ATP (adenosine triphosphate) detection nano particle based on up-conversion luminescent material as well as preparation method and application of ATP detection nano particle

Abstract

The invention discloses ATP (adenosine triphosphate) detection nanoparticles based on an up-conversion luminescent material as well as a preparation method and application of the ATP detection nanoparticles. Through intelligent structural design and function integration, the nano particle successfully breaks through many limitations in the prior art, and a new technical scheme is provided for ATP related research and clinical application. The invention not only solves the problems in the existing ATP detection method, but also establishes a systematic mesoporous material optimization method, and provides important theoretical and technical basis for developing a new generation of intelligent diagnosis and treatment technology.

Description

Technical Field

[0001] The present invention relates to the field of nanomaterials, and in particular to an ATP detection nanoparticle based on up-conversion luminescent materials, and a preparation method and application thereof. Background Art

[0002] Adenosine triphosphate (ATP), as one of the most important energy substances and signaling molecules in organisms, plays a key role in life processes such as cell metabolism, neurotransmission, and signal transduction. The steady-state balance of intracellular ATP concentration is crucial to maintaining normal physiological functions, and its imbalance is often closely related to a variety of major diseases, such as neurodegenerative diseases, cardiovascular diseases, and mental illnesses. Therefore, the precise monitoring and regulation of ATP concentration has important scientific significance and clinical application value. However, although there are many types of nanomaterials currently used for ATP detection and regulation, there are still many technical problems that need to be solved.

[0003] Traditional analytical nanomaterials show significant limitations in ATP detection. Taking metal nanoparticles as an example, although they have high sensitivity, they have poor selectivity and are easily interfered by other metal ions. At the same time, they only have detection functions but cannot achieve active regulation of ATP, and they have poor stability and are prone to aggregation and inactivation. Although quantum dot nanoprobes have good optical properties, they suffer from severe photobleaching, and the detection process is irreversible, making it difficult to achieve continuous monitoring. In addition, although carbon-based nanomaterials (such as carbon nanotubes, graphene, etc.) have unique physical and chemical properties, they have poor biocompatibility, long-term accumulation may cause toxicity, and lack specific recognition sites, insufficient detection selectivity, and too single function.

[0004] Existing multifunctional nanoplatforms still face many challenges in terms of structural design and functional integration. First, the design of the core-shell structure is generally arbitrary and lacks systematic optimization. The synergy between the functional modules is poor, and the shell thickness is difficult to accurately control, which seriously affects the energy transfer efficiency. Secondly, the recognition mechanism of ATP mostly relies on a single recognition method, the design of specific recognition groups lacks innovation, the recognition process is poorly reversible, and it is difficult to meet the needs of dynamic monitoring. More importantly, most nanoplatforms only have passive detection functions, lack of organic integration with advanced technologies such as optogenetics, and cannot achieve precise regulation of ATP concentration.

[0005] In terms of system integration, existing technologies also face severe challenges. The first is the problem of insufficient integration of functional modules. The detection and regulation functions are often separated, and there is a lack of a unified excitation-response mechanism, which leads to high system complexity and cumbersome operation. Secondly, there are obvious deficiencies in intelligent response, the response process is difficult to control, there is a lack of real-time feedback mechanism, and the system stability and repeatability need to be improved. In addition, in terms of biological applications, there are also problems such as insufficient tissue penetration depth, poor biocompatibility, and poor long-term stability, which seriously restrict the practical application of nanomaterials in the field of ATP monitoring and regulation. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide an ATP detection nanoparticle based on upconversion luminescent materials.

[0007] Another object of the present invention is to provide a method for preparing the above-mentioned ATP detection nanoparticles based on up-conversion luminescent materials.

[0008] Another object of the present invention is to provide an application of the above-mentioned ATP detection nanoparticles based on up-conversion luminescent materials.

[0009] The purpose of the present invention is achieved through the following technical solutions:

[0010] A method for preparing ATP detection nanoparticles based on upconversion luminescent materials comprises the following steps:

[0011] (1) Preparation of crude probe

[0012] The rhodamine was dissolved in ethanol, and an amino compound was added, and the mixture was stirred and refluxed for reaction. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the mixture was redissolved in dichloromethane and washed. The solvent was removed by distillation under reduced pressure again to obtain a crude probe;

[0013] (2) Purification of rhodamine probe

[0014] The crude probe is redispersed with hydrochloric acid and stirred, a sodium hydroxide solution is added to precipitate, the aqueous phase is removed, the obtained precipitate is dispersed in dichloromethane, washed, and dried to obtain a rhodamine probe;

[0015] (3) Preparation of UCNPs

[0016] Yttrium acetate tetrahydrate, ytterbium acetate tetrahydrate and erbium acetate tetrahydrate are dissolved in octadecene, oleic acid and octadecene oleic acid are added, and water and oxygen are removed by heating under vacuum, and then reflux reaction is carried out under argon protection, and UCNPs are obtained by centrifugation, and UCNPs solution is obtained after redispersing in cyclohexane;

[0017] (4) Mesoporous silica coating

[0018] Dissolve CTAB in water, stir to react, add UCNPs solution, continue to stir to react, obtain CTAB-stabilized UCNPs aqueous solution, then add CTAB-stabilized UCNPs aqueous solution to alkaline mixed solution, heat and mix, then drop TEOS, continue to stir to react, then add ethanol to terminate the reaction, centrifuge, wash, and obtain mesoporous silicon nanoparticles;

[0019] (5) Removal of template

[0020] Dispersing mesoporous silicon nanoparticles in a hydrochloric acid ethanol solution, stirring and refluxing the solution, centrifuging the solution, and then repeating the dispersion, refluxing, and centrifuging to obtain mesoporous silica-coated upconversion nanoparticles;

[0021] (6) Preparation of ATP detection nanoparticles based on upconversion luminescent materials

[0022] The mesoporous silica-coated upconversion nanoparticles obtained in step (5) are dispersed in an ethanol solution containing the rhodamine probe prepared in step (2), stirred at room temperature, centrifuged after the reaction is completed, and washed to obtain ATP detection nanoparticles based on upconversion luminescent materials.

[0023] The molar ratio of rhodamine to the amino compound in step (1) is 3-5:80-100; preferably 4.2:92.

[0024] The amino compound described in step (1) includes at least one of diethylenetriamine, ethylamine, butylamine, naphthalenediamine and methylbutylenediamine.

[0025] The stirring reflux reaction conditions of step (1) are 75-85° C. and 1000-1500 rpm for 20-28 hours.

[0026] The concentration of the hydrochloric acid in step (2) is 0.8-1.2M.

[0027] The concentration of the sodium hydroxide solution in step (2) is 0.8-1.2M.

[0028] The molar ratio of yttrium acetate tetrahydrate, ytterbium acetate tetrahydrate and erbium acetate tetrahydrate in step (3) is 30-50:5-15:1-2, preferably 39:10:1.

[0029] The volume ratio of oleic acid to octadecene oleic acid in step (3) is 1-2:4-6.

[0030] The reflux reaction conditions of step (3) are 250-350° C. for 0.5-2 h.

[0031] The stirring reaction conditions of step (4) are 65-75° C. and 700-800 rpm for 8-12 min.

[0032] The mass ratio of CTAB to UCNPs in the UCNPs solution in step (4) is 2 to 4:1, preferably 3:1.

[0033] The conditions for continuing the stirring reaction in step (4) are stirring the reaction at 65-75° C. and 700-800 rpm for 3-5 hours.

[0034] The volume ratio of the CTAB-stabilized UCNPs aqueous solution to the alkaline mixed solution in step (4) is 1-2:2-4; preferably 10:23.15.

[0035] The ratio of the alkaline mixed solution in step (4) is water: ethanol: sodium hydroxide solution = 200: 30: 1.5, wherein the concentration of the sodium hydroxide solution is 2M.

[0036] The heating in step (4) is heating to 65-75°C.

[0037] The volume ratio of the TEOS to CTAB-stabilized UCNPs aqueous solution in step (4) is 15-44:2000.

[0038] The conditions for continuing the stirring reaction in step (4) are stirring the reaction at 65-75° C. and 700-800 rpm for 0.5-2 h.

[0039] The pH of the hydrochloric acid ethanol solution in step (5) is 1.4 to 1.5.

[0040] The stirring reflux reaction conditions of step (5) are 55-65° C. and 700-800 rpm for 2-4 hours.

[0041] The number of repetitions in step (5) is 2 to 4 times.

[0042] The ratio of the mesoporous silica-coated upconversion nanoparticles to the rhodamine probe in step (6) is 10-30 μg:1 μmol, preferably 20 μg:1 μmol.

[0043] The stirring condition in step (6) is stirring for 20 to 30 hours.

[0044] The washing in step (6) is performed using ethanol.

[0045] The centrifugal condition is 7000-9000 rpm for 5-15 min.

[0046] An ATP detection nanoparticle based on up-conversion luminescent material is prepared according to the above preparation method.

[0047] The application of the ATP detection nanoparticles based on upconversion luminescent materials in detecting ATP concentration.

[0048] Compared with the prior art, the present invention has the following advantages and effects:

[0049] The present invention proposes a multifunctional integrated platform based on upconversion nanomaterials. Through intelligent structural design and functional integration, it successfully breaks through many limitations of existing technologies and provides a new technical solution for ATP-related research and clinical applications. The present invention not only solves the problems existing in existing ATP detection methods, but also establishes a systematic mesoporous material optimization method, providing an important theoretical and technical basis for the development of a new generation of intelligent diagnosis and treatment technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 are the structural formulas and mass spectrometry results of different rhodamine probes prepared in Example 1;

[0051] Figure 2 is a transmission electron microscope photograph of the composite nanoparticles UCNPs@mSiO2 / RB-Bu prepared in Example 3;

[0052] Figure 3 is a graph showing the results of the nanoparticle selectivity test in Example 5;

[0053] Figure 4 This is a diagram of the reversibility test results in Example 5.

[0054] Figure 5 It is a diagram of the kinetic test results in Example 5. DETAILED DESCRIPTION

[0055] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0056] If no specific experimental conditions are specified in the following embodiments, conventional experimental conditions or experimental conditions recommended by the reagent company are generally followed. The materials and reagents used, unless otherwise specified, are all reagents and materials obtained from commercial sources.

[0057] Example 1

[0058] 1.1 Synthesis of ethylenediamine rhodamine probe (RB-EA)

[0059] (1) Rhodamine B (2.0 g, 4.2 mmol) was dissolved in 50 mL of ethanol, diethylenetriamine (10 mL, 92 mmol) was added to the above solution, and the mixture was vigorously stirred (1200 rpm) and refluxed at 80°C for 24 hours. The solvent was removed by distillation under reduced pressure to obtain a dark yellow oily product. The oily product was redissolved in dichloromethane and then treated with saturated NaHCO 3 and washed with saturated NaCl solution; the solvent was removed by distillation under reduced pressure to obtain a light orange solid;

[0060] (2) The solid was redispersed with 1M HCl (50 mL), and 1M NaOH was gradually added dropwise under gentle stirring until no light pink solid precipitated; the aqueous phase was removed, and the light pink precipitate was dispersed in dichloromethane (100 mL) and repeatedly washed with deionized water; the dichloromethane was removed to obtain a dark orange solid (RB-EA); the product was dried under vacuum to obtain the ethylenediamine rhodamine probe (RB-EA), which was stored at -20°C in the dark. Before use, RB-EA was dissolved in ethanol to prepare a 10 mM stock solution.

[0061] Mass spectrometry characterization: [RB-EA+H]+C32H42N5O2+, m / z calculated value 528.4.

[0062] 1.2 Synthesis of ethylaminorhodamine probe (RB-Et)

[0063] Refer to the preparation method in 1.1, except that diethylenetriamine is replaced by the same molar amount of ethylamine.

[0064] Mass spectrometry characterization: [RB-Et+H]+C32H41N4O2+, m / z calculated value 513.2.

[0065] 1.3 Synthesis of butylamino rhodamine probe (RB-Bu)

[0066] The preparation method is as described in 1.1, except that diethylenetriamine is replaced by the same molar amount of butylamine.

[0067] Mass spectrometry characterization: [RB-Bu+H]+C34H45N4O2+, m / z calculated value 541.3.

[0068] 1.4 Synthesis of naphthyl diamine rhodamine probe (RB-ND)

[0069] The preparation method is as described in 1.1, except that diethylenetriamine is replaced by the same molar amount of naphthalenediamine.

[0070] Mass spectrometry characterization: [RB-ND+H]+C41H45N4O2+, m / z calculated value 625.2.

[0071] 1.5 Synthesis of methylbutylene diamine rhodamine probe (RB-MD)

[0072] The preparation method is as described in 1.1, except that diethylenetriamine is replaced by the same molar amount of methylbutylene diamine.

[0073] Mass spectrometry characterization: [RB-MD+H]+C33H43N4O2+, m / z calculated value 527.3.

[0074] 1.6 Probe selectivity test

[0075] In order to evaluate the selectivity of different RB-R probes (RB-EA, RB-Et, RB-Bu, RB-ND, RB-MD) for ATP, a series of selectivity test experiments were designed and implemented. First, the solutions of each probe were prepared, and ATP and ADP solutions were prepared respectively. The specific experimental methods are as follows:

[0076] During sample preparation, 1970 μL of deionized water was added to a 2 mL cuvette, followed by 20 μL of probe solution and 10 μL of ATP or ADP solution, and then gently shaken to mix evenly. The final concentration of the probe was 10 μM, and the final concentration of ATP or ADP was 100 μM. The excitation wavelength of all samples was set at about 550 nm, and the emission wavelength was set at about 580 nm to ensure the accuracy of fluorescence measurement.

[0077] The fluorescence intensity of each sample was measured using a fluorescence photometer. The instrument was first calibrated, and a blank control containing only deionized water and the probe was used for baseline correction. Each sample was measured at least three times to ensure the repeatability and accuracy of the data. The response of each probe under different conditions was evaluated by calculating the fluorescence enhancement factor, that is, the ratio of the fluorescence intensity in the presence of ATP or ADP to the fluorescence intensity of the blank control. The experimental results showed that the fluorescence enhancement of the RB-Bu probe was about 25 times in the presence of ATP, while the change in the presence of ADP was only about 3 times, indicating that it is highly selective for ATP. The fluorescence responses of other probes in the presence of ATP and ADP are relatively balanced, and the selectivity is not as significant as that of the RB-Bu probe.

[0078] 1.7 Probe reaction rate test

[0079] In this experiment, in order to investigate the reaction rate of RB-EA and RB-Bu probes in the presence of ATP and ADP, the reaction system was prepared according to the experimental method in 1.6. After adding the probes, the change in fluorescence intensity was recorded once per second and continuously monitored until the fluorescence signal reached stability.

[0080] The experimental results show that when the RB-Bu probe reacts with ATP, it can achieve a stable fluorescence response in about 60 seconds. After the addition of ADP, the reaction rate is significantly reduced, and it takes about 120 seconds for a significant fluorescence change to appear. In contrast, the overall response speed of the RB-EA probe is slightly lower than that of RB-Bu, but it also shows a trend of rapid response to ATP. This result shows that in practical applications, the RB-Bu probe can more quickly monitor the dynamic changes of ATP concentration and effectively inhibit interference with ADP, thereby having better response speed and selectivity in biological systems.

[0081] 1.8 Probe reversibility test

[0082] In order to evaluate the reversibility of different RB-R probes (RB-EA, RB-Et, RB-Bu), this experiment focused on the stability of the fluorescence signal of the RB-Bu probe during multiple cycles of use.

[0083] Prepare the reaction system and detect the fluorescence intensity according to the experimental method in 1.6. When the probe fluorescence signal reaches stability, add apyrase (1U) to decompose ATP and observe the decrease of fluorescence intensity with ATP consumption. Then add ATP (final concentration 100μM) again to restore it to the initial fluorescence intensity. Repeat the cycle of "adding ATP-enzyme catalyzing ATP degradation" for at least 5 times.

[0084] The experimental results show that both RB-EA and RB-Et organic molecules show a significant increase in fluorescence intensity after the addition of ATP, but after the removal of ATP, the fluorescence intensity recovery is limited and shows a gradual decay trend in multiple cycles, indicating that its reversibility is poor. In contrast, the RB-Bu organic molecule can achieve a relatively complete fluorescence recovery in each cycle, and the fluorescence intensity remains basically stable after the addition and removal of ATP, showing good reversible response performance. The fluorescence change range of the RB-Bu probe after each cycle is kept within ±5%, which fully demonstrates its excellent reversibility and reusability. In contrast, other organic probes cannot maintain such a stable response in multiple cycle tests. This result provides an important basis for the real-time monitoring and regulation of ATP concentration in a closed-loop system, and further demonstrates the potential advantages of the RB-Bu probe in sensor devices or biological system applications.

[0085] Example 2 Preparation of mesoporous silica-coated upconversion nanoparticles

[0086] 2.1 Preparation of mesoporous silica-coated upconversion nanoparticles (5 nm)

[0087] (1) Preparation of core-shell upconversion nanoparticles (UCNPs)

[0088] Using octadecene as solvent, 0.78 mmol Y(CH 3 COO 3 ·4H 2 O(yttrium acetate tetrahydrate), 0.2mmol Yb(CH 3 COO 3 ·4H 2 O(ytterbium acetate tetrahydrate) and 0.02mmol Er(CH 3 COO 3 ·4H 2 O (erbium acetate tetrahydrate) was mixed and dissolved, 6 mL of oleic acid and 15 mL of octadecene oleic acid were added as surfactants, heated to 100 ° C under vacuum conditions and maintained for 30 minutes to remove water and oxygen, and then heated to 300 ° C under argon protection and refluxed for 1 hour to obtain UCNPs. After purification by centrifugation at 8000 rpm for 10 min, the resulting mixture was dispersed in cyclohexane to prepare a 10 mg / mL UCNPs solution.

[0089] (2) Mesoporous silica coating

[0090] 60 mg of CTAB was dissolved in 20 mL of water and stirred at 750 rpm in a 70°C water bath for 10 minutes; 2 mL of the cyclohexane solution containing UCNPs prepared in step (1) was added and the stirring was continued at 750 rpm for about 4 hours until the cyclohexane was completely evaporated; 10 mL of the UCNPs aqueous solution stabilized by CTAB was added to an alkaline mixed solution containing 20 mL of water, 3 mL of ethanol and 150 μL of 2M NaOH and stirred at 70°C; 75 μL of tetraethoxysilane (TEOS) was slowly added dropwise at 70°C and stirred for 1 hour; 30 mL of ethanol was added to terminate the reaction; the product was collected by centrifugation (8000 rpm, 10 minutes) and washed with ethanol several times to obtain mesoporous silicon nanoparticles UCNPs@mSiO 2 .

[0091] (3) Template removal

[0092] The UCNPs@mSiO 2 The nanoparticles were dispersed in 40 mL of hydrochloric acid ethanol solution with a pH of 1.4-1.5, magnetically stirred and refluxed at 60 °C for 3 h, and centrifuged at 8000 rpm for 10 min to remove the etched CTAB. The above nanoparticles were dispersed in hydrochloric acid ethanol, stirred and refluxed for 3 times to obtain mesoporous silica-coated upconversion nanoparticles (UCNPs@mSiO 2 ); finally, the solid was dispersed in PBS solution and stored at 4°C. The thickness of the mesoporous layer was about 5 nm.

[0093] 2.2 Preparation of mesoporous silica-coated upconversion nanoparticles (10 nm)

[0094] Referring to the preparation method in 2.1, the difference is that in step (2) the mesoporous silica coating step, the amount of TEOS is changed to 150 μL, and the other conditions remain unchanged; transmission electron microscopy (TEM) characterization confirms that the thickness of the mesoporous layer is about 10 nm.

[0095] 2.3 Preparation of mesoporous silica-coated upconversion nanoparticles (20 nm)

[0096] The preparation method in 2.1 was referred to, except that the amount of TEOS in the mesoporous silica coating step (2) was changed to 220 μL, and the other conditions remained unchanged. Transmission electron microscopy (TEM) characterization confirmed that the thickness of the mesoporous layer was about 20 nm.

[0097] Example 3 Preparation of ATP detection nanoparticles based on upconversion luminescent materials

[0098] 400 μg of the 5 nm mesoporous silica-coated upconversion nanoparticles prepared in 2.1 of Example 2 were dispersed in 2 mL of an ethanol solution containing 10 mM RB-Bu probe prepared in 1.1 of Example 1, stirred at room temperature for 24 hours to achieve sufficient loading of the dye, centrifuged and repeatedly washed with ethanol to remove unloaded dye molecules, to obtain 5 nm ATP detection nanoparticles based on upconversion luminescent materials, which were dispersed in PBS buffer (pH 7.4) for later use.

[0099] Referring to the above method, 10 nm ATP detection nanoparticles based on upconversion luminescent materials and 20 nm ATP detection nanoparticles based on upconversion luminescent materials were prepared.

[0100] Example 4 Study on fluorescence quenching efficiency of different mesoporous silica layer thicknesses

[0101] 4.1 Fluorescence quenching efficiency test

[0102] A series of 100 μM ATP solutions were prepared; three UCNPs@mSiO2 with different mesoporous layer thicknesses loaded with RB-Bu prepared in Example 3 were added. 2 The nanoparticles were added to the above ATP solution respectively; under 980nm laser excitation, the change of fluorescence intensity at 540nm was measured; and the fluorescence quenching efficiency ΔF / F was calculated.

[0103] 4.2 Experimental Results

[0104] The experimental results show that the sample with a mesoporous layer thickness of 10 nm exhibits the best fluorescence quenching efficiency, reaching the maximum value (ΔF / F≈0.8) when the Rb-Bu concentration is 50 μM; the sample with a mesoporous layer thickness of 5 nm exhibits a moderate fluorescence quenching efficiency (ΔF / F≈0.4); the sample with a mesoporous layer thickness of 20 nm shows the lowest fluorescence quenching efficiency (ΔF / F≈0.2); the fluorescence quenching efficiency of all samples first increases and then tends to be stable with the increase of ATP concentration.

[0105] 4.3 Conclusion Analysis

[0106] Experimental results show that the thickness of the mesoporous layer significantly affects the fluorescence quenching efficiency of the probe. A mesoporous layer thickness of 10 nm provides the most suitable spatial configuration, which can ensure sufficient dye loading and maintain good ATP molecule diffusion and binding efficiency. A too thin mesoporous layer (5 nm) may lead to insufficient dye molecule loading; a too thick mesoporous layer (20 nm) may affect the diffusion and accessibility of ATP molecules, thereby reducing detection sensitivity.

[0107] Example 5

[0108] 5.1 pH stability test

[0109] In order to evaluate the pH stability of the nanoparticles, the 10 nm ATP detection nanoparticles based on upconversion luminescent materials prepared in Example 3 were dispersed in HEPES buffer solutions of different pH values ​​(pH 4.0, 5.0, 6.5, 7.4, 8.0 and 9.0, 10.0), and tested for their reaction with ATP under different pH conditions. The experiment showed that it has good stability in detecting ATP in a wide pH range.

[0110] 5.2 Light stability test

[0111] To evaluate the photostability of the nanoparticles, a PBS buffer solution (pH 7.4) containing 10 nm upconversion luminescent material-based ATP detection nanoparticles was prepared and illuminated at room temperature with a 980 nm near-infrared (NIR) laser (power density 2 W / cm 2 ) was irradiated continuously for 30 minutes. The up-conversion fluorescence spectrum was recorded every 5 minutes to observe the intensity change of the green fluorescence peak.

[0112] The test results show that during 30 minutes of continuous illumination, the fluorescence intensity only decreased by about 5%, indicating that NaYbF 4 :2% Er@NaYF 4 UCNPs@mSiO 2 -RB-Bu nanoparticles have excellent light stability and can maintain stable fluorescence output under long-term illumination.

[0113] 5.3 Selective Testing

[0114] In order to evaluate the UCNPs@mSiO 2 -RB-Bu nanoparticles selectivity for ATP, and designed a series of selectivity test experiments, which were set up with reference to the experiment in 1.6. Specifically, 10μM concentration of RB-Bu probe was used to test ATP, ADP and AMP at 100μM concentration, and other potential interfering substances, including Cu, were tested at 10mM concentration. 2+ 、SO 4 2- , Glutamate (Glu), HCO 4 - , Cl - , Glucose, CO 3 2- , Glycine (Gly), HPO 4 - 、Zn 2+ and Fe 2+ Equal volumes of each interfering substance solution were added to the pre-prepared UCNPs@mSiO 2 -RB-Bu nanoparticle solution was allowed to stand at room temperature for 30 minutes to ensure sufficient binding. Subsequently, a 980 nm near-infrared (NIR) laser (power density 2 W / cm 2 ) to excite the nanoparticles and record the changes in the fluorescence response of each component.

[0115] The experimental results are as follows Figure 3 As shown, UCNPs@mSiO 2 -RB-Bu nanoparticles have a significantly increased fluorescence intensity of about 70 times in the presence of ATP, which is significantly enhanced compared with other nucleotides and interfering substances. 2+ 、SO 4 2- , Glu, HCO 4 - , Cl - , glucose, CO 3 2- , Gly, HPO 4 - 、Zn 2+ and Fe 2+ The change in fluorescence ratio caused by the 10 mM concentration was very small, not exceeding 10 times.

[0116] 5.4 Cytotoxicity test

[0117] HEK-293 cells and astrocytes were cultured in modified Dulbecco's Eagle Medium (DMEM), with 100 mg / L puromycin, 10% fetal bovine serum and 100 U / mL penicillin added to the culture medium at 37 ° C and 5% CO2. HEK-293 / astrocytes were seeded into 96-well plates with approximately 10,000 cells per well. After 24 hours of cell culture, medium containing 0, 50, 100, 200, 400 and 600 μg / mL UCNP@mSiO2 / RB-Bu was added and incubated for 24 hours or 48 hours. Subsequently, 20 μL of 5 mg / mL MTT solution was added and incubated for another 4 hours. After discarding the culture medium, it was replaced with 150 μL of dimethyl sulfoxide (DMSO) solution, and the cell proliferation activity was calculated by the following formula: Cell activity (%) = (average A value of the treatment group / average A value of the control group) × 100%. All experiments were carried out in dark conditions.

[0118] The results showed that the prepared nanoparticles had little effect on cell proliferation and therefore had high safety.

[0119] 5.5 Reversibility test

[0120] In order to evaluate the reversible response ability of different ethylenediamine rhodamine probes (RB-EA, RB-Et, RB-Bu) and their nanoprobes in ATP detection, a series of experiments of cyclic addition and removal of ATP were designed. The specific experimental steps are as follows:

[0121] Referring to the experimental settings of 1.8, 10 μM concentration of NaYbF prepared in Example 3 was added to 4 :2%Er@NaYF 4 UCNPs@mSiO 2 -RB-Bu nanoparticles were prepared into a 10 mg / mL solution. The solutions were divided into sample pools and cyclic addition and removal of ATP was performed. In each cycle, 100 μM ATP was first added to the probe solution and incubated for 10 minutes to promote the binding of the RB probe and ATP, and the corresponding fluorescence response was recorded. Subsequently, an equal volume of 2M NaOH solution was added to neutralize the system, and ATP was degraded by adding apyrase (1U), the system was restored to the initial state, and the recovery of fluorescence intensity was recorded. The whole process was repeated three times to verify the reversible response characteristics of the probe.

[0122] The results are as follows Figure 4 As shown in the figure, the experimental results show that when the RB-Bu probe is integrated into the NaYbF 4 :2% Er@NaYF 4UCNPs@mSiO 2 After being placed in the nanocarrier, the nanoprobe showed a more excellent reversible response characteristic in the experiment of cyclic addition and removal of ATP. The fluorescence intensity can quickly and completely recover to the initial level in each cycle, and maintain a highly consistent response curve in multiple cycles, indicating that the nanocarrier effectively improves the reversibility and stability of the RB-Bu probe. These results show that the RB-Bu probe, especially with the assistance of the nanocarrier, has excellent reversible response ability and is suitable for long-term, repeated ATP dynamic monitoring applications.

[0123] 5.6 Dynamics test

[0124] Referring to the experimental settings of 1.8, 10 μM concentration of NaYbF prepared in Example 3 was added to 4 :2%Er@NaYF 4 UCNPs@mSiO 2 -RB-Bu nanoparticles were prepared into a 10 mg / mL solution. The solution was dispensed into sample pools and ATP was immediately added to the sample pools, with a final ATP concentration of 100 μM. Thereafter, the fluorescence spectrometer was used to record the NaYbF 4 :2% Er@NaYF 4 UCNPs@mSiO 2 The fluorescence intensity changes of -RB-Bu at different time points were recorded every second, ranging from 0 to 600 seconds.

[0125] The results are as follows Figure 5 As shown, the fluorescence intensity of the nanoparticles did not change much during the test, proving that they have good stability.

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing ATP detection nanoparticles based on upconversion luminescent materials, characterized in that The steps include: (1) Preparation of rough probe The rhodamine was dissolved in ethanol, and an amino compound was added, and the mixture was stirred and refluxed for reaction. After the reaction was completed, the solvent was removed by distillation under reduced pressure, and the mixture was redissolved in dichloromethane and washed. The solvent was removed by distillation under reduced pressure again to obtain a crude probe; (2) Purification of rhodamine probe The crude probe is redispersed with hydrochloric acid and stirred, a sodium hydroxide solution is added to precipitate, the aqueous phase is removed, the obtained precipitate is dispersed in dichloromethane, washed, and dried to obtain a rhodamine probe; (3) Preparation of UCNPs Yttrium acetate tetrahydrate, ytterbium acetate tetrahydrate and erbium acetate tetrahydrate are dissolved in octadecene, oleic acid and octadecene oleic acid are added, and water and oxygen are removed by heating under vacuum, and then reflux reaction is carried out under argon protection, and UCNPs are obtained by centrifugation, and UCNPs solution is obtained after redispersing in cyclohexane; (4) Mesoporous silica coating Dissolve CTAB in water, stir to react, add UCNPs solution, continue to stir to react, obtain CTAB-stabilized UCNPs aqueous solution, then add CTAB-stabilized UCNPs aqueous solution to alkaline mixed solution, heat and mix, then drop TEOS, continue to stir to react, then add ethanol to terminate the reaction, centrifuge, wash, and obtain mesoporous silicon nanoparticles; (5) Removal of template Dispersing mesoporous silicon nanoparticles in a hydrochloric acid ethanol solution, stirring and refluxing the solution, centrifuging the solution, and then repeating the dispersion, refluxing, and centrifuging to obtain mesoporous silica-coated upconversion nanoparticles; (6) Preparation of ATP detection nanoparticles based on upconversion luminescent materials The mesoporous silica-coated upconversion nanoparticles obtained in step (5) are dispersed in an ethanol solution containing the rhodamine probe prepared in step (2), stirred at room temperature, centrifuged after the reaction is completed, and washed to obtain ATP detection nanoparticles based on upconversion luminescent materials.

2. The preparation method according to claim 1, characterized in that: The molar ratio of rhodamine to the amino compound in step (1) is 3-5:80-100; The amino compound described in step (1) includes at least one of diethylenetriamine, ethylamine, butylamine, naphthalenediamine and methylbutylenediamine.

3. The preparation method according to claim 1, characterized in that: The stirring reflux reaction conditions of step (1) are 75-85° C. and 1000-1500 rpm for 20-28 hours; The concentration of the hydrochloric acid in step (2) is 0.8 to 1.2 M; The concentration of the sodium hydroxide solution in step (2) is 0.8-1.2M.

4. The preparation method according to claim 1, characterized in that: The molar ratio of yttrium acetate tetrahydrate, ytterbium acetate tetrahydrate and erbium acetate tetrahydrate in step (3) is 30-50:5-15:1-2; The volume ratio of oleic acid to octadecene oleic acid in step (3) is 1-2:4-6.

5. The preparation method according to claim 1, characterized in that: The reflux reaction in step (3) is carried out at 250-350° C. for 0.5-2 h; The stirring reaction conditions of step (4) are 65-75° C. and 700-800 rpm for 8-12 min; The conditions for continuing the stirring reaction in step (4) are 65-75° C. and 700-800 rpm for 3-5 hours; The heating in step (4) is heating to 65-75° C. The conditions for continuing the stirring reaction in step (4) are stirring the reaction at 65-75° C. and 700-800 rpm for 0.5-2 h.

6. The preparation method according to claim 1, characterized in that: The mass ratio of CTAB to UCNPs in the UCNPs solution in step (4) is 2 to 4:1; The volume ratio of the CTAB-stabilized UCNPs aqueous solution to the alkaline mixed solution in step (4) is 1-2:2-4; The ratio of the alkaline mixed solution in step (4) is water: ethanol: sodium hydroxide solution = 200: 30: 1.5, wherein the concentration of the sodium hydroxide solution is 2M; The volume ratio of the TEOS to CTAB-stabilized UCNPs aqueous solution in step (4) is 15-44:2000.

7. The preparation method according to claim 1, characterized in that: The pH of the hydrochloric acid ethanol solution in step (5) is 1.4 to 1.5; The stirring reflux reaction conditions of step (5) are 55-65° C. and 700-800 rpm for 2-4 hours; The number of repetitions in step (5) is 2 to 4 times; The stirring condition in step (6) is 20 to 30 hours; The washing in step (6) is washing with ethanol; The centrifugal condition is 7000-9000 rpm for 5-15 min.

8. The preparation method according to claim 1, characterized in that: The ratio of the mesoporous silica-coated upconversion nanoparticles to the rhodamine probe in step (6) is 10 to 30 μg.

9. An ATP detection nanoparticle based on up-conversion luminescent material, prepared according to any one of the preparation methods of claims 1 to 8.

10. Use of the ATP detection nanoparticles based on upconversion luminescent materials according to claim 9 in detecting ATP concentration.

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