Method for detecting internal temperature of plant body based on p-AuNP-coated HTT

Abstract

The invention relates to the field of plant body internal temperature measurement, and discloses a method for detecting the internal temperature of a plant body based on p-AuNP-coated HTT, and the method comprises the steps: preparing a temperature-sensitive probe p-AuNP-coated HTT; injecting the temperature-sensitive probe into a plant body to obtain a plant body containing the temperature-sensitive probe; the temperature-sensitive probe is excited, so that the temperature-sensitive probe emits a light-emitting signal; collecting the light-emitting signal in a delayed manner to obtain a sample signal; determining the temperature in the plant according to the sample signal; wherein the time delay is shorter than the luminescence life of the temperature-sensitive probe p-AuNP-coated HTT and is longer than the luminescence life of autofluorescence of the plant body. In the embodiment of the invention, the temperature-sensitive probe is input into the plant body to detect the light-emitting signal so as to obtain the internal temperature, so that the nondestructive detection of the plant body is realized. Through delayed detection, the influence of autofluorescence of plants is filtered out. The temperature-sensitive probe p-AuNP-coated HTT in the embodiment of the invention has good temperature sensitivity, pH stability and calcium salt stability, and can improve the accuracy of the internal temperature of the plant body.

Description

Technical Field

[0001] The present invention relates to the field of measuring the internal temperature of plants. More specifically, the present invention relates to a method for detecting the internal temperature of plants based on p-AuNP@HTT. Background Art

[0002] Temperature is a key environmental factor for plant growth and development, as well as crop yield and quality. The internal temperature of plants reflects the temperature conditions of roots, stems, leaves, flowers, and fruits, and directly affects life activities such as biochemical reactions, cell membrane movement, and transpiration within the plants. Therefore, accurately measuring the internal temperature of plants is crucial for studying plant temperature adaptability and regulatory mechanisms, helping to deeply understand the response laws and molecular mechanisms of plants to temperature, and thus contributing to improving food yield and quality.

[0003] However, current temperature measurement technologies such as infrared thermal imaging can only detect the surface temperature of plants and cannot accurately measure the internal temperature of plants; temperature measurement technologies such as thermocouples can easily change the measured environment and cause damage to the measured part due to direct contact with the measured part, affecting the accuracy of the temperature measurement results. Due to the lack of effective internal temperature measurement means, scientists currently can only use the external environmental temperature to replace the internal temperature of plants, which leads to large errors or even mistakes, seriously restricting the research progress in the field of plant temperature response.

[0004] Detecting the internal temperature of plants faces the following difficulties: First, plants are living organisms with highly dynamic physiological activities, so internal temperature measurement must be non-destructive and the interference with the normal physiological processes of plants should be minimized. Second, there are a large number of substances such as proteins and small molecules inside plants, and they usually exhibit autofluorescence, and these fluorescence spectra are widely distributed in the visible and near-infrared regions. Finally, the biochemical environment inside plants (such as pH value and ion concentration) may seriously interfere with non-invasive detection signals such as light emission.

[0005] Currently, traditional methods for eliminating autofluorescence interference include sequential scanning and spectral scanning. However, for the case where the excitation and emission spectra overlap, sequential scanning cannot be effectively processed. And spectral scanning is also difficult to completely remove the influence of complex plant autofluorescence, which has become a bottleneck for high-quality imaging of traditional fluorescence microscopy.

[0006] Therefore, there is an urgent need to provide a method for non-destructively measuring the internal temperature of plants, which can exclude the interference of autofluorescence and biochemical environment to meet the urgent needs of plant temperature research. Summary of the Invention

[0007] To at least solve one or more of the above-mentioned technical problems, the present invention provides a method for detecting the internal temperature of a plant body based on p-AuNP@HTT, including: preparing a temperature-sensitive probe p-AuNP@HTT; injecting the temperature-sensitive probe p-AuNP@HTT into the plant body to obtain a plant body containing the temperature-sensitive probe p-AuNP@HTT; exciting the temperature-sensitive probe p-AuNP@HTT to make the temperature-sensitive probe p-AuNP@HTT emit a light signal; collecting the light signal with a time delay to obtain a sample signal; determining the temperature inside the plant body according to the sample signal; wherein the time delay is less than the luminescence lifetime of the temperature-sensitive probe p-AuNP@HTT and greater than the luminescence lifetime of the autofluorescence of the plant body.

[0008] According to an embodiment of the present invention, the light signal is the luminescence intensity.

[0009] According to an embodiment of the present invention, the luminescence lifetime of the temperature-sensitive probe p-AuNP@HTT > 1000 ns.

[0010] According to an embodiment of the present invention, the time delay is set to ≥ 10 ns.

[0011] According to an embodiment of the present invention, the time delay is set to 100 ns.

[0012] According to an embodiment of the present invention, the temperature-sensitive probe p-AuNP@HTT is prepared by the following method: mixing a solution of PMMA-co-MAA copolymer in tetrahydrofuran with a solution of AuNP@HTT in tetrahydrofuran to obtain a first mixture; dropping the first mixture into ultrapure water drop by drop while continuously stirring during the dropping process to obtain a second mixture; allowing the second mixture to stand for 10 minutes to obtain a third mixture; removing the tetrahydrofuran in the third mixture to obtain an aqueous solution of p-AuNP@HTT.

[0013] According to an embodiment of the present invention, during the process of dropping the first mixture into ultrapure water, the temperature is maintained at 35 - 40 °C.

[0014] According to an embodiment of the present invention, during the process of removing the tetrahydrofuran in the third mixture, the temperature is maintained at 40 - 50 °C.

[0015] According to an embodiment of the present invention, the AuNP@HTT is prepared by the following method: Prepare solution A: an ethanol solution of HAuCl4, solution B: an ethanol solution of NaOH, and solution C: a chloroform solution of HTT respectively; mix solution A, solution B, and solution C, and stir for 15 minutes to obtain a first mixture; irradiate the first mixture with a UV lamp having a wavelength of 390 nm and a power of 8 w for 60 hours to obtain a second mixture; add the second mixture to cold ethanol at -20°C to obtain a third mixture; store the third mixture at 4°C until no yellow precipitate is formed in the third mixture to obtain a fourth mixture; separate the precipitate in the fourth mixture to obtain AuNP@HTT.

[0016] According to an embodiment of the present invention, the concentration of solution A is 10 mM, the concentration of solution B is 0.2 M, and the concentration of solution C is 20 μmol / L; the volume ratio of solution A, solution B, and solution C is 5:1:45.

[0017] In the embodiments of the present invention, by inputting a temperature-sensitive probe into a plant body to detect a luminescence signal to obtain an internal temperature, non-destructive detection of the plant body is achieved. By performing delayed detection, the influence of the autofluorescence of the plant body is filtered out. The temperature-sensitive probe p-AuNP@HTT in the embodiments of the present invention has good temperature sensitivity, pH stability, and calcium salt stability, and can improve the accuracy of the internal temperature of the plant body. Description of the Drawings

[0018] By reading the following detailed description with reference to the drawings, the above and other objects, features, and advantages of the exemplary embodiments of the present invention will become readily understood. In the drawings, several embodiments of the present invention are shown in an exemplary rather than restrictive manner, and the same or corresponding reference numerals represent the same or corresponding parts, wherein:

[0019] Figure 1 A schematic diagram of the method steps for detecting the internal temperature of a plant body according to an embodiment of the present invention is shown;

[0020] Figure 2 A schematic diagram of the photoluminescence spectrum of p-AuNP@HTT according to an embodiment of the present invention is shown;

[0021] Figure 3 A transmission electron microscope image of p-AuNP@HTT according to an embodiment of the present invention is shown;

[0022] Figure 4 A statistical histogram of the particle distribution of p-AuNP@HTT according to an embodiment of the present invention is shown;

[0023] Figure 5Shows the schematic diagram of the full-wavelength luminescence intensity of p-AuNP@HTT varying with temperature according to an embodiment of the present invention;

[0024] Figure 6 Shows the schematic diagram of the fitting curve of the relative luminescence intensity at the emission peak of p-AuNP@HTT varying with temperature according to an embodiment of the present invention;

[0025] Figure 7 Shows the schematic diagram of the relative luminescence intensity of p-AuNP@HTT varying with temperature under the acquisition condition of delay (100 ns) according to an embodiment of the present invention;

[0026] Figure 8 Shows the schematic diagram of the luminescence lifetime of p-AuNP@HTT according to an embodiment of the present invention;

[0027] Figure 9 Shows the schematic diagram of the stability of the luminescence intensity of p-AuNP@HTT in the plant physiological pH range according to an embodiment of the present invention;

[0028] Figure 10 Shows the luminescence intensity of p-AuNP@HTT varying with Ca 2+ concentration according to an embodiment of the present invention;

[0029] Figure 11 Shows the schematic diagram of the luminescence intensity of p-AuNP@HTT varying with Na + concentration according to an embodiment of the present invention;

[0030] Figure 12 Shows the schematic diagram of the luminescence intensity of p-AuNP@HTT varying with K + concentration according to an embodiment of the present invention;

[0031] Figure 13 Shows the schematic diagram of the luminescence intensity of p-AuNP@HTT varying with Mg 2+ concentration according to an embodiment of the present invention;

[0032] Figure 14 Shows the schematic diagram of the luminescence intensity of the p-AuNP@HTT temperature-sensitive probe inside tobacco varying with the ambient temperature under high-temperature stress according to an embodiment of the present invention;

[0033] Figure 15 Shows the schematic diagram of the correspondence between the temperature change amount inside tobacco and the ambient temperature change amount under high-temperature stress according to an embodiment of the present invention;

[0034] Figure 16 Shows the schematic diagram of the luminescence intensity of the p-AuNP@HTT temperature-sensitive probe inside tobacco varying with the ambient temperature under low-temperature stress according to an embodiment of the present invention;

[0035] Figure 17 A schematic diagram showing the correspondence between the change amount of the internal temperature of tobacco and the change amount of the ambient temperature under low temperature stress according to an embodiment of the present invention. Detailed implementation manners

[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts fall within the protection scope of the present invention.

[0037] It should be understood that the terms "comprising" and "including" used in the specification and claims of the present invention indicate the presence of the described features, wholes, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, wholes, steps, operations, elements, components, and / or their combinations.

[0038] It should also be understood that the terms used in the specification of the present invention are only for the purpose of describing specific embodiments, and are not intended to limit the present invention. As used in the specification and claims of the present invention, unless otherwise clearly specified in the context, the singular forms "a", "an", and "the" are intended to include the plural forms. It should also be further understood that the term " / and" used in the specification and claims of the present invention refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0039] Next, the detailed implementation manners of the present invention will be described in detail in conjunction with the accompanying drawings.

[0040] Figure 1 A schematic diagram showing the method steps for detecting the internal temperature of a plant body according to an embodiment of the present invention.

[0041] As Figure 1 shown, the method 100 for detecting the internal temperature of a plant body includes step S101 of injecting a temperature-sensitive probe into the plant body to obtain a plant body containing the temperature-sensitive probe; step S102 of exciting the temperature-sensitive probe to make the temperature-sensitive probe emit a light signal; step S103 of collecting the light signal with a time delay to obtain a sample signal; and step S104 of determining the internal temperature of the plant body according to the sample signal, where the time delay is less than the luminescence lifetime of the temperature-sensitive probe and greater than the luminescence lifetime of the autofluorescence of the plant body.

[0042] To detect the internal temperature of a plant, a commonly used method is to inject a temperature-sensitive probe into the plant, then activate the temperature-sensitive probe, and determine the internal temperature of the plant by detecting the luminescence signal emitted by the temperature-sensitive probe.

[0043] The inventors recognized that in addition to different spectral parameters, different types of fluorescence also have differences in luminescence lifetime. For example, the luminescence lifetime of the autofluorescence of a plant is usually short, generally less than 10 ns. Therefore, when performing luminescence detection, it is possible to consider separating the luminescence signal from the time dimension, so as to remove the interference of background noises such as autofluorescence, reflected light, and stray light.

[0044] Table 1 shows the luminescence lifetime table of some luminescent materials.

[0045] Table 1.

[0046] Name of Luminescent Material Luminescence Lifetime (ns) Fluorescein 4.3 ns Autofluorescence of Plants < 10 ns Quantum Dots (QDs) 26.2 - 160 ns Green Fluorescent Protein (GFP) 2.8 ns Rare Earth Luminescent Materials 62 - 820 μs Gold Nanoclusters (AuNCs) > 100 ns Luminescent Gold Nanoparticles (L - AuNPs) 10 - 720000 ns

[0047] As can be seen from Table 1, the luminescence lifetime of the autofluorescence of a plant is short. In order to remove the interference of autofluorescence, the temperature-sensitive probe is prepared from a luminescent material with a luminescence lifetime greater than that of the autofluorescence of the plant. The luminescence lifetime of the temperature-sensitive probe refers to the luminescence lifetime of the luminescent material contained in the temperature-sensitive probe.

[0048] Specifically, a pulsed laser is used, combined with a long-lifetime fluorescent probe. The fluorescent probe is injected into the plant. When the fluorescent substance in the fluorescent probe is excited, the fluorescent signal is not immediately collected, but waits for the fluorescence to decay for a period of time (such as 20 ns or 50 ns). After the background noises such as autofluorescence are completely decayed, the luminescence signal of the long-lifetime fluorescent probe is collected. This technique is called time-gated fluorescence imaging technology. By not detecting the short-lifetime signals and only detecting the long-lifetime signals, high-contrast imaging of long-lifetime fluorescent (or luminescent) substances can be obtained. Then, by using multi-period signals to increase the signal-to-noise ratio, the imaging quality can be improved and the interference of short-lifetime background fluorescence (or other luminescence) on the image can be reduced.

[0049] Based on this, in an embodiment of the present invention, the selection criterion for the temperature-sensitive probe is: the luminescence lifetime of the temperature-sensitive probe is greater than the lifetime of the autofluorescence of the plant. For example, the luminescent gold nanoparticles p-AuNP@HTT with a surface ligand of HTT are selected as the temperature-sensitive probe, and its luminescence lifetime is greater than 1000 ns. The temperature-sensitive probe in this embodiment is composed of gold nanoparticles combined with the surface ligand HTT and embedded in PMMA-co-MAA to form p-AuNP@HTT. After injecting the temperature-sensitive probe p-AuNP@HTT into the plant, it needs to be cultured for a period of time, such as 7-10 hours. Preferably, it is cultured for 8 hours to make the temperature-sensitive probe p-AuNP@HTT evenly distributed in the plant to be measured.

[0050] Then, enter step S102, where the excitation light excites the thermosensitive probe in the plant body, causing the plant body containing the thermosensitive probe to emit a luminescence signal, and the luminescence signal is the luminescence intensity.

[0051] In the present invention, existing excitation devices and acquisition devices can be used, such as a laser confocal microscope. The excitation light can be pulsed laser, and the wavelength can be set according to the type of thermosensitive probe. The type of the luminescence signal emitted by the whole plant body will change with the passage of time. For example, during the period from the initial moment to the end of the luminescence lifetime of the plant's autofluorescence, the luminescence signal includes the plant's autofluorescence and the light emitted by the thermosensitive probe, and the two are mixed together and difficult to distinguish. When the time exceeds the luminescence lifetime of the plant's autofluorescence, the plant's autofluorescence decays, and only the light of the thermosensitive probe with a luminescence lifetime longer than that of the plant's autofluorescence remains in the luminescence signal.

[0052] Based on this, in step S103, the luminescence signal is collected with a time delay. By collecting the luminescence signal with a time delay, the interference of the plant's autofluorescence can be filtered out. The length of the time delay is set to be greater than or equal to the luminescence lifetime of the plant's autofluorescence and less than the luminescence lifetime of the thermosensitive probe. For example, the length of the time delay is set to be delay ≥ 10 ns. Specifically, the delay is set to 50 ns, 100 ns, 300 ns or 500 ns.

[0053] In the research on thermosensitive materials, the inventors found that gold nanostructures have excellent biocompatibility, are easy to synthesize, have adjustable sizes and unique physical and chemical properties. Gold nanostructures with luminescence properties usually have a particle size less than 2 nm, and such nanomaterials are also called gold nanoclusters (AuNCs).

[0054] Gold nanostructures with a size greater than 2 nm are also called gold nanoparticles (AuNPs), and the particle size ranges from 2 to 100 nm. Due to the presence of surface plasmon resonance (SPR) on their surfaces, almost no photoluminescence properties are observed. However, with the development of technology and in-depth research, it is found that some gold nanoparticles with a size greater than 2 nm can also have photoluminescence properties. Such gold nanoparticles with photoluminescence properties are called luminescent gold nanoparticles, that is, L-AuNPs. The screening of luminescent gold nanoparticles can be obtained through a simple photoluminescence test, which will not be elaborated in the present invention.

[0055] Example 1.

[0056] Prepare the thermosensitive probe p-AuNP@HTT, which includes two main steps:

[0057] Step 1, Preparation of the precursor AuNP@HTT of the thermosensitive probe:

[0058] Prepare an ethanol solution of HAuCl4 (10 mM) (Solution A), an ethanol solution of NaOH (0.2 M) (Solution B), and a chloroform solution of HTT (20 μmol / L) (Solution C) respectively.

[0059] At 25 °C, mix Solution A (1.0 mL), Solution B (200 μL), and Solution C (9.0 mL), and stir magnetically (100 rpm) for 15 minutes.

[0060] Irradiate with an ultraviolet lamp (390 nm, 8 W) for 60 hours, add the above reaction mixture to cold ethanol at -20 °C, and store it in a refrigerator (4 °C) until no more yellow precipitate forms.

[0061] After the reaction, carefully collect the precipitate by centrifugation at 4000 rpm for 15 minutes, and wash it three times with anhydrous ethanol to obtain the final product AuNP@HTT.

[0062] Among them, the surface ligand HTT is 2-n-Hexylthio-1,3,4-thiadiazole-5-thiol.

[0063] Step 2, Preparation of the thermosensitive probe p-AuNP@HTT:

[0064] Mix 1.0 mL of a solution of PMMA-co-MAA copolymer in tetrahydrofuran (concentration 1.0 mg / mL) and 1.0 mL of a solution of AuNP@HTT in tetrahydrofuran (concentration 1.0 g / mL) to obtain a mixture.

[0065] Slowly add the above mixture drop by drop to 20 mL of ultrapure water at 37 °C, keep stirring continuously during the dropping process, and then let it stand and react for 10 minutes.

[0066] Remove tetrahydrofuran by a vacuum rotary evaporator at 45 °C, and centrifuge through an ultrafiltration tube and make up the volume to obtain p-AuNP@HTT with a certain concentration.

[0067] Among them, the PMMA-co-MAA copolymer, Polymethylmethacrylate-co-methacrylic acid, is called poly(methyl methacrylate-co-methacrylic acid) in Chinese.

[0068] Example 2.

[0069] Measure the spectrum of p-AuNP@HTT:

[0070] Figure 2 Schematic diagram showing the photoluminescence spectrum of p-AuNP@HTT according to an embodiment of the present invention.

[0071] The measurement temperature of this photoluminescence spectrum is 25 °C. The abscissa is the wavelength in nm, and the ordinate is the relative luminescence intensity (dimensionless).

[0072] From the photoluminescence spectrum of p-AuNP@HTT, it can be seen that the wavelength corresponding to the maximum luminescence intensity is located at 543 nm.

[0073] Example 3.

[0074] Measure the particle size of p-AuNP@HTT:

[0075] Perform transmission electron microscopy imaging on p-AuNP@HTT. Drop a p-AuNP@HTT solution with a concentration of 0.01 mg / mL onto a copper mesh of an ultra-thin microgrid membrane, dry it in the dark and naturally. Use a TECNAI F30 field emission transmission electron microscope for imaging. Statistically analyze the size distribution of the gold core particles of p-AuNP@HTT.

[0076] Figure 3 Shows the transmission electron micrograph of p-AuNP@HTT according to an embodiment of the present invention.

[0077] Figure 4 Shows the statistical histogram of the particle distribution of p-AuNP@HTT according to an embodiment of the present invention.

[0078] As Figure 3 and Figure 4 shown, the size of the gold core of p-AuNP@HTT is 23.89 ± 4.08 nm (N = 100), and the particle morphology distribution is uniform and the crystal structure is clear and complete.

[0079] Example 4.

[0080] Measure the temperature sensitivity of the luminescence intensity of p-AuNP@HTT:

[0081] Figure 5 Shows the schematic diagram of the change of the full-wavelength luminescence intensity of p-AuNP@HTT with temperature according to an embodiment of the present invention. The abscissa is the wavelength in nanometers (nm), and the ordinate is the photoluminescence intensity. Different curves represent the change of the photoluminescence intensity of p-p-AuNP@HTT at different temperatures in the range from 5 °C to 60 °C, and the temperature gradient is 5 °C.

[0082] As Figure 5As shown, the change in the luminescence intensity of p-AuNP@HTT with temperature (excitation wavelength: 405 nm, slit width Ex: 5 nm, Em: 2.5 nm). When the temperature is increased from 5 °C to 60 °C, the luminescence peak of the temperature-sensitive probe p-AuNP@HTT does not shift significantly, but its luminescence intensity decreases correspondingly with the temperature increase process.

[0083] Figure 6 The schematic fitting curve of the relative luminescence intensity at the emission peak of p-AuNP@HTT according to an embodiment of the present invention with respect to temperature is shown. The fitting function is y = -1.22x + 104.42, and the correlation coefficient between the relative luminescence intensity and temperature is R 2 = 0.99.

[0084] As Figure 6 shown, the fitting curve of the luminescence intensity of p-AuNP@HTT with temperature. When the temperature is increased from 5 °C to 60 °C, the luminescence intensity of p-AuNP@HTT gradually decreases and shows a linear relationship with the temperature change: for every 1 °C increase in temperature, the luminescence intensity decreases by 1.22%. It can be seen that the luminescence intensity of p-AuNP@HTT has good temperature sensitivity.

[0085] Figure 7 The schematic diagram of the change in the relative luminescence intensity of p-AuNP@HTT with temperature under the delay (100 ns) acquisition condition according to an embodiment of the present invention is shown. The abscissa is the ambient temperature, with the unit of °C, and the ordinate is the relative luminescence intensity (dimensionless relative quantity).

[0086] As Figure 7 shown, in the delay acquisition mode of the time-gated imaging system, after a delay of 100 ns, when the temperature is increased from 20 °C to 55 °C, the luminescence intensity of p-AuNP@HTT gradually decreases with the increase in ambient temperature and still shows a good linear relationship: the fitting function is y = -1.23x + 130.92, and the correlation coefficient between the relative luminescence intensity and temperature is R 2 = 0.99. For every 1 °C increase in temperature, the luminescence intensity decreases by 1.23%. Therefore, p-AuNP@HTT is suitable for detecting temperature in time-gated imaging.

[0087] Example 6.

[0088] Measure the luminescence lifetime of p-AuNP@HTT:

[0089] Figure 8 The schematic diagram of the luminescence lifetime of p-AuNP@HTT according to an embodiment of the present invention is shown.

[0090] As Figure 8As shown, the luminescence lifetime curve of p-AuNP@HTT at 25 °C. After triple-exponential fitting, its luminescence lifetimes are τ1 = 38.56 ns (5.80%); τ2 = 295.81 ns (25.25%); τ3 = 1472.71 ns (68.95%), and the average fluorescence lifetime is 1092.36 ns. Thus, the luminescence lifetime of p-AuNP@HTT is much greater than the lifetime of plant autofluorescence, and time-gated imaging can be used to remove the interference of plant autofluorescence.

[0091] Example 7.

[0092] Measure the pH stability of p-AuNP@HTT:

[0093] To explore the stability of the luminescence intensity of p-AuNP@HTT in the plant physiological pH range, the pH of the p-AuNP@HTT solution was adjusted to six gradients of 5 - 10 with a buffer solution, and the luminescence intensity of p-AuNP@HTT under different pH conditions was measured.

[0094] Figure 9 The schematic diagram shows the stability of the luminescence intensity of p-AuNP@HTT in the plant physiological pH range according to the embodiment of the present invention.

[0095] As Figure 9 shown, in the range of pH 5 - 10, with the increase of pH, the luminescence intensity of p-AuNP@HTT is relatively stable. The pH inside the plant is in the range of 5 - 8. Therefore, the luminescence intensity of p-AuNP@HTT will not be interfered by pH within the plant physiological range, ensuring the accuracy of the measurement results.

[0096] Example 8.

[0097] Measure the Ca 2+ stability of p-AuNP@HTT:

[0098] Ca 2+ acts as a second messenger to participate in the response of plants to environmental temperature changes during temperature stress. Therefore, excluding the influence of Ca 2+ ion concentration changes on the luminescence intensity of p-AuNP@HTT is crucial for the accuracy of the acquisition results. Measure the change of the luminescence intensity of p-AuNP@HTT with Ca 2+ concentration (10 -8 -10 -2 mol / L).

[0099] Figure 10 The schematic diagram shows the change of the luminescence intensity of p-AuNP@HTT with Ca 2+ concentration according to the embodiment of the present invention.

[0100] As Figure 10 shown, within the range of calcium ion concentration from 10 -8 - 10 -2 mol / L, the luminescence intensity of p - AuNP@HTT remains stable. According to the literature reports, the concentration of free calcium ions in plants is between 10 -7 - 10 -3 mol / L, and the concentration of free calcium ions at the cell wall position can reach 10 -4 - 10 -3 mol / L. Therefore, within this concentration range, it will not affect the luminescence intensity of p - AuNP@HTT, ensuring the accuracy of the measurement results.

[0101] Example 9.

[0102] Measure the Na + , K + , Mg 2+ ion stability of p - AuNP@HTT:

[0103] Figure 11 shows the schematic diagram of the change of the luminescence intensity of p - AuNP@HTT according to the embodiment of the present invention with the concentration of Na + .

[0104] Figure 12 shows the schematic diagram of the change of the luminescence intensity of p - AuNP@HTT according to the embodiment of the present invention with the concentration of K + .

[0105] Figure 13 shows the schematic diagram of the change of the luminescence intensity of p - AuNP@HTT according to the embodiment of the present invention with the concentration of Mg 2+ .

[0106] It can be seen that within the plant physiological range, the luminescence intensity of p - AuNP@HTT does not change with the concentrations of Na + , K + and Mg 2+ , showing very good ion stability of p - AuNP@HTT and ensuring the accuracy of the measurement results.

[0107] Example 10.

[0108] Measure the internal temperature of tobacco leaves with the p - AuNP@HTT thermosensitive probe:

[0109] Inject 50 μl of p-AuNP@HTT with a concentration of 80 μg / ml into tobacco leaves using a syringe. After 8 hours, place the tobacco in a temperature-controlled chamber. Use a PI-MAX4 time-gated imaging system to completely filter out the autofluorescence background of the plant at a delay of 100 ns. While changing the ambient temperature, collect and record in real-time the luminescence intensity of p-AuNP@HTT in the tobacco leaves and the change in ambient temperature. After the collection is completed, analyze the correspondence between the change in internal temperature of the tobacco leaves and the change in ambient temperature.

[0110] Figure 14 Figure showing the change in the luminescence intensity of the p-AuNP@HTT thermosensitive probe inside tobacco under high-temperature stress with the change in ambient temperature according to an embodiment of the present invention.

[0111] Figure 15 Figure showing the correspondence between the change in internal temperature of tobacco and the change in ambient temperature under high-temperature stress according to an embodiment of the present invention.

[0112] Figure 16 Figure showing the change in the luminescence intensity of the p-AuNP@HTT thermosensitive probe inside tobacco under low-temperature stress with the change in ambient temperature according to an embodiment of the present invention.

[0113] Figure 17 Figure showing the correspondence between the change in internal temperature of tobacco and the change in ambient temperature under low-temperature stress according to an embodiment of the present invention.

[0114] As Figure 14 shown, the luminescence intensity (relative fluorescence intensity, dimensionless) of the p-AuNP@HTT thermosensitive probe inside tobacco under high-temperature stress changes with the change in ambient temperature: as the ambient temperature increases, the luminescence intensity of the probe decreases, and vice versa. In the range of 0 - 300 s, the luminescence intensity of the probe remains constant with the ambient temperature; in the range of 300 - 800 s, the luminescence intensity of the probe gradually decreases as the ambient temperature increases. The change in the luminescence intensity of the probe is synchronous with the change in ambient temperature, indicating that the p-AuNP@HTT thermosensitive probe has a fast time response to temperature changes and can be used for detecting temperature rapid change events / phenomena.

[0115] As Figure 15 shown, the amplitude of the change in ambient temperature is higher than that of the change in internal temperature of the plant, reflecting that there may be a temperature adaptation mechanism in tobacco to "resist" the change in ambient temperature, which may be related to the self-protection mechanism of the plant.

[0116] Figure 16 and Figure 17 Figures showing the correspondence between the change in internal temperature of tobacco and the change in ambient temperature under low-temperature stress. Similar to the results of high-temperature stress, there may be a low-temperature stress adaptation mechanism in tobacco to "resist" the change in ambient low temperature.

[0117] In an embodiment of the present invention, a method for detecting the internal temperature by inputting a temperature-sensitive probe into a plant body to detect a luminescence signal realizes non-destructive detection of the plant body. Through delayed detection, the influence of the autofluorescence of the plant body is filtered out. The temperature-sensitive probe p-AuNP@HTT in the embodiment of the present invention has good temperature sensitivity, pH stability and calcium salt stability, and can improve the accuracy of the internal temperature of the plant body.

[0118] Although multiple embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes and alternative forms may be contemplated by those skilled in the art without departing from the spirit and scope of the present invention. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed in practicing the present invention. The appended claims are intended to define the scope of the present invention and thus cover equivalents or alternatives within the scope of these claims.

Claims

1. A method for detecting the internal temperature of a plant body based on p-AuNP@HTT, characterized in that, Including: Preparing a temperature-sensitive probe p-AuNP@HTT; Injecting the temperature-sensitive probe p-AuNP@HTT into a plant to obtain a plant body containing the temperature-sensitive probe p-AuNP@HTT; Exciting the temperature-sensitive probe p-AuNP@HTT to make the temperature-sensitive probe p-AuNP@HTT emit a luminescence signal; Delayed acquisition of the luminescence signal to obtain a sample signal; Determining the temperature in the plant according to the sample signal; Wherein, the delay is less than the luminescence lifetime of the temperature-sensitive probe p-AuNP@HTT and greater than the luminescence lifetime of the autofluorescence of the plant body.

2. The method according to claim 1, wherein The luminescence signal is luminescence intensity.

3. The method according to claim 1, wherein The luminescence lifetime of the temperature-sensitive probe p-AuNP@HTT > 1000 ns.

4. The method according to claim 1, wherein The delay is set to ≥ 10 ns.

5. The method according to claim 4, wherein The delay is set to 100 ns.

6. The method according to claim 1, wherein The temperature-sensitive probe p-AuNP@HTT is prepared by the following method: Mixing a solution of PMMA-co-MAA copolymer in tetrahydrofuran with a solution of AuNP@HTT in tetrahydrofuran to obtain a first mixture; Dropwise adding the first mixture into ultrapure water while continuously stirring during the dropping process to obtain a second mixture; Letting the second mixture stand to obtain a third mixture; Removing tetrahydrofuran from the third mixture to obtain an aqueous solution of p-AuNP@HTT.

7. The method according to claim 6, wherein During the process of dropwise adding the first mixture into ultrapure water, the temperature is maintained at 35 - 40 °C.

8. The method according to claim 6, wherein During the process of removing tetrahydrofuran from the third mixture, the temperature is maintained at 40 - 50 °C.

9. The method according to claim 6, wherein The AuNP@HTT is prepared by the following method: Respectively preparing solution A: an ethanol solution of HAuCl4, solution B: an ethanol solution of NaOH, and solution C: a chloroform solution of HTT; Mixing solution A, solution B, and solution C and stirring for 15 minutes to obtain a first mixture; Irradiating the first mixture with a UV lamp having a wavelength of 390 nm and a power of 8 w for 60 hours to obtain a second mixture; Adding the second mixture to cold ethanol at -20 °C to obtain a third mixture; Storing the third mixture at 4 °C until no yellow precipitate is formed in the third mixture to obtain a fourth mixture; Separating the precipitate in the fourth mixture to obtain AuNP@HTT.

10. The method according to claim 9, wherein The concentration of solution A is 10 mM, the concentration of solution B is 0.2 M, and the concentration of solution C is 20 μmol / L; The volume ratio of solution A, solution B, and solution C is 5:1:45.

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