 610 nm, forming a clear I-wavelength emission band. 610 / I 465 Ratio fluorescence signal. The ratio detection mode eliminates interference from external factors through a self-calibration mechanism, significantly improving the accuracy and reproducibility of detection results. It solves the problem that existing technologies rely on fluorescence intensity changes at a single wavelength ("quenching-recovery"), which are susceptible to light source fluctuations, probe concentration differences, and background fluorescence interference, resulting in poor signal reliability.

[0126] VI. Anti-interference test

[0127] Experimental steps:

[0128] The present invention employs copper pre-structured QDs@HOF (Cu) 2+ Cu obtained in the preparation of QDs@HOF composite material 2+ The -QDs@HOF system was used to detect fluorescence in the following substances: Blank control, Citric acid (CA), Glutamic acid (Glu), Ammonium chloride (NH4Cl), Thiodiacetic acid (TDGA), Glycine (Gly), Creatine (creatine), Serine (Ser), Uric acid (UA), Aspartic acid (Asp), Sodium chloride (NaCl), Cysteine ​​(Cys), Urea (Urea), Homocysteine ​​(Hcy), Ascorbic acid (AA), and Mg. 2+ K + Ca 2+ .

[0129] Specifically:

[0130] 1. Place Cu 2+ - The QDs@HOF system was prepared into a stock solution of 1 mg / mL with deionized water.

[0131] 2. Prepare 0.5 mmol / L solutions of the different substances mentioned above with deionized water; the blank control is: take 1 mL of Cu 2+ -QDs@HOF stock solution + 1 mL deionized water.

[0132] 3. Take 18 centrifuge tubes and take 1 mL of Cu. 2+ The stock solution of the -QDs@HOF system was placed in different centrifuge tubes, and then 1 mL of solutions of different substances were placed in different centrifuge tubes. The solutions were mixed evenly and reacted at 25℃±1℃ in the dark for 10 min.

[0133] 4. The detection process is the same as step 4 in section IV, Selective Detection Test. The results are as follows: Figure 6 As shown.

[0134] Experimental results:

[0135] Depend on Figure 6 It can be seen that the Cu constructed in this invention 2+ The QDs@HOF system effectively distinguishes GSH from other amino acids and thiols, exhibiting significantly better anti-interference capabilities than the simple mixed control system. This demonstrates that the present invention pre-organizes copper ions at the QDs@HOF composite interface, utilizing the ordered channels and abundant functional groups of HOF to achieve confinement and stable anchoring of copper sites, forming a well-defined local coordination environment. This "pre-organized" structure not only enhances the stability of copper sites but also endows them with specific recognition capabilities for GSH. It solves the problem of free Cu in existing technologies. 2+It is susceptible to non-specific competitive interference from multiple coexisting substances in complex systems, leading to technical problems of poor selectivity.

[0136] VII. Establishment of Standard Curve

[0137] 1. Preparation of GSH concentration gradient solutions

[0138] 1.1. Preparation of stock solution: Accurately weigh an appropriate amount of glutathione (GSH, MW = 307.32 g / mol), dissolve it in deionized water and make up to volume to prepare a GSH stock solution with a concentration of 0.01 mol / L.

[0139] 1.2. Take 5 clean 1 mL volumetric flasks (or stoppered colorimetric tubes) and number them 1 to 5.

[0140] Accurately transfer 40 μL, 50 μL, 80 μL, 100 μL and 150 μL of stock solution into each volumetric flask, then dilute to 1.0 mL with deionized water, stopper and shake well.

[0141] The final GSH concentrations for each gradient are calculated as shown in Table 1:

[0142] Table 1

[0143] 1.3. Take 5 centrifuge tubes and take 1 mL of Cu 2+ The stock solution of the QDs@HOF system was placed in different centrifuge tubes, and then 1 mL of GSH solution with different concentration gradients was placed in different centrifuge tubes. The mixtures were mixed evenly and reacted at 25℃±1℃ in the dark for 10 min.

[0144] 2. The detection process is the same as step 4 in section IV, Selective Detection Test. The results are as follows: Figure 7 As shown.

[0145] 3. Standard curve results:

[0146] I can be established through the above. 610 / I 465 The standard curve between the ratio and GSH concentration enables quantitative analysis with a wide linear range (0.4-1.5 mmol / L) and low detection limit. The detection process is rapid and easy to operate, requiring no complex sample pretreatment.

[0147] In summary, this invention constructs Cu 2+ The -QDs@HOF composite system has successfully overcome key technical challenges in existing GSH fluorescence detection technologies, such as single response dimension, poor stability in complex environments, and fuzzy structure-signal correlation, achieving significant technological progress.

[0148] The above-disclosed embodiments are merely preferred embodiments of the present invention and should not be construed as limiting the scope of the invention. Those skilled in the art will understand that implementing all or part of the above-described embodiments and making equivalent changes in accordance with the claims of the present invention are still within the scope of the invention.

Claims

1. A copper pre-organized quantum dot / hydrogen-bonded organic framework composite system, characterized in that: The composite system comprises quantum dots as a light-emitting core, a hydrogen-bonded organic framework as a confinement matrix of the quantum dots, and an interface-regulated copper species, the quantum dots are compounded with the hydrogen-bonded organic framework, and the copper species is preorganized and anchored at the compound interface of the quantum dots and the hydrogen-bonded organic framework; the quantum dots are ZnSe / ZnS core-shell quantum dots, the hydrogen-bonded organic framework is self-assembled from trimesic acid and melamine, and the copper species is a copper ion; The copper species, the quantum dots, and the hydrogen-bonded organic framework together form a reconfigurable local coordination environment, and in the presence of glutathione, the reconfigurable local coordination environment can induce redistribution of a light-emitting channel of the composite system to generate a ratio fluorescence response.

2. A method for preparing the copper pre-organized quantum dot / hydrogen-bonded organic framework composite system according to claim 1, characterized by: The method comprises the following steps: S1: preparing core-shell quantum dots rich in carboxyl functional groups on the surface; S2: co-assembling the core-shell quantum dots and a hydrogen-bonded organic framework precursor to construct a quantum dot / hydrogen-bonded organic framework composite material by in-situ encapsulation and chemical bonding; S3: reacting the quantum dot / hydrogen-bonded organic framework composite material with a copper salt solution, centrifuging and washing after the reaction, and preorganizing and anchoring the copper ions in the interface and channels of the composite material by coordination to obtain a copper-preorganized quantum dot / hydrogen-bonded organic framework composite system.

3. The preparation method of the copper pre-organized quantum dot / hydrogen-bonded organic framework composite system according to claim 2, characterized in that: In the step S2, the co-assembling of the core-shell quantum dots and the hydrogen-bonded organic framework precursor to construct the quantum dot / hydrogen-bonded organic framework composite material by in-situ encapsulation and chemical bonding specifically comprises the following steps: S21: dispersing the core-shell quantum dots in water, then adding EDC and NHS aqueous solutions, and stirring and reacting at room temperature to activate the core-shell quantum dots to obtain an activated quantum dot solution; S22: dissolving trimesic acid and melamine in DMF respectively to obtain a trimesic acid DMF solution and a melamine DMF solution, and then adding the trimesic acid DMF solution and the melamine DMF solution into the activated quantum dot solution in sequence, mixing, and reacting to obtain a quantum dot / hydrogen-bonded organic framework composite material.

4. The preparation method of the copper pre-organized quantum dot / hydrogen-bonded organic framework composite system according to claim 3, characterized in that: The reaction time of the reaction of the activated quantum dot solution with the trimesic acid DMF solution and the melamine DMF solution is 20-28 hours, and the reaction temperature is 55-65°C.

5. The method for preparing the copper pre-organized quantum dot / hydrogen-bonded organic framework composite system according to claim 3, characterized in that: The mass ratio of the trimesic acid to the melamine is 3:1-2.

6. The method for preparing the copper pre-organized quantum dot / hydrogen-bonded organic framework composite system as described in claim 3, characterized in that: The mass ratio of the quantum dot / hydrogen-bonded organic framework composite material to the copper salt solution is 1:1-1.

5.

7. The method for preparing the copper pre-organized quantum dot / hydrogen-bonded organic framework composite system according to any one of claims 2-6, characterized in that: In the step S3, the reaction temperature is room temperature, and the reaction time is 3.5-4.5 hours.

8. A method for the ratiometric fluorescence detection of glutathione, characterized by: The method comprises the following steps: S1: mixing and incubating a sample to be detected with the copper-preorganized quantum dot / hydrogen-bonded organic framework composite system of claim 1 to obtain a detection system; S2: exciting the detection system with excitation light, and detecting the fluorescence intensity of the detection system at a short-wave emission peak and a long-wave emission peak; S3: calculating the fluorescence intensity ratio of the two emission peaks, and determining the concentration of glutathione in the sample to be detected according to a preset standard curve.

9. The copper-preorganized quantum dot / hydrogen-bonded organic framework composite system of claim 1 in the preparation of a kit for detecting a glutathione-related disease.

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