High-sensitivity ellipsoidal rhodamine B-gelatin fluorescent nanometer thermometer and preparation method thereof

Abstract

The preparation method comprises the following steps: firstly, dissolving gelatin in deionized water to form a gelatin solution, adding rhodamine B and a catalyst, and carrying out amidation reaction to chemically bond amino groups of the gelatin with carboxyl groups of the rhodamine B; then adding a cross-linking agent to cross-link the rhodamine B-gelatin conjugate into nano particles, and enabling the rhodamine B-gelatin fluorescent nano thermometer to be ellipsoidal by utilizing centrifugal force formed by high-speed stirring in the cross-linking process. The rhodamine B is firmly loaded on the gelatin nanoparticles, the loading capacity of the rhodamine B is greatly increased, the residence time of the rhodamine B in an organism is prolonged, the photobleaching effect of the rhodamine B is overcome, the rhodamine B-gelatin fluorescent nanoparticles are prepared into ellipsoids through uneven stress under the action of centrifugal force when aggregated particles are formed, and the rhodamine B-gelatin fluorescent nanoparticles are prepared into the ellipsoids. The preparation method is simple, the temperature measurement sensitivity is high, the photobleaching phenomenon of rhodamine B is effectively weakened, and the biomedical utilization prospect is good.

Description

Technical Field

[0001] The present invention relates to the technical field of nanothermometers, and particularly relates to a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer and a preparation method thereof. Background Art

[0002] Temperature is one of the most fundamental physical quantities, and temperature measurement is an indispensable technology in industrial production, daily life and scientific research. Traditional temperature measurement techniques mainly include two methods: contact type and non-contact type. Contact temperature measurement mainly determines the temperature by directly contacting the temperature detector with the object to be measured so that heat exchange occurs between the two. Common contact temperature measurement techniques include thermocouples, thermal resistors, glass liquid thermometers, bimetallic thermometers, etc. Non-contact temperature measurement techniques do not require direct contact with the object to be measured, but determine its temperature by measuring the radiation energy emitted by the object. Common non-contact temperature measurement techniques include infrared thermometry, laser thermometry, microwave thermometry, fiber optic thermometry, etc. Each of the above temperature measurement methods has its own characteristics and is suitable for different usage scenarios, but they are all difficult to measure the temperature changes in micro-areas, such as the organs of living organisms and the temperature changes in the micro-regions inside cells, and these changes have important effects on the growth and development of living organisms. Therefore, as nanoparticles containing temperature-sensitive materials, nanothermometers have become an important tool for measuring the temperature changes in micro-areas of living organisms.

[0003] Current nanothermometers mainly measure temperature changes at the micro- and nano-scales based on different principles such as thermal reflection, Raman light, optical interference, or fluorescence. Among them, fluorescence temperature sensing has become an important method in current biomedical research. For example, Zhu Chunlei et al. constructed a ratiometric fluorescence nanothermometer by using phase change materials to regulate the aggregation-caused quenching (ACQ) and aggregation-induced emission (AIE) behaviors of fluorescent molecules and applied it to intracellular temperature detection (Publication No.: CN118063335A). There are also Zhou Hong et al. who invented a doped rare-earth fluorescence nanothermometer that can be used for temperature detection in microscopic environments (Publication No.: CN105092079A). Existing fluorescence nanothermometers mainly consist of quantum dots, inorganic nanomaterials, and organic fluorescent dyes. Quantum dot nanothermometers have high sensitivity and low usage, but usually have strong cytotoxicity and poor biocompatibility. Inorganic nanothermometers have good reproducibility, but low sensitivity, strong cytotoxicity, and there are also problems with difficult metabolism in organisms. Organic fluorescent dyes such as fluorescein, coumarin, and rhodamine, etc., have low usage, good biocompatibility, and can be completely metabolized by organisms, and have a wide range of applications in biological fields such as cell imaging, protein labeling, and DNA detection, and are excellent materials for nanothermometers in living systems. Rhodamine B is a kind of rhodamine-based organic fluorescent dye, and its fluorescence intensity will decrease with the increase of temperature. Therefore, temperature information can be obtained by measuring the change of its fluorescence intensity. The temperature-sensitive property of rhodamine B mainly stems from the xanthene chromophore in the molecular structure, and the movement of its diethylamino substituent has a significant response to temperature. Rhodamine B has been widely used in fluorescence measurement research due to its unique fluorescence properties. For example, Zhang Huidan et al. used rhodamine B as a light conversion material based on its good fluorescence performance and stable chemical properties to prepare a light conversion film that can efficiently convert green light into red light, as detailed in the patent application with Publication No. CN118791768A. Feng Lei et al. used rhodamine B as a precursor to prepare a carbonized polymer with both high-temperature resistance surface activity and solid-state luminescence, which can not only emit high-brightness red light, have a high solid quantum yield, but also have good biocompatibility, as detailed in the patent application with Publication No.: CN119118107A. There are also people who used rhodamine B to prepare a visual ratiometric fluorescence probe that can be applied to antibiotic detection, as detailed in the patent with Publication No.: CN118813247A. Yang Tingting et al. developed a temperature-sensitive fluorescence probe with strong anti-interference ability and good reproducibility by using the temperature-sensitive property of rhodamine B and applied it to mercury ion detection, as detailed in the patent application with Publication No.: CN118852711A. The above inventions all utilize the fluorescence properties of rhodamine B, and there are many inventions using rhodamine B as a fluorescence probe. However, there are almost no inventions using rhodamine B to make a nanothermometer for detecting intracellular temperature. The main reasons are that rhodamine B has strong water solubility, obvious photobleaching effect, fast metabolism in organisms, and difficult loading of nanoparticles, etc., which limit its measurement of organism temperature.In addition, it is found that ellipsoidal nanoparticles have better fluorescence response performance than spherical nanoparticles. When made into a nanothermometer, they should have better temperature measurement performance. Moreover, ellipsoidal nanoparticles are more likely to enter the interior of cells and are very suitable for measuring the temperature of cells. Summary of the Invention

[0004] In order to overcome the defects of the above-mentioned prior art, the purpose of the present invention is to provide a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer and its preparation method, which firmly load rhodamine B on gelatin nanoparticles, greatly increase the loading amount of rhodamine B, increase the retention time in the organism and overcome its photobleaching effect. In addition, in the present invention, when forming aggregated particles, the rhodamine B-gelatin fluorescent nanoparticles are prepared into an ellipsoidal shape by making the acting force uneven through centrifugal force. Not only is the temperature measurement sensitivity significantly improved, but also the ellipsoidal nanoparticles are more likely to enter the interior of cells and are very suitable for measuring the temperature of cells, and have important application prospects in the temperature measurement in the field of biomedicine.

[0005] In order to achieve the above purpose, the technical solution of the present invention is as follows:

[0006] A highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer, which is an ellipsoidal nanoparticle, and the carboxyl group of rhodamine B is combined with the amino group on gelatin to load rhodamine B on gelatin nanoparticles. The mass ratio of the raw materials of rhodamine B to gelatin is (1-3):(10-50).

[0007] A preparation method of a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer, comprising the following steps:

[0008] Step 1: Dissolve 10-50 g of gelatin in 1000-1600 mL of deionized water at a temperature of 40-65 °C to prepare a gelatin solution; simultaneously add 1-3 g of rhodamine B and 0.68-1.7 g of a catalyst to the prepared gelatin solution, and stir at 300 revolutions per minute for 48-96 hours to form a rhodamine B-gelatin conjugate solution;

[0009] The catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or N-hydroxysuccinimide, or a mixture of the two with a mass ratio of (1-4):1;

[0010] Step 2: Add 800-1600 mL of a precipitant to the above rhodamine B-gelatin conjugate solution at one time, discard the supernatant after precipitation for 0.5-1.5 hours; redissolve the precipitate in 1000 mL of deionized water, and add 600-1600 mL of the precipitant again at a rate of 1-5 drops per minute; after the reaction solution becomes turbid, keep the reaction temperature in the range of 20-50 °C and continue to stir for 1-5 hours;

[0011] The precipitant is acetone or isopropanol, or a mixture of equal volumes of acetone and isopropanol;

[0012] Step 3: Add 10 - 30 mL of crosslinking agent to the final solution of Step 2 at a temperature of 20 - 50 °C, and then stir the mixed solution at a speed of 600 - 1200 revolutions per minute for 0.5 - 1 hour; during the formation of aggregated particles, ellipsoidal nanoparticles are produced by making the particles subject to uneven forces through centrifugal force, thus obtaining the fluorescent nanothermometer;

[0013] The crosslinking agent is glutaraldehyde or glyceraldehyde, or a mixture of equal volumes of glutaraldehyde and glyceraldehyde;

[0014] The obtained fluorescent nanothermometer is loaded into a dialysis bag with a molecular weight cut-off of 3000 Da, and magnetically stirred at 300 revolutions per minute, and dialyzed in 5000 mL of deionized water for 1 - 3 days. After removing the incompletely reacted small molecule compounds, the sample is collected and stored at 4 °C.

[0015] Compared with the prior art, the advantages of the present invention are as follows:

[0016] 1. In the present invention, the carboxyl group of rhodamine B is combined with the amino group on gelatin in Step 1 to load rhodamine B on gelatin nanoparticles. This process makes full use of the high temperature sensitivity of the fluorescent dye rhodamine B and the excellent biocompatibility of the biological macromolecule gelatin, effectively solving the problem that rhodamine B is prone to leakage from the carrier due to its excessive water solubility. Therefore, the present invention significantly increases the loading amount of rhodamine B, prolongs its residence time in the organism, and overcomes its photobleaching effect.

[0017] 2. In Step 3 of the present invention, high-speed centrifugation is used to promote the full stretching of gelatin in the horizontal direction, thereby increasing the crosslinking probability in the horizontal direction. Finally, the rhodamine B - gelatin fluorescent nanoparticles are in an ellipsoidal shape. Ellipsoidal nanoparticles are more likely to enter the interior of cells, so they can monitor the temperature changes in tiny regions of organisms that are difficult to measure by current contact or non-contact temperature measurement techniques.

[0018] In summary, the present invention has developed a nanothermometer that can be used for precise measurement of temperature in biological microregions. After considering various factors such as temperature measurement sensitivity, anti-environmental interference, and biocompatibility, the fluorescent dye rhodamine B was selected as the temperature-sensitive molecule, and gelatin with good biocompatibility, low cytotoxicity, and easy surface modification was selected as the carrier of rhodamine B. The fluorescent dye rhodamine B was connected to gelatin, a natural product, by amidation reaction and then prepared into ellipsoidal nanoparticles as the nanothermometer, which has the advantages of high measurement sensitivity, good stability, good biocompatibility, long retention time in the body, and anti-photobleaching effect. It can overcome the problems existing in rhodamine B, such as easy photobleaching, susceptibility to solution environment, and strong cytotoxicity; cell experiments have proved that the maximum fluorescence intensity changes by an average of 7.6% per degree Celsius within the temperature measurement range. Description of the Drawings

[0019] Figure 1 It is a schematic diagram of the preparation process of ellipsoidal rhodamine B-gelatin fluorescent nanothermometer.

[0020] Figure 2 It is a transmission electron microscope picture of the ellipsoidal rhodamine B-gelatin fluorescent nanothermometer synthesized in Example 8.

[0021] Figure 3 It is the fluorescence property characterization of the ellipsoidal rhodamine B-gelatin fluorescent nanothermometer synthesized in Example 8. Among them, Figure 3 A in it is the fluorescence emission spectrum of the nanothermometer changing with temperature; Figure 3 B in it is the change of the maximum fluorescence intensity of the nanothermometer with temperature from 25°C to 75°C; Figure 3 C in it is the change of the fluorescence emission intensity of the nanothermometer during repeated heating and cooling processes; Figure 3 D in it is the change of the maximum fluorescence intensity of the nanothermometer after being stored outdoors for different times.

[0022] Figure 4 It is the application of the ellipsoidal rhodamine B-gelatin fluorescent nanothermometer synthesized in Example 8 in cell temperature measurement. Among them, Figure 4 A in it is the laser confocal microscopy picture of the nanothermometer in cells from 28°C to 40°C; Figure 4 B in it is the change of the maximum fluorescence intensity of the nanothermometer in cells with temperature from 28°C to 40°C. Specific Embodiments

[0023] The present invention will be described below in conjunction with specific drawings and specific embodiments.

[0024] Example 1

[0025] The fluorescent nanothermometer of this example is an ellipsoidal nanoparticle, which is composed of rhodamine B loaded on gelatin nanoparticles, and the raw material mass ratio of rhodamine B to gelatin is 1:40.

[0026] Step 1: dissolving 40 g of gelatin in 1000 mL of deionized water at 40° C. to prepare a gelatin solution; adding 1 g of rhodamine B and 0.7 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the prepared gelatin solution, the contents of which are 0.35 g and 0.35 g, respectively; stirring at 300 rpm for 72 hours to form a rhodamine B-gelatin conjugate solution;

[0027] Step 2: Add 1000 mL of acetone to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 0.5 hours; redissolve the precipitate in 1000 mL of deionized water, and add 800 mL of mixed precipitant, including 400 mL of acetone and 400 mL of isopropanol, at a rate of 1 drop per minute; after the reaction solution becomes turbid, keep the reaction temperature at 50°C and continue stirring for 1 hour;

[0028] Step 3: Add 10 mL of mixed crosslinking agent, including 5 mL of 10% glutaraldehyde and 5 mL of 10% glyceraldehyde, to the final solution of the above step 2 at a temperature of 50°C, and then stir the mixed solution at a speed of 600 revolutions per minute for 0.5 hours; in the process of forming aggregated particles, the particles are subjected to uneven force through centrifugal force to produce ellipsoidal nanoparticles, that is, a fluorescent nanothermometer is obtained.

[0029] The obtained fluorescent nanothermometer was placed in a dialysis bag with a molecular weight cutoff of 3000 Daltons, magnetically stirred at 300 rpm, and dialyzed in 5000 mL of deionized water for 3 days. After removing the incompletely reacted small molecule compounds, the samples were collected and stored at 4°C.

[0030] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and monodisperse. The maximum fluorescence intensity changes by an average of 5.8% per degree Celsius between 25°C and 75°C. The determination coefficient R between the maximum fluorescence intensity and temperature is 2 It is 0.975, which has a good linear relationship; after repeated heating and cooling and long-term storage, the maximum fluorescence intensity of the nanothermometer can always remain relatively stable.

[0031] Example 2

[0032] The fluorescent nanothermometer of this example is an ellipsoidal nanoparticle, which is composed of rhodamine B loaded on gelatin nanoparticles, and the raw material mass ratio of rhodamine B to gelatin is 3:10.

[0033] This example includes the following steps:

[0034] Step 1: dissolving 10 g of gelatin in 1000 mL of deionized water at 45° C. to prepare a gelatin solution; adding 3 g of rhodamine B and 1.05 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, 0.7 g and 0.35 g respectively, to the prepared gelatin solution; then stirring at 300 rpm for 48 hours to form a rhodamine B-gelatin conjugate solution;

[0035] Step 2: Add 800 mL of acetone to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 1 hour; redissolve the precipitate in 1000 mL of deionized water, and add 600 mL of acetone again at a rate of 3 drops per minute; after the reaction solution becomes turbid, keep the reaction temperature at 37°C and continue stirring for 1 hour;

[0036] Step 3: Add 10 mL of 10% glyceraldehyde to the final solution of step 2 at 37°C, and stir the mixed solution at 1200 revolutions per minute for 0.75 hours; in the process of forming aggregated particles, centrifugal force is used to make the particles unevenly stressed to produce ellipsoidal nanoparticles, thus obtaining a fluorescent nanothermometer.

[0037] The obtained fluorescent nanothermometer was placed in a dialysis bag with a molecular weight cutoff of 3000 Daltons, magnetically stirred at 300 rpm, and dialyzed against 5000 mL of deionized water for 2 days. After removing the incompletely reacted small molecule compounds, the sample was collected and stored at 4°C.

[0038] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and monodisperse. The maximum fluorescence intensity changes by an average of 5.5% per degree Celsius between 25°C and 75°C. The determination coefficient R between the maximum fluorescence intensity and temperature is 2 It is 0.969, which has a good linear relationship; after repeated heating and cooling and long-term storage, the maximum fluorescence intensity of the nanothermometer can always remain relatively stable.

[0039] Example 3

[0040] The fluorescent nanothermometer of this example is an ellipsoidal nanoparticle, which is composed of rhodamine B loaded on gelatin nanoparticles, and the raw material mass ratio of rhodamine B to gelatin is 3:40.

[0041] This example includes the following steps:

[0042] Step 1: dissolving 40 g of gelatin in 1000 mL of deionized water at 65° C. to prepare a gelatin solution; adding 3 g of rhodamine B and 0.7 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the prepared gelatin solution, the contents of which are 0.35 g and 0.35 g, respectively; stirring at 300 rpm for 72 hours to form a rhodamine B-gelatin conjugate solution;

[0043] Step 2: Add 1600 mL of acetone to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 1.5 hours; redissolve the precipitate in 1000 mL of deionized water, and add 800 mL of acetone again at a rate of 5 drops per minute; after the reaction solution becomes turbid, keep the reaction temperature at 37°C and continue stirring for 1 hour;

[0044] Step 3: Add 20 mL of mixed crosslinking agent, including 10 mL of 10% glutaraldehyde and 10 mL of 10% glyceraldehyde, to the final solution of the above step 2 at a temperature of 37°C, and then stir the mixed solution at a speed of 800 revolutions per minute for 1 hour; in the process of forming aggregated particles, the particles are subjected to uneven force through centrifugal force to produce ellipsoidal nanoparticles, that is, a fluorescent nanothermometer is obtained.

[0045] The obtained fluorescent nanothermometer was placed in a dialysis bag with a molecular weight cutoff of 3000 Daltons, magnetically stirred at 300 rpm, and dialyzed against 5000 mL of deionized water for 3 days. After removing the incompletely reacted small molecule compounds, the sample was collected and stored at 4°C.

[0046] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and monodisperse. The maximum fluorescence intensity changes by an average of 6.7% per degree Celsius between 25°C and 75°C. The determination coefficient R between the maximum fluorescence intensity and temperature is 2 It is 0.983, which has a good linear relationship; after repeated heating and cooling and long-term storage, the maximum fluorescence intensity of the nanothermometer can always remain relatively stable.

[0047] Example 4

[0048] The fluorescent nanothermometer of this example is an ellipsoidal nanoparticle, which is composed of rhodamine B loaded on gelatin nanoparticles, and the raw material mass ratio of rhodamine B to gelatin is 3:50.

[0049] This example includes the following steps:

[0050] Step 1: Dissolve 50 g of gelatin in 1300 mL of deionized water at a temperature of 45 °C to prepare a gelatin solution; simultaneously add 3 g of rhodamine B and 1 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the prepared gelatin solution, 0.8 g and 0.2 g respectively; then stir at 300 revolutions per minute for 96 hours to form a rhodamine B-gelatin conjugate solution;

[0051] Step 2: Add 800 mL of isopropanol to the above rhodamine B-gelatin conjugate solution at one time, discard the supernatant after precipitation for 0.5 hours; redissolve the precipitate in 1000 mL of deionized water, and add 1600 mL of a mixed precipitant at a rate of 1 drop per minute, including 800 mL of acetone and 800 mL of isopropanol; after the reaction solution becomes turbid, keep the reaction temperature at 20 °C and continue to stir for 3 hours;

[0052] Step 3: Add 20 mL of 10% glutaraldehyde to the final solution of Step 2 at a temperature of 20 °C, and then stir the mixed solution at a speed of 600 revolutions per minute for 0.5 hours; generate ellipsoidal nanoparticles by making the particles experience uneven forces through centrifugal force during the formation of aggregated particles, that is, obtain a fluorescent nanothermometer.

[0053] The obtained fluorescent nanothermometer is loaded into a dialysis bag with a molecular weight cut-off of 3000 Da, magnetically stirred at 300 revolutions per minute, dialyzed in 5000 mL of deionized water for 1 day, the unreacted small molecule compounds are removed, the sample is collected and stored at 4 °C.

[0054] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and shows monodispersity. The average change of the maximum fluorescence intensity per degree Celsius is 7.1% between 25 °C and 75 °C, and the determination coefficient R 2 between the maximum fluorescence intensity and the temperature is 0.984, showing a good linear relationship; the maximum fluorescence intensity of the nanothermometer can remain relatively stable after repeated heating and cooling and long-term storage.

[0055] Example 5

[0056] The fluorescent nanothermometer of this example is an ellipsoidal nanoparticle, with rhodamine B loaded on gelatin nanoparticles, and the mass ratio of rhodamine B to gelatin raw materials is 2:10.

[0057] This example includes the following steps:

[0058] Step 1: dissolving 10 g of gelatin in 1000 mL of deionized water at a temperature of 45° C. to prepare a gelatin solution; adding 2 g of rhodamine B and 1.2 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, 0.9 g and 0.3 g respectively, to the prepared gelatin solution; then stirring at 300 rpm for 52 hours to form a rhodamine B-gelatin conjugate solution;

[0059] Step 2: add 1000 mL of isopropanol to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 1 hour; redissolve the precipitate in 1000 mL of deionized water, and add 1600 mL of isopropanol again at a rate of 2 drops per minute; after the reaction solution becomes turbid, maintain the reaction temperature at 20°C and continue stirring for 5 hours;

[0060] Step 3: Add 20 mL of 10% glyceraldehyde to the final solution of step 2 at a temperature of 20°C, and stir the mixed solution at a speed of 1200 revolutions per minute for 0.5 hours; in the process of forming aggregated particles, the particles are subjected to uneven force by centrifugal force to produce ellipsoidal nanoparticles, that is, a fluorescent nanothermometer is obtained.

[0061] The obtained fluorescent nanothermometer was placed in a dialysis bag with a molecular weight cutoff of 3000 Daltons, magnetically stirred at 300 rpm, and dialyzed against 5000 mL of deionized water for 2 days. After removing the incompletely reacted small molecule compounds, the sample was collected and stored at 4°C.

[0062] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and monodisperse. The maximum fluorescence intensity changes by an average of 6.8% per degree Celsius between 25°C and 75°C. The determination coefficient R between the maximum fluorescence intensity and temperature is 2 It is 0.988, which has a good linear relationship; after repeated heating and cooling and long-term storage, the maximum fluorescence intensity of the nanothermometer can always remain relatively stable.

[0063] Example 6

[0064] The fluorescent nanothermometer of this example is an ellipsoidal nanoparticle, which is composed of rhodamine B loaded on gelatin nanoparticles, and the raw material mass ratio of rhodamine B to gelatin is 1:20.

[0065] This example includes the following steps:

[0066] Step 1: dissolving 40 g of gelatin in 1300 mL of deionized water at 48° C. to prepare a gelatin solution; adding 2 g of rhodamine B and 0.8 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide, 0.6 g and 0.2 g respectively, to the prepared gelatin solution; then stirring at 300 rpm for 50 hours to form a rhodamine B-gelatin conjugate solution;

[0067] Step 2: Add 1600 mL of isopropanol to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 0.65 hours; redissolve the precipitate in 1000 mL of deionized water, and add 600 mL of isopropanol again at a rate of 1 drop per minute; after the reaction solution becomes turbid, maintain the reaction temperature at 30°C and continue stirring for 5 hours;

[0068] Step 3: Add 30 mL of mixed crosslinking agent, including 15 mL of 10% glutaraldehyde and 15 mL of 10% glyceraldehyde, to the final solution of the above step 2 at a temperature of 30°C, and then stir the mixed solution at a speed of 1200 revolutions per minute for 0.5 hours; in the process of forming aggregated particles, the particles are subjected to uneven force through centrifugal force to produce ellipsoidal nanoparticles, that is, a fluorescent nanothermometer is obtained.

[0069] The obtained fluorescent nanothermometer was placed in a dialysis bag with a molecular weight cutoff of 3000 Daltons, magnetically stirred at 300 rpm, and dialyzed against 5000 mL of deionized water for 1 day. After removing the incompletely reacted small molecule compounds, the sample was collected and stored at 4°C.

[0070] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and monodisperse. The maximum fluorescence intensity changes by an average of 7.1% per degree Celsius between 25°C and 75°C. The determination coefficient R between the maximum fluorescence intensity and temperature is 2 It is 0.991, which has a good linear relationship; after repeated heating and cooling and long-term storage, the maximum fluorescence intensity of the nanothermometer can always remain relatively stable.

[0071] Example 7

[0072] The fluorescent nanothermometer of this example is an ellipsoidal nanoparticle, which is composed of rhodamine B loaded on gelatin nanoparticles, and the raw material mass ratio of rhodamine B to gelatin is 1:50.

[0073] This example includes the following steps:

[0074] Step 1: Dissolve 50 g of gelatin in 1600 mL of deionized water at a temperature of 40 °C to prepare a gelatin solution; add 1 g of rhodamine B and a mixture of 1 g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the prepared gelatin solution, with 0.6 g and 0.4 g respectively; then stir at 300 revolutions per minute for 48 hours to form a rhodamine B-gelatin conjugate solution;

[0075] Step 2: Add 800 mL of a mixed precipitant, including 400 mL of acetone and 400 mL of isopropanol, to the above rhodamine B-gelatin conjugate solution at one time, discard the supernatant after precipitation for 1.5 hours; redissolve the precipitate in 1000 mL of deionized water, and add 600 mL of a mixed precipitant, including 300 mL of acetone and 300 mL of isopropanol, again at a rate of 1 drop per minute; after the reaction solution becomes turbid, keep the reaction temperature at 50 °C and continue to stir for 3 hours;

[0076] Step 3: Add 30 mL of 10% glutaraldehyde to the final solution of Step 2 at a temperature of 50 °C, and then stir the mixed solution at a high speed of 800 revolutions per minute for 0.75 hours; generate ellipsoidal nanoparticles by making the particles experience uneven forces through centrifugal force during the formation of aggregated particles, that is, obtain a fluorescent nanothermometer.

[0077] Put the obtained fluorescent nanothermometer into a dialysis bag with a molecular weight cut-off of 3000 Da, stir magnetically at 300 revolutions per minute, and dialyze in 5000 mL of deionized water for 3 days. After removing the unreacted small molecule compounds, collect the sample and store it at 4 °C.

[0078] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and shows monodispersity. The average change of the maximum fluorescence intensity per degree Celsius is 7.4% between 25 °C and 75 °C, and the determination coefficient R 2 between the maximum fluorescence intensity and temperature is 0.989, showing a good linear relationship; the maximum fluorescence intensity of the nanothermometer can always remain relatively stable after repeated heating and cooling and long-term storage.

[0079] Example 8

[0080] The fluorescent nanothermometer in this example is an ellipsoidal nanoparticle, with rhodamine B loaded on gelatin nanoparticles, and the mass ratio of the raw materials of rhodamine B to gelatin is 2:50.

[0081] This example includes the following steps:

[0082] Step 1: dissolving 50 g of gelatin in 1600 mL of deionized water at 65° C. to prepare a gelatin solution; adding 2 g of rhodamine B and 0.68 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the prepared gelatin solution, the mixture being 0.48 g and 0.2 g respectively; and stirring at 300 rpm for 50 hours to form a rhodamine B-gelatin conjugate solution;

[0083] Step 2: add 1000 mL of mixed precipitant, including 500 mL of acetone and 500 mL of isopropanol, to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 0.85 hours; redissolve the precipitate in 1000 mL of deionized water, and add 800 mL of isopropanol again at a rate of 4 drops per minute; after the reaction solution becomes turbid, maintain the reaction temperature at 20°C and continue stirring for 5 hours;

[0084] Step 3: Add 30 mL of 10% glyceraldehyde to the final solution of step 2 at a temperature of 20°C, and stir the mixed solution at a speed of 1200 revolutions per minute for 1 hour; in the process of forming aggregated particles, the particles are subjected to uneven force through centrifugal force to produce ellipsoidal nanoparticles, that is, a fluorescent nanothermometer is obtained.

[0085] The obtained fluorescent nanothermometer was placed in a dialysis bag with a molecular weight cutoff of 3000 Daltons, magnetically stirred at 300 rpm, and dialyzed against 5000 mL of deionized water for 2 days. After removing the incompletely reacted small molecule compounds, the sample was collected and stored at 4°C.

[0086] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and monodisperse. The maximum fluorescence intensity changes by an average of 6.3% per degree Celsius between 25°C and 75°C. The determination coefficient R between the maximum fluorescence intensity and temperature is 2 It is 0.979, which has a good linear relationship; after repeated heating and cooling and long-term storage, the maximum fluorescence intensity of the nanothermometer can always remain relatively stable.

[0087] Example 9

[0088] The fluorescent nanothermometer of this example is an ellipsoidal nanoparticle, which is composed of rhodamine B loaded on gelatin nanoparticles, and the raw material mass ratio of rhodamine B to gelatin is 1:10.

[0089] This example includes the following steps:

[0090] Step 1: dissolving 20 g of gelatin in 1000 mL of deionized water at 45° C. to prepare a gelatin solution; adding 2 g of rhodamine B and 1.7 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the prepared gelatin solution, the mixture being 1 g and 0.7 g respectively; and stirring at 300 rpm for 72 hours to form a rhodamine B-gelatin conjugate solution;

[0091] Step 2: add 1600 mL of mixed precipitant, including 800 mL of acetone and 800 mL of isopropanol, to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 0.5 hours; redissolve the precipitate in 1000 mL of deionized water, and add 1600 mL of acetone again at a rate of 3 drops per minute; after the reaction solution becomes turbid, keep the reaction temperature at 37°C and continue stirring for 1 hour;

[0092] Step 3: Add 10 mL of 10% glutaraldehyde to the final solution of step 2 at 37°C, and stir the mixed solution at 1200 rpm for 0.5 hours; in the process of forming aggregated particles, the particles are subjected to uneven force by centrifugal force to produce ellipsoidal nanoparticles, thus obtaining a fluorescent nanothermometer.

[0093] The obtained fluorescent nanothermometer was placed in a dialysis bag with a molecular weight cutoff of 3000 Daltons, magnetically stirred at 300 rpm, and dialyzed in 5000 mL of deionized water for 3 days. After removing the incompletely reacted small molecule compounds, the samples were collected and stored at 4°C.

[0094] The rhodamine B-gelatin fluorescent nanothermometer synthesized in this example is ellipsoidal and monodisperse. The maximum fluorescence intensity changes by an average of 7.6% per degree Celsius between 25°C and 75°C. The determination coefficient R between the maximum fluorescence intensity and temperature is 2 It is 0.996, which has a good linear relationship; after repeated heating and cooling and long-term storage, the maximum fluorescence intensity of the nanothermometer can always remain relatively stable.

[0095] Figure 2 The transmission electron microscope image of the Rhodamine B-gelatin fluorescent nanothermometer synthesized in this example shows that the nanothermometer is ellipsoidal. The ellipsoidal Rhodamine B-gelatin fluorescent nanothermometer synthesized in Example 8 has excellent optical properties, such as Figure 3 shown. Figure 3 A in the figure is the fluorescence emission spectrum of the nanothermometer as it changes with temperature. The maximum fluorescence intensity changes by an average of 7.6% per degree Celsius between 25°C and 75°C. Figure 3In which, B is the change of the maximum fluorescence intensity of the nanothermometer with temperature from 25 °C to 75 °C. It can be seen that there is a good linear relationship between the maximum fluorescence intensity and the temperature; Figure 3 In which, C is the change of the fluorescence emission intensity of the nanothermometer during repeated heating and cooling processes. The results show that the nanothermometer has good cyclic temperature measurement stability; Figure 3 In which, D is the change of the maximum fluorescence intensity of the nanothermometer after being stored outdoors for different times. The results show that the nanothermometer can still maintain its temperature measurement stability after long-term storage. Figure 4 This is the application of the ellipsoidal rhodamine B-gelatin fluorescent nanothermometer synthesized in Example 8 in cell temperature measurement. The fluorescence emission intensity of the nanothermometer in cells decreases continuously with the increase of temperature from 28 °C to 40 °C, and there is a good linear relationship between the maximum fluorescence intensity and the temperature. The determination coefficient R 2 reaches 0.998.

[0096] Experiments prove that the ellipsoidal rhodamine B-gelatin fluorescent nanothermometer synthesized in this example has good application prospects in the temperature measurement of biological micro-regions.

[0097] In summary, in the present invention, the biocompatible gelatin amino group is chemically combined with the carboxyl group of rhodamine B, and then they are crosslinked with glutaraldehyde and stirred at high speed. When forming aggregated particles, the particles are unevenly stressed by centrifugal force to generate ellipsoidal nanoparticles, forming a highly sensitive ellipsoidal fluorescent nanothermometer. This thermometer has the characteristics of a large rhodamine B loading capacity, high measurement sensitivity, strong anti-photobleaching ability, and good biocompatibility. It is easily taken up by cells or tissues, can measure the temperature changes of subcellular structures or specific tissues, and has important application prospects in the biomedical field. The preparation method of the present invention is simple and efficient, and the obtained product has strong stability, good anti-photobleaching ability, high temperature measurement sensitivity, and strong biological adaptability. The cytotoxicity is significantly reduced compared with rhodamine B; the elliptical shape makes it easier to enter cells than other shaped nanoparticles, so the usage amount is lower. The rhodamine B-gelatin ellipsoidal fluorescent nanothermometer prepared by the present invention has important application prospects in the temperature measurement of biological micro-regions.

Claims

1. A highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer, characterized in that: The nanoparticles are ellipsoidal in shape. The carboxyl group of rhodamine B is combined with the amino group on gelatin so that rhodamine B is loaded on the gelatin nanoparticles. The raw material mass ratio of rhodamine B to gelatin is (1-3):(10-50).

2. A high-sensitivity ellipsoidal rhodamine B-gelatin fluorescent nanothermometer according to claim 1, characterized in that: The nanoparticles are ellipsoidal in shape, and are composed of rhodamine B loaded on gelatin nanoparticles, with the raw material mass ratio of rhodamine B to gelatin being 1:

10.

3. A method for preparing a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer, characterized in that: The following steps are involved: Step 1: dissolving 10-50 g of gelatin in 1000-1600 mL of deionized water at a temperature of 40-65° C. to prepare a gelatin solution; adding 1-3 g of rhodamine B and 0.68-1.7 g of a catalyst to the prepared gelatin solution, stirring at 300 rpm for 48-96 hours to form a rhodamine B-gelatin conjugate solution; Step 2: add 800-1600 mL of precipitant to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 0.5-1.5 hours; redissolve the precipitate in 1000 mL of deionized water, and add 600-1600 mL of precipitant again at a rate of 1-5 drops per minute; after the reaction solution becomes turbid, maintain the reaction temperature in the range of 20-50° C. and continue stirring for 1-5 hours; Step 3: Add 10-30 mL of a crosslinking agent to the final solution of step 2 at a temperature of 20-50°C, and stir the mixed solution at a speed of 600-1200 revolutions per minute for 0.5-1 hour; in the process of forming aggregated particles, the particles are subjected to uneven force by centrifugal force to produce ellipsoidal nanoparticles, thus obtaining a fluorescent nanothermometer.

4. The method for preparing a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer according to claim 3, characterized in that: The catalyst is 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride or N-hydroxysuccinimide, or a mixture of the two in a mass ratio of (1-4):

1.

5. The method for preparing a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer according to claim 3, characterized in that: The precipitant is acetone or isopropanol, or a mixture of acetone and isopropanol in equal volumes.

6. The method for preparing a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer according to claim 3, characterized in that: The cross-linking agent is glutaraldehyde or glyceraldehyde, or a mixture of glutaraldehyde and glyceraldehyde in equal volumes.

7. The method for preparing a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer according to claim 3, characterized in that: The fluorescent nano-thermometer was placed in a dialysis bag with a molecular weight cutoff of 3000 Daltons, magnetically stirred at 300 rpm, and dialyzed in 5000 mL of deionized water for 1-3 days. After removing the incompletely reacted small molecule compounds, the sample was collected and stored at 4°C.

8. The method for preparing a highly sensitive ellipsoidal rhodamine B-gelatin fluorescent nanothermometer according to claim 3, characterized in that: The following steps are involved: Step 1: dissolving 20 g of gelatin in 1000 mL of deionized water at 45° C. to prepare a gelatin solution; adding 2 g of rhodamine B and 1.7 g of a mixture of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide to the prepared gelatin solution, the mixture being 1 g and 0.7 g respectively; and stirring at 300 rpm for 72 hours to form a rhodamine B-gelatin conjugate solution; Step 2: add 1600 mL of mixed precipitant, including 800 mL of acetone and 800 mL of isopropanol, to the above rhodamine B-gelatin conjugate solution at one time, and discard the supernatant after precipitation for 0.5 hours; redissolve the precipitate in 1000 mL of deionized water, and add 1600 mL of acetone again at a rate of 3 drops per minute; after the reaction solution becomes turbid, keep the reaction temperature at 37°C and continue stirring for 1 hour; Step 3: Add 10 mL of 10% glutaraldehyde to the final solution of step 2 at 37°C, and stir the mixed solution at 1200 rpm for 0.5 hours; in the process of forming aggregated particles, the particles are subjected to uneven force by centrifugal force to produce ellipsoidal nanoparticles, thus obtaining a fluorescent nanothermometer.

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