A ratiometric fluorescent nanothermometer, preparation method and application thereof

Abstract

The application discloses a ratio type fluorescent nanometer thermometer and a preparation method and application, and is prepared as follows: a polybasic saturated fatty acid is heated and melted into a liquid state, and after cooling, a solid is formed; the solid is ground into powder; the powder is heated and melted into a liquid to obtain A liquid; ACQ fluorescent molecules and an AIE molecule with an aggregation-induced emission property are dispersed in the A liquid, and after cooling, a solid is obtained; the solid is ground into powder to obtain powder B; the powder B is dissolved in dimethyl sulfoxide to prepare a B solution; the B solution is added dropwise into an aqueous solution of a non-intercalated surfactant; after the dropwise addition is completed, stirring is continued for 20-40 min; and excess non-intercalated surfactant is removed by dialysis; the thermometer has a temperature detection range of 25-48 DEG C, effectively covering the physiological temperature of 37 DEG C; the thermometer has excellent sensitivity and accuracy, and the maximum relative thermal sensitivity thereof in a water environment is as high as 63.66% / DEG C, which is much higher than that of a previously reported ratio type fluorescent nanometer thermometer.

Description

Technical Field

[0001] This invention belongs to the field of biological detection and relates to a ratiometric fluorescent nanothermometer, its preparation method, and its application. Background Technology

[0002] Temperature is one of the most fundamental and important physiological parameters of living organisms, playing a crucial role in regulating various biochemical processes, such as gene expression, signal transduction, and thermogenesis in brown adipose tissue. Abnormal temperatures are often associated with a series of pathological changes; for example, the inflammation and tumor microenvironment typically exhibit higher temperatures. Therefore, achieving highly sensitive and accurate temperature detection at the subcellular level is of great significance for understanding and revealing various life processes. Among various temperature detection systems, fluorescent nanothermometers have attracted attention due to their advantages such as high sensitivity, good spatiotemporal resolution, non-invasiveness, and real-time response. Examples include fluorescent proteins, organic molecular compounds, thermoresponsive polymers, quantum dots, lanthanide ion-doped nanoparticles, and vacancy-containing nanodiamonds. Organic / polymer nanothermometers, in particular, have good biocompatibility and have thus been widely developed. In temperature detection systems, temperature changes are usually quantitatively correlated with changes in one or more fluorescence parameters, thereby indirectly reflecting subtle temperature variations, such as peak position, emission intensity, fluorescence lifetime, emission intensity ratio, fluorescence polarization anisotropy, electron spin resonance, or optical detection magnetic resonance. However, accurately detecting minute changes in intracellular temperature remains a challenge. Therefore, developing a novel fluorescent nanothermometer for ultrasensitive localization and detection of intracellular temperature is of great significance.

[0003] Compared to signal readout methods that rely solely on a single fluorescence parameter, ratiometric measurement methods inherently offer advantages such as signal self-calibration and high signal-to-background ratio, enabling ultrasensitive and more reliable temperature detection. Currently, there are two main design principles for ratiometric detectors: one involves introducing two independent structures into a single nanoparticle, each generating a temperature-dependent signal and a temperature-independent signal (i.e., a reference signal); the other integrates two related structures into a single nanoparticle, both generating temperature-dependent signals with opposite signal changes. These opposite signal changes amplify the net difference between signals, which helps improve detector sensitivity. However, the temperature detection range and sensitivity of current ratiometric fluorescent nanothermometers still require further improvement.

[0004] Phase transition materials (PCMs) are a novel class of thermoresponsive materials, typically possessing well-defined molecular structures, narrow melting point ranges, and reversible solid-liquid phase transition properties. Among various PCMs, naturally derived saturated fatty acids have attracted researchers' attention due to their low cost, good biocompatibility, and biodegradability. Furthermore, the high crystallinity of saturated fatty acids allows them to load various fluorescent molecules and to reversibly control the aggregation (in solid PCMs) and dispersion (in molten PCMs) states of fluorophores. Organic fluorescent molecules with aggregation-induced quenching (ACQ) properties exhibit strong fluorescence in dissolved or dispersed states; however, in aggregated states, the fluorescence signal weakens or even disappears due to intermolecular π-π interactions. Conversely, organic fluorescent molecules with aggregation-induced emission (AIE) properties exhibit weak or no emission in dilute solutions; however, in aggregated and solid states, they emit strong fluorescence due to restricted intramolecular motion. Currently, there are no reports on constructing ratiometric fluorescent nanothermometers by controlling the luminescence behavior of ACQ and AIE fluorescent molecules using PCMs for ultrasensitive intracellular temperature detection. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide an AIE molecule with aggregation-induced emission properties.

[0006] The second objective of this invention is to provide a method for preparing AIE molecules with aggregation-induced emission properties.

[0007] The third objective of this invention is to provide a ratiometric fluorescent nanothermometer.

[0008] The fourth objective of this invention is to provide a method for preparing a ratiometric fluorescent nanothermometer.

[0009] The fifth objective of this invention is to provide an application of a ratiometric fluorescent nanothermometer in intracellular temperature detection.

[0010] The technical solution of this invention is summarized as follows:

[0011] An AIE molecule with aggregation-induced emission properties has the structure shown in Formula V;

[0012]

[0013] Where R1 is -(CH2) n CH3, n = 3-11.

[0014] The method for preparing the above-mentioned AIE molecule with aggregation-induced emission properties includes the following steps:

[0015] 1) Place zinc powder in a reaction vessel, evacuate and purge with dry nitrogen, add dry tetrahydrofuran as solvent, cool to -78℃, add titanium tetrachloride dropwise, heat the reaction system under reflux for 2-4 hours, add pyridine, dry tetrahydrofuran solution of compound I and compound II, heat under reflux, monitor by thin-layer chromatography, and obtain compound III;

[0016] Preferably, the molar ratio of zinc powder, titanium tetrachloride, pyridine, compound I, and compound II is 5:2.6:(1-1.5):1.3:1;

[0017] 2) Place compound III in a reaction vessel, evacuate and purge with dry nitrogen, add dry dichloromethane as solvent, cool to -78°C, add boron tribromide dichloromethane solution dropwise, raise the reaction system to room temperature, stir, monitor by thin-layer chromatography, and obtain compound IV;

[0018] Preferably, the molar ratio of compound III to boron tribromide is 1:(3-5);

[0019] 3) Compound IV and cesium carbonate were placed in a reaction vessel, evacuated and purged with dry nitrogen, R1I was added, N,N-dimethylformamide was added as solvent, stirred at room temperature, and monitored by thin-layer chromatography to obtain an AIE molecule (V) with aggregation-induced emission properties.

[0020] Preferably, the molar ratio of compound IV, cesium carbonate and R1 I is 1:(3-5):4;

[0021] The reaction formula is as follows:

[0022]

[0023] Where R1 is -(CH2) n CH3, n = 3-11;

[0024] A method for preparing a ratiometric fluorescent nanothermometer includes the following steps:

[0025] 1) Heat polysaturated fatty acids to melt them into a liquid state, cool them down to solid state, grind them into powder, and then heat the powder to melt it into a liquid state to obtain liquid A;

[0026] 2) Disperse ACQ fluorescent molecules and the aforementioned AIE molecule with aggregation-induced emission properties in liquid A, cool to obtain a solid, grind into powder, and obtain powder B; dissolve powder B in dimethyl sulfoxide to prepare solution B; the mass ratio of the ACQ fluorescent molecules, the AIE molecules and liquid A is (0.1-1):(0.5-5):100; dissolve the non-intercalating surfactant in water to prepare aqueous solution C;

[0027] 3) The solution B was uniformly added dropwise to the stirred aqueous solution C at 0-4℃ using a syringe pump. After the addition was complete, stirring was continued for 20-40 minutes. Excess non-intercalated surfactant was removed by dialysis to prepare a ratio-type fluorescent nanothermometer.

[0028] The mass ratio of powder B to non-embedded surfactant is 1:(5-20);

[0029] The polysaturated fatty acids are selected from at least two of decaic acid, lauric acid, myristic acid, palmitic acid, and stearic acid.

[0030] The preferred ACQ fluorescent molecules are Nile Red, Cyanide Cy5, or Rhodamine B.

[0031] Non-embedded surfactants are preferably polyvinyl alcohol, polystyrene, or polyethylene glycol diacrylamide.

[0032] A ratiometric fluorescent nanothermometer was prepared using the above method.

[0033] The above-mentioned ratiometric fluorescent nanothermometer is used for intracellular temperature detection.

[0034] Beneficial effects:

[0035] (1) The temperature detection range of the ratio fluorescent nano thermometer of the present invention is 25-48℃, which effectively covers the physiological temperature range (37℃); it has excellent sensitivity and accuracy, and its maximum relative thermal sensitivity in the water environment is as high as 63.66% / ℃, which is much higher than the ratio fluorescent nano thermometers previously reported.

[0036] (2) The ratio-type fluorescent nano thermometer of the present invention is mainly prepared by nanoprecipitation method based on non-embedded surfactant. Non-embedded surfactant can improve the colloidal stability of nanoparticles and maximize the ability of poly-saturated fatty acids to undergo multiple phase transitions. The synthesis steps are simple, the yield is high, and it is easy to purify.

[0037] (3) The ratiometric fluorescent nanothermometer of the present invention exhibits excellent sensitivity and accuracy in intracellular temperature detection, with a maximum relative thermal sensitivity of up to 44.01% / ℃, which is also higher than the maximum relative thermal sensitivity of previously reported ratiometric fluorescent nanothermometers in intracellular temperature detection. In addition, the ratiometric fluorescent nanothermometer can also detect minute changes in intracellular temperature under chemical stimulation with ultrasensitivity. Attached Figure Description

[0038] Figure 1 The fluorescence properties of the AIE molecule with aggregation-induced emission properties synthesized in Example 3 and the fluorescence properties of the Nile Red ACQ molecule are characterized.

[0039] (A) Fluorescence emission spectra of AIE molecules in different water contents.

[0040] (B) A graph showing the fluorescence intensity of AIE molecules at 515 nm as a function of water content.

[0041] (C) Normalized absorption and emission of AIE molecules (solid line) and normalized absorption and emission of Nile Red dye (dashed line).

[0042] Figure 2 The phase transition properties of standard and non-standard dibasic saturated fatty acids and the temperature detection range of the constructed nanothermometer were tested.

[0043] (A) Schematic diagram of the solid-liquid phase of a binary saturated fatty acid.

[0044] (B) Differential scanning calorimetry curves of standard (black) and non-standard (gray) binary saturated fatty acids.

[0045] (C) Schematic diagram of the preparation and temperature detection mechanism of the fluorescent nano thermometer.

[0046] (D) Fluorescence spectrum of a nano thermometer based on standard dibasic fatty acids (Example 5) as a function of temperature.

[0047] (E) Fluorescence spectrum of a nano thermometer based on non-standard dibasic fatty acids (Example 4) as a function of temperature.

[0048] Figure 3 This describes the morphology, particle size, and temperature detection performance of a ratiometric fluorescent nanothermometer prepared in Example 4.

[0049] (A) Ultraviolet absorption spectrum of a ratiometric fluorescent nanothermometer.

[0050] (B) Transmission electron microscope image of the morphology of a ratiometric fluorescent nanothermometer.

[0051] (C) Particle size distribution of a ratiometric fluorescent nanothermometer.

[0052] (D) Fluorescence spectrum of ratiometric fluorescent nanothermometer as a function of temperature.

[0053] (E) A graph showing the ratio of fluorescence emission intensity at 500 nm to 615 nm as a function of temperature for a ratiometric fluorescent nanothermometer.

[0054] (F) Graph showing the fluorescence emission intensity ratio of a ratiometric fluorescent nanothermometer changing repeatedly between 25-48℃.

[0055] Figure 4 This is a ratiometric fluorescent nanothermometer prepared in Example 4 used for temperature detection within cells.

[0056] (A) CLSM images of the AIE and ACQ channels of a ratiometric fluorescent nanothermometer at different temperatures.

[0057] (B) Average fluorescence intensity data output of the AIE and ACQ channels of the ratiometric fluorescent nanothermometer at different temperatures.

[0058] (C) Temperature calibration curve of ratiometric fluorescent nanothermometer in cells.

[0059] (D) Changes in intracellular temperature under the stimulation of calcium iontophoresis.

[0060] (E) Changes in intracellular temperature under CCCP stimulation. Detailed Implementation

[0061] The present invention will be further described below through specific embodiments.

[0062] Example 1:

[0063] Synthesis of Compound III

[0064] Zinc powder (1.58 g, 25 mmol) was placed in a 250 mL three-necked round-bottom flask equipped with a reflux condenser. The flask was evacuated and purged with nitrogen, and the mixture was circulated three times. Then, 20 mL of dry THF was added as a solvent. The mixture was cooled to -78 °C, and TiCl4 (1.45 mL, 13 mmol) was added dropwise. After the addition was complete, the mixture was heated under reflux for 2 h (or 3 h or 4 h). Pyridine (0.6 mL, 7.5 mmol, or 5 mmol or 6 mmol) was added. After stirring for 10 min, an ultra-dry THF solution (10 mL of THF) of 4,4'-bis(N,N-dimethylamino)benzophenone (compound I) (1.75 g, 6.5 mmol) and 4-methoxybenzophenone (compound II) (1.1 g, 5 mmol) was added, and the mixture was heated under reflux.

[0065] Thin-layer chromatography was used for monitoring. After the reaction was complete, the reaction was quenched with a 10 wt% aqueous solution of K₂CO₃, and a large amount of water was added until the solid in the solution turned gray. The mixture was extracted three times with DCM, and the organic phases were combined and washed twice with saturated brine. The crude product was evaporated under reduced pressure and then purified by silica gel column chromatography (ethyl acetate / petroleum ether = 1:20, v / v) to give a yellow solid, which was compound III, in 36% yield.

[0066] 1H NMR (400MHz, CDCl3) δ 7.16-6.98 (m, 6H), 6.99-6.81 (m, 6H), 6.69-6.61 (m, 2H), 6.54-6.37 (m, 4H), 3.74 (s, 3H), 2.88 (d, J = 7.0Hz, 12H). 13 C NMR (100MHz, CDCl3) δ157.52,148.79,145.60,140.49,137.83,136.59,132.61, 131.68,129.53,127.64,125.56,113.11,111.49,55.15,40.52.HRMS(ESI,m / z,C 31 H 32 N2O,[M+H + ]):calcd,449.2575; found,449.2589.

[0067] Example 2:

[0068] Synthesis of Compound IV

[0069] Compound III (0.45 g, 1 mmol) was placed in a 25 mL round-bottom reaction flask, evacuated, and purged with nitrogen three times. Then, 10 mL of dry dichloromethane was added as a solvent. The mixture was cooled to -78 °C, and a dichloromethane solution of BBr3 (4 mL, containing 4 mmol of BBr3, or 3 mmol or 5 mmol could be used) was added dropwise. After the addition was complete, the mixture was brought back to room temperature and stirred for 12 h. Thin-layer chromatography was used for monitoring. After the reaction was complete, the reaction was quenched with ice water, and the mixture was extracted three times with DCM. The organic phase was dried over anhydrous Na2SO4. The crude product was purified by silica gel column chromatography, eluting with a mixture of petroleum ether and ethyl acetate (6:1 v / v) to give compound IV in 42% yield.

[0070] 1 H NMR (400MHz, CDCl3) δ7.13-6.96(m,5H),6.96-6.82(m,6H),6.58-6.40(m,6H),4.64(s,1H),2.88(d,J=6.4Hz,12H). 13 C NMR (100MHz, CDCl3) δ153.69,148.77,145.43,140.31,137.63,136.80,133. 18,132.82,132.57,131.62,127.62,125.60,114.71,111.98,111.78,40.71. HRMS(ESI,m / z,C 30 H30 N2O,[M+H + ]):calcd,435.2418; found,435.2435.

[0071] Example 3:

[0072] Synthesis of compound V-1

[0073] Compound IV (87 mg, 0.2 mmol) and cesium carbonate (260 mg, 0.8 mmol, or 0.6 mmol or 1.0 mmol could be added) were added to a Schlenk reaction tube, which was then evacuated under nitrogen and circulated three times. 1-Iodohexane (170 μL, 0.8 mmol) and N,N-dimethylformamide (DMF, 3 mL) were then added, and the mixture was stirred at room temperature for 12 h. Thin-layer chromatography was used for monitoring. After the reaction was complete, the reaction was quenched with water, extracted three times with DCM, and the organic phase was dried over anhydrous Na₂SO₄. The crude product was purified by silica gel column chromatography, eluted with a mixture of petroleum ether and ethyl acetate (10:1 v / v) to give AIE molecules with aggregation-induced emission properties, V⁻¹, in 73% yield.

[0074] 1 H NMR (400MHz, CDCl3) δ7.11-7.05(m,6H),6.92-6.86(m,6H),6.63(d,J=8.3Hz,2H),6.48-6.43(m,4H ),3.88(m,2H),2.89(s,12H),1.73(m,2H),1.44-1.41(m,2H),1.38-1.28(m,4H),0.93-0.86(m,3H). 13 C NMR (100MHz, CDCl3) δ157.06,148.68,145.55,140.27,137.53,132.57,132.53,131.62,127.54,1 25.47,113.60,111.56,111.46,67.83,40.49,31.69,29.39,25.82,22.66,14.10.HRMS(ESI,m / z,C 36 H 42 N2O,[M+H + ]):calcd,519.3357; found,519.3370.

[0075] The reaction formula is as follows:

[0076]

[0077] The 1-iodohexane of this example was replaced with 1-iodobutane, 1-iodopentane, 1-iodoheptane, 1-iodooctane, 1-iodononane; 1-iododecane, 1-iodoundecane, and 1-iodododecane, and otherwise prepared in the same manner as in this example.

[0078] Compound V-2 (R1 is -(CH2)3CH3);

[0079] Compound V-3 (R1 is -(CH2)4CH3);

[0080] Compound V-4 (R1 is -(CH2)6CH3);

[0081] Compound V-5 (R1 is -(CH2)7CH3);

[0082] Compound V-6 (R1 is -(CH2)8CH3);

[0083] Compound V-7 (R1 is -(CH2)9CH3);

[0084] Compound V-8 (R1 is -(CH2)) 10 CH3);

[0085] Compound V-9 (R1 is -(CH2)) 11 CH3).

[0086] Example 4:

[0087] A method for preparing a ratiometric fluorescent nanothermometer (named AIE / ACQ@PVANPs) includes the following steps:

[0088] 1) Lauric acid (LA) and palmitic acid (PA) are heated and melted into a liquid state at a mass ratio of 55:45. After cooling, they become solid non-standard di-saturated fatty acids, which are then ground into powder and heated and melted into a liquid state to obtain liquid A1.

[0089] 2) Disperse Nile Red molecules and AIE molecules V-1 with aggregation-induced emission properties in liquid A1 at a mass ratio of 0.6:1:100, cool to obtain a solid, grind into powder to obtain powder B1; dissolve powder B1 in dimethyl sulfoxide to prepare B1 solution; dissolve non-intercalating surfactant polyvinyl alcohol (weight average molecular weight of 120,000) in water to prepare C1 aqueous solution (PVA aqueous solution);

[0090] 3) According to the mass ratio of powder B1 to PVA of 1:10, the B solution was uniformly added dropwise to the stirred PVA aqueous solution at 0℃ using a syringe pump. After the addition was completed, stirring was continued for 30 minutes. Excess PVA was removed by dialysis to prepare the ratio-type fluorescent nano thermometer 1.

[0091] Example 5: (Comparative Example)

[0092] A method for preparing a ratiometric fluorescent nanothermometer (using a standard dibasic fatty acid to prepare liquid A) includes the following steps:

[0093] 1) Lauric acid (LA) and palmitic acid (PA) were heated and melted into a liquid at a mass ratio of 77.5:22.5. After cooling, they became solid standard di-saturated fatty acids, which were ground into powder and then heated and melted into a liquid to obtain control liquid A.

[0094] 2) Disperse Nile Red molecules and AIE molecules V-1 with aggregation-induced emission properties in control liquid A at a mass ratio of 0.6:1:100, cool to obtain a solid, grind into powder to obtain control powder B; dissolve control powder B in dimethyl sulfoxide to prepare control B solution; dissolve non-intercalating surfactant polyvinyl alcohol (weight average molecular weight of 120,000) in water to prepare C1 aqueous solution (PVA aqueous solution);

[0095] 3) According to the mass ratio of powder B control to PVA of 1:10, the B control solution was uniformly added dropwise to the stirred PVA aqueous solution at 0℃ using a syringe pump. After the addition was completed, stirring was continued for 30 minutes. Excess PVA was removed by dialysis to prepare the ratio-type fluorescent nano thermometer control.

[0096] Note: Lauric acid and palmitic acid in a mass ratio of 77.5:22.5 are standard dibasic saturated fatty acids.

[0097] Example 6:

[0098] A method for preparing a ratiometric fluorescent nanothermometer includes the following steps:

[0099] 1) Non-standard di-saturated fatty acids, such as deca-acid (CA) and myristic acid (MA) in a mass ratio of 30:70, are heated and melted into a liquid state. After cooling, they become solid. They are ground into powder and then heated and melted into a liquid state to obtain liquid A2.

[0100] 2) Disperse cyanine Cy5 molecules and AIE molecules (compound V-2) with aggregation-induced emission properties in liquid A2 at a ratio of 0.1:0.5:100, cool to obtain a solid, grind into powder to obtain powder B2; dissolve powder B2 in dimethyl sulfoxide to prepare B2 solution; dissolve non-intercalating surfactant polystyrene (weight average molecular weight of 180,000) in water to prepare C2 aqueous solution (PS aqueous solution);

[0101] 3) According to the mass ratio of powder B2 to PS of 1:5, the B2 solution was uniformly added dropwise to the PS aqueous solution at 4℃ and stirred by a syringe pump. After the addition was completed, stirring was continued for 40 min. Excess PS was removed by dialysis to prepare the ratio-type fluorescent nano thermometer 2.

[0102] Example 7:

[0103] A method for preparing a ratiometric fluorescent nanothermometer includes the following steps:

[0104] 1) Lauric acid (LA), palmitic acid (PA) and stearic acid (SA) are heated and melted into a liquid state in a mass ratio of 65:25:10. After cooling, they become solid non-standard ternary saturated fatty acids, which are then ground into powder and heated and melted into a liquid state to obtain liquid A3.

[0105] 2) Disperse Rhodamine B molecules and AIE molecules (compound V-9) with aggregation-induced emission properties in liquid A3 at a ratio of 1:5:100, cool to obtain a solid, grind into powder to obtain powder B3; dissolve powder B3 in dimethyl sulfoxide to prepare B3 solution; dissolve non-intercalating surfactant polyethylene glycol diacrylamide (weight average molecular weight of 10,000) in water to prepare C3 aqueous solution (PEG-ACA aqueous solution);

[0106] 3) According to the mass ratio of powder B3 to PEG-ACA of 1:20, the B3 solution was uniformly added dropwise to the stirred PEG-ACA aqueous solution at 2℃ using a syringe pump. After the addition was completed, the mixture was stirred for 20 min. The excess PEG-ACA was removed by dialysis to prepare the ratio-type fluorescent nano thermometer 3.

[0107] Using compounds V-3 (R1 is -(CH2)4CH3); compound V-4 (R1 is -(CH2)6CH3); compound V-5 (R1 is -(CH2)7CH3); compound V-6 (R1 is -(CH2)8CH3); compound V-7 (R1 is -(CH2)9CH3); compound V-8 (R1 is -(CH2) 10 By replacing compound V-9 of this embodiment with CH3, and otherwise the same as in this embodiment, the following can be obtained in sequence: ratio-type fluorescent nanothermometer 4, ratio-type fluorescent nanothermometer 5, ratio-type fluorescent nanothermometer 6, ratio-type fluorescent nanothermometer 7, ratio-type fluorescent nanothermometer 8, and ratio-type fluorescent nanothermometer 9.

[0108] Example 8:

[0109] Example 3 synthesized an aggregation-induced emission (AIE) molecule (TPE-Al, compound V-1) exhibiting typical AIE phenomena, such as... Figure 1As shown in Figure A (fluorescence emission spectra of AIE molecules at different water contents), at different water contents (f w In a DMSO / H2O mixture, when f w When the concentration is ≥80%, the fluorescence intensity is significantly enhanced. Figure 1 B shows the fluorescence intensity of the AIE molecule at 515 nm as a function of water content, further confirming the increase in fluorescence intensity. Nile red (NR) was selected as the ACQ molecule. Figure 1 The normalized absorption and emission of AIE molecules (solid line) and Nile Red dye (dashed line) are shown. The maximum absorption and maximum emission in water for TPE-Al are 385 nm and 515 nm, respectively, while the maximum absorption and maximum emission in DMSO for NR are 550 nm and 630 nm, respectively. Both molecules have broad absorption spectra, and fluorescence from both molecules can be simultaneously excited at 405 nm. Therefore, it is possible to simultaneously excite the fluorescence spectra of AIE and ACQ molecules under the same excitation (Ex = 405 nm), thereby improving the sensitivity and accuracy of ratiometric detection.

[0110] from Figure 2 As shown in the schematic diagram of the solid-liquid phases of the binary saturated fatty acids in Figure A, it can be observed that, compared to the standard binary eutectic mixture which only has one phase transition, the non-standard binary eutectic mixture has two phase transition points: the eutectic point and a point on the liquidus line. The solid non-standard binary eutectic mixture undergoes three main stages during heating: complete solidification (solid A + B), partial melting (solid A + liquid), and complete melting (liquid A + B), with a significantly expanded phase transition temperature range. To verify this hypothesis, differential scanning calorimetry (DSC) curves were measured for the standard eutectic mixture of LA and PA (see step 1 of Example 5) and the non-standard eutectic mixture (see step 1 of Example 4), as shown below. Figure 2 As shown in Figure B, the standard eutectic mixture exhibits only one sharp exothermic peak, while the non-standard eutectic mixture exhibits multiple exothermic peaks, indicating that the strategy of using non-standard eutectic mixtures to expand the phase transition temperature range is feasible. To further prove this hypothesis, a ratiometric fluorescent nanothermometer was constructed, and its fabrication and temperature detection mechanism are illustrated in the following diagram. Figure 2 As shown in Figure C. Subsequently, the temperature detection range of the nanothermometers prepared in Examples 4 and 5 was compared. It was found that the AIE fluorescence and ACQ fluorescence of the two thermometers showed completely opposite trends with increasing temperature. The AIE fluorescence gradually decreased with increasing temperature, while the ACQ fluorescence gradually increased with increasing temperature. However, in the nanothermometer prepared in Example 5, this change only lasted until 40°C. Figure 2 D), while the temperature detection range of the nano thermometer prepared in Example 4 can be extended to 48°C ( Figure 2E). Therefore, nano thermometers prepared using non-standard eutectic saturated fatty acids can broaden the temperature detection range.

[0111] Next, the AIE / ACQ@PVANPs synthesized in Example 4 were characterized in a series of ways. First, the prepared nanoparticles were broken down with DMSO, and the dissolved AIE and ACQ molecules were measured by UV absorption spectroscopy. Figure 3 As shown in Figure A, absorption peaks of the two molecules were observed, indicating that the two fluorescent molecules were successfully loaded into the nanoparticles. The encapsulation efficiencies of AIE and ACQ molecules were 40.38% and 37.50%, respectively, with corresponding loading amounts of 0.10% and 0.07%. Transmission electron microscopy revealed that AIE / ACQ@PVANPs exhibited a spherical structure with an average diameter of approximately 120 nm. Figure 3 B); Dynamic light scattering revealed that the hydrated diameter of the nanoparticles ranged from 90 to 400 nm, with an average diameter of approximately 140 nm. Figure 3 C). We then further tested the temperature detection capability of AIE / ACQ@PVANP by narrowing the temperature range, such as... Figure 3 As shown in Figure D, as the temperature increases from 25℃ to 48℃, the fluorescence intensity of the AIE molecule gradually decreases, while the fluorescence intensity of the ACQ molecule gradually increases. Furthermore, the fluorescence emission intensity ratio of the AIE molecule to the ACQ molecule drops sharply from 1.67 to 0.08, with a net difference of approximately 20 times. Figure 3 E). We further calculated the relative thermal sensitivity and temperature resolution of the ratiometric fluorescent nanothermometer and found that the maximum relative thermal sensitivity of the ratiometric fluorescent nanothermometer was 63.66% °C. -1 The corresponding temperature resolution was 0.02℃ (37℃), significantly higher than previously reported organic fluorescent nanothermometers. To test the reversibility of AIE / ACQ@PVANPs during repeated heating and cooling, we monitored the fluorescence intensity ratio of AIE / ACQ@PVANPs at 25℃ and 48℃ for 5 cycles. Figure 3 As shown in Figure F, the temperature detection performance of the nanoparticles was almost unaffected, indicating that AIE / ACQ@PVANPs possess excellent cycling stability and reversibility. These results demonstrate that AIE / ACQ@PVANPs exhibit a reversible and ultrasensitive signal response to temperature changes.

[0112] To verify the feasibility of using AIE / ACQ@PVANPs for intracellular temperature detection, we incubated AIE / ACQ@PVANPs with cells, and then collected and quantitatively analyzed fluorescence signals at different temperatures using laser confocal scanning microscopy (CLSM). Figure 4As shown in Figures A and 4B, when the ambient temperature is set to 25°C, the AIE signal dominates the overall acquired signal. When the temperature rises to 37°C, the AIE signal begins to decrease, while the ACQ signal increases, reaching almost the same level as the AIE signal. When the temperature is further increased to 42°C, the inverse signal change becomes more pronounced, with the ACQ signal becoming dominant. This demonstrates the potential of AIE / ACQ@PVA NPs as fluorescent nanothermometers for intracellular temperature detection.

[0113] To achieve accurate intracellular temperature detection, we collected CLSM images at different temperatures, established a calibration curve for intracellular temperature, and correlated the fluorescence emission intensity ratio with temperature. The emission intensity ratio (IL) was calculated by extracting the average emission intensities of the AIE and ACQ channels. AIE / I ACQ ), and use this to plot the calibration curve ( Figure 4 C). Based on the calibration curve, we calculated the maximum relative thermal sensitivity of AIE / ACQ@PVA NPs in cells to be 44.01%℃. -1 The corresponding temperature resolution is 0.03℃ (40℃). Due to the excellent intracellular temperature detection performance of AIE / ACQ@PVA NPs, they can be used to monitor changes in intracellular temperature under external chemical stimuli. It has been reported that Ca... 2+ It can promote ion pumping, accelerate respiratory reactions, and lead to an increase in intracellular temperature. Calcium ionomycin can directly stimulate the calcium pool, regulate calcium influx, and increase intracellular calcium levels. 2+ Content. We quantified the changes in intracellular temperature before and after stimulation with calcium ionomycin using an intracellular temperature calibration curve, such as... Figure 4 As shown in Figure D, at 10 min, the intracellular temperature of the cells increased by approximately 1 °C after stimulation, and this temperature change persisted until 25 min. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP), an uncoupling agent for mitochondrial oxidative phosphorylation, also stimulated an increase in intracellular temperature. Quantitatively based on the calibration curve, after CCCP stimulation, the intracellular temperature sustained an increase of 1.07 °C over 25 min. Figure 4 E). Therefore, AIE / ACQ@PVA NPs exhibit strong detection stability in physiological environments, enabling the monitoring of minute temperature changes under chemical stimulation. In summary, a ratiometric fluorescent nanothermometer can serve as an ultrasensitive analytical tool to detect various thermal behaviors at the subcellular level, showing great promise in revealing complex physiological processes in biological systems.

[0114] Experiments have shown that,

[0115] Any one of the ratiometric fluorescent nanothermometers 2 to 9 has the ability to detect extracellular and intracellular temperatures similar to that of the ratiometric fluorescent nanothermometer 1.

Claims

1. A method for preparing a ratiometric fluorescent nanothermometer, characterized in that: Includes the following steps: 1) Polysaturated fatty acids are heated and melted into a liquid state, cooled to become a solid state, ground into powder, and then the powder is heated and melted into a liquid state to obtain liquid A; 2) Disperse ACQ fluorescent molecules and an AIE molecule with aggregation-induced emission properties in liquid A, cool to obtain a solid, grind into powder, and obtain powder B; dissolve powder B in dimethyl sulfoxide to prepare solution B; the mass ratio of the ACQ fluorescent molecules, the AIE molecules and liquid A is (0.1-1):(0.5-5):100; dissolve a non-intercalating surfactant in water to prepare aqueous solution C; The aforementioned AIE molecule with aggregation-induced emission properties has the structure shown in formula (V); （V）； Wherein, R1 is -(CH2). n CH3, n = 3-11; 3) The solution B was uniformly added dropwise to the stirred aqueous solution C at 0-4 °C using a syringe pump. After the addition was complete, stirring was continued for 20-40 min. Excess non-intercalated surfactant was removed by dialysis to prepare a ratio-type fluorescent nanothermometer. The mass ratio of powder B to non-embedded surfactant is 1:(5-20). The polysaturated fatty acids are selected from at least two of decaic acid, lauric acid, myristic acid, palmitic acid, and stearic acid.

2. The preparation method according to claim 1, characterized in that: The ACQ fluorescent molecule is Nile Red, Cyanide Cy5, or Rhodamine B.

3. The preparation method according to claim 1, characterized in that: The non-embedded surfactant is polyvinyl alcohol, polystyrene, or polyethylene glycol diacrylamide.

4. A ratiometric fluorescent nanothermometer prepared by the preparation method according to any one of claims 1-3.

5. The application of a ratiometric fluorescent nanothermometer of claim 4 in intracellular temperature detection.

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