Method for detecting internal temperature of plant body based on L-AuNP-coated TMT and temperature-sensitive probe

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 L-AuNP (at) TMT and a temperature-sensitive probe, and the method comprises the steps: preparing the temperature-sensitive probe L-AuNP (at) TMT; 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 L-AuNP-TMT and 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 L-AuNP (at) TMT in the embodiment of the invention has good temperature sensitivity, relatively good pH stability and calcium salt stability, and can improve the accuracy of the internal temperature of a 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 and a temperature-sensitive probe for detecting the internal temperature of plants based on L-AuNP@TMT. Background Art

[0002] Temperature is a key environmental factor for plant growth and development, 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 inside plants. Therefore, accurately measuring the internal temperature of plants is crucial for studying plant temperature adaptability and regulatory mechanisms, helps to deeply understand the response rules and molecular mechanisms of plants to temperature, and further helps to improve food yield and quality and ensure food security.

[0003] However, currently used 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, due to direct contact with the measured part, easily change the measured environment, cause damage to the measured part, and affect the accuracy of the temperature measurement result. 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, and their physiological activities have highly dynamic changes. Therefore, internal temperature measurement must be non-destructive, and the interference with the normal physiological process 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 light and near-infrared light 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 L-AuNP@TMT, including: preparing a temperature-sensitive probe L-AuNP@TMT with a particle size range of 2.42±0.53 nm; injecting the temperature-sensitive probe L-AuNP@TMT into the plant body to obtain a plant body containing the temperature-sensitive probe L-AuNP@TMT; exciting the temperature-sensitive probe L-AuNP@TMT to make the temperature-sensitive probe L-AuNP@TMT emit a luminescence signal; collecting the luminescence 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 L-AuNP@TMT and greater than the luminescence lifetime of the autofluorescence of the plant body.

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

[0009] According to an embodiment of the present invention, the luminescence lifetime of the temperature-sensitive probe L-AuNP@TMT > 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, preparing a temperature-sensitive probe L-AuNP@TMT with a particle size range of 2.42±0.53 nm includes: dropping an aqueous solution of TMT into distilled water to obtain a first mixture; dropping an aqueous solution of sodium hydroxide into the first mixture, and continuously stirring during the dropping process to obtain a second mixture; dropping an aqueous solution of chloroauric acid into the second mixture, and continuously stirring during the dropping process to obtain a third mixture; irradiating the third mixture with an ultraviolet lamp to obtain a fourth mixture; separating the precipitate in the fourth mixture to obtain the temperature-sensitive probe L-AuNP@TMT.

[0013] According to an embodiment of the present invention, the wavelength of the ultraviolet lamp is 390 nm, the power is 8 w, and the irradiation time is 10 minutes.

[0014] According to an embodiment of the present invention, during the dropping of sodium hydroxide and the dropping of chloroauric acid, the temperature is controlled at 25°C.

[0015] According to an embodiment of the present invention, the molar ratio of TMT, sodium hydroxide, and chloroauric acid is 10:1:5.

[0016] According to another aspect of the present invention, a temperature-sensitive probe for detecting the internal temperature of a plant body is provided. The particle size range of the temperature-sensitive probe is 2.42 ± 0.53 nm, and it is prepared by the following photocatalytic reduction method: Drop the aqueous solution of TMT into distilled water to obtain a first mixture, then drop the aqueous solution of sodium hydroxide into the first mixture while continuously stirring during the dropping process to obtain a second mixture; Drop the aqueous solution of chloroauric acid into the second mixture while continuously stirring during the dropping process to obtain a third mixture; Irradiate the third mixture with an ultraviolet lamp to obtain a fourth mixture; Separate the precipitate in the fourth mixture to obtain the temperature-sensitive probe L-AuNP@TMT.

[0017] In an embodiment of the present invention, non-destructive detection is achieved by inputting the temperature-sensitive probe into the plant body to detect the fluorescence signal. Through delayed detection, the influence of autofluorescence is filtered out. The temperature-sensitive probe L-AuNP@TMT has good temperature sensitivity of luminescence intensity and can accurately detect the internal temperature measurement result of the plant body. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] By reading the following detailed description with reference to the accompanying 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 Shows a schematic diagram of the method steps for detecting the internal temperature of a plant body according to an embodiment of the present invention;

[0020] Figure 2 Shows a schematic diagram of the photoluminescence spectrum of L-AuNP@TMT according to an embodiment of the present invention;

[0021] Figure 3 Shows a transmission electron microscope image of L-AuNP@TMT according to an embodiment of the present invention;

[0022] Figure 4 Shows a statistical histogram of the particle distribution of L-AuNP@TMT according to an embodiment of the present invention;

[0023] Figure 5 Shows a schematic diagram of the change in the full-wavelength luminescence intensity of L-AuNP@TMT with temperature according to an embodiment of the present invention;

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

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

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

[0027] Figure 9 Shows the schematic diagram of the luminescence intensity of L-AuNP@TMT varying with pH according to an embodiment of the present invention;

[0028] Figure 10 Shows the schematic diagram of the luminescence intensity of L-AuNP@TMT varying with the concentration of Ca 2+ ;

[0029] Figure 11 Shows the schematic diagram of the luminescence intensity of the L-AuNP@TMT thermosensitive probe inside tobacco varying with the ambient temperature under high temperature stress according to an embodiment of the present invention;

[0030] Figure 12 Shows the schematic diagram of the corresponding relationship 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;

[0031] Figure 13 Shows the schematic diagram of the luminescence intensity of the L-AuNP@TMT thermosensitive probe inside tobacco varying with the ambient temperature under low temperature stress according to an embodiment of the present invention;

[0032] Figure 14 Shows the schematic diagram of the corresponding relationship between the temperature change amount inside tobacco and the ambient temperature change amount under low temperature stress according to an embodiment of the present invention. Detailed implementation manners

[0033] 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 of 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.

[0034] It should be understood that the terms "including" and "comprising" 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.

[0035] 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 the context clearly indicates otherwise, 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.

[0036] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0037] Figure 1 The 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.

[0038] 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 delaying the acquisition of the light signal to obtain a sample signal; step S104 of determining the internal temperature of the plant body according to the sample signal; wherein the 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.

[0039] A relatively common method for detecting the internal temperature of a plant body is to inject a temperature-sensitive probe into the plant body, and then excite the temperature-sensitive probe to determine the internal temperature of the plant body by detecting the light signal emitted by the temperature-sensitive probe.

[0040] The inventors have 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 body 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 to remove the interference of background noises such as autofluorescence, reflected light and stray light.

[0041] Table 1 shows the luminescence lifetime table of some luminescent materials.

[0042] Table 1.

[0043]

[0044] It can be seen from Table 1 that the luminescence lifetime of the autofluorescence of the plant body is short. In order to remove the interference of the autofluorescence, the temperature-sensitive probe is prepared from a luminescent material with a luminescence lifetime greater than that of the autofluorescence of the plant body.

[0045] Specifically, a pulsed laser is used in combination with a long-lived 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 collected immediately. Instead, it waits for the fluorescence to decay for a period of time (such as 20 ns or 50 ns). After the background noise such as autofluorescence has completely decayed, the luminescence signal of the long-lived fluorescent probe is collected. This technique is called time-gated fluorescence imaging. By not detecting short-lived signals and only detecting long-lived signals, high-contrast imaging of long-lived fluorescent (or luminescent) substances can be obtained. Then, by using multi-cycle signals to increase the signal-to-noise ratio, the imaging quality can be improved and the interference of short-lived background fluorescence (or other luminescence) on the image can be reduced.

[0046] Based on this, in an embodiment of the present invention, the selection criterion for the temperature-sensitive probe is that the luminescence lifetime of the temperature-sensitive probe is greater than the lifetime of the autofluorescence of the plant body. The luminescence lifetime of the temperature-sensitive probe refers to the luminescence lifetime of the luminescent material contained in the temperature-sensitive probe. For example, the luminescent gold nanoparticles L-AuNP@TMT with a surface ligand of TMT are selected as the temperature-sensitive probe, and its luminescence lifetime is greater than 1000 ns. After injecting the temperature-sensitive probe L-AuNP@TMT into the plant body, 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 L-AuNP@TMT evenly distributed in the plant body to be measured.

[0047] Then, step S102 is entered, and the temperature-sensitive probe in the plant body is excited by the excitation light, so that the plant body containing the temperature-sensitive probe emits a luminescence signal, and the luminescence signal is the luminescence intensity.

[0048] In the present invention, existing excitation equipment and acquisition equipment can be used. For example, a laser confocal microscope can be used. The excitation light can be a pulsed laser, and the wavelength can be set according to the type of the temperature-sensitive 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 autofluorescence of the plant body, the luminescence signal includes the autofluorescence of the plant body and the light emitted by the temperature-sensitive probe, and the two are mixed together and difficult to distinguish. When the time exceeds the luminescence lifetime of the autofluorescence of the plant body, the autofluorescence of the plant body decays, and only the light of the temperature-sensitive probe with a luminescence lifetime greater than the luminescence lifetime of the autofluorescence of the plant body remains in the luminescence signal.

[0049] 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 autofluorescence of the plant body can be filtered out. The time length of the time delay is set to be greater than or equal to the luminescence lifetime of the autofluorescence of the plant body and less than the luminescence lifetime of the temperature-sensitive probe. For example, the length of the time delay is set to be time delay ≥ 10 ns. Specifically, the time delay is set to 50 ns, 100 ns, 300 ns, or 500 ns.

[0050] In the research on temperature-sensitive 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 luminescent properties usually have a particle size of less than 2 nm, and such nanomaterials are also called gold nanoclusters (AuNCs).

[0051] Gold nanostructures with a size greater than 2 nm, also called gold nanoparticles (AuNPs), have a particle size range of 2 - 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 has been 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 this invention.

[0052] In the embodiment of the present invention, the temperature-sensitive probe L-AuNP@TMT has a particle size of about 2.4 nm, which is greater than 2 nm and belongs to the category of luminescent gold nanoparticles (L-AuNPs).

[0053] According to an embodiment of the present invention, the temperature-sensitive probe L-AuNP@TMT is prepared by the following method: dropping an aqueous solution of the surface ligand TMT into distilled water to obtain a first mixture; dropping an aqueous solution of sodium hydroxide into the first mixture and continuously stirring during the dropping process to obtain a second mixture; dropping an aqueous solution of chloroauric acid into the second mixture and continuously stirring during the dropping process to obtain a third mixture; reacting the third mixture for 7 min, then irradiating the third mixture with an ultraviolet lamp and continuing to react for 10 minutes to obtain a fourth mixture; separating the precipitate in the fourth mixture, such as by centrifugation using an ultrafiltration tube, washing it three times with distilled water, and making up the volume, etc., to obtain the temperature-sensitive probe L-AuNP@TMT.

[0054] According to an embodiment of the present invention, the temperature detection range inside plants is 0 - 60 °C

[0055] In the embodiment of the present invention, a method for non-destructively detecting plants is achieved by inputting the temperature-sensitive probe into the plants and detecting the luminescence signal to obtain the internal temperature. By delaying the detection, the influence of the autofluorescence of the plants is filtered out.

[0056] Example 1.

[0057] Preparation of the temperature-sensitive probe L-AuNP@TMT:

[0058] First, prepare solutions of TMT (5-mercapto-1,3,4-thiadiazole-2-ylthio) acetic acid (20 mM) (Solution A),

[0059] NaOH solution (500 mM) (Solution B) and an aqueous solution of HAuCl4 (10 mM) (Solution C) respectively;

[0060] Next, at 25 °C, add dropwise Solution A (1.0 mL), Solution B (200 μL) and Solution C (1.0 mL) successively to 7.8 mL of distilled water, and stir magnetically (300 rpm) for 7 minutes;

[0061] Then, irradiate with an ultraviolet lamp (390 nm, 8 W) for 10 minutes;

[0062] After the reaction is completed, centrifuge the above mixed solution through a filter sieve at 4000 rpm for 60 minutes, wash the precipitate three times with distilled water, and make up the volume to obtain the final product L-AuNP@TMT.

[0063] Among them, @ represents a core-shell structure, with the former being the core and the latter being the shell. L represents luminescence, luminescent.

[0064] The surface ligand TMT is: (5-mercapto-1,3,4-thiadiazole-2-ylthio) acetic acid, and its English name is (5-Mercapto-1,3,4-thiadiazole-2-ylthio)acetic Acid.

[0065] Example 2.

[0066] Measure the photoluminescence spectrum of L-AuNP@TMT:

[0067] Figure 2 The schematic diagram of the photoluminescence spectrum of L-AuNP@TMT according to the embodiment of the present invention is shown.

[0068] 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).

[0069] It can be seen from the photoluminescence spectrum of L-AuNP@TMT that the wavelength corresponding to the maximum luminescence intensity is located at 543 nm.

[0070] Example 3.

[0071] Measure the particle size of L-AuNP@TMT:

[0072] For the transmission electron microscopy (TEM) imaging of L-AuNP@TMT, a solution of L-AuNP@TMT with a concentration of 0.01 mg / mL was dropped onto a copper mesh of an ultrathin microgrid membrane, dried naturally in the dark, and imaged using a TECNAI F30 field emission transmission electron microscope. The size distribution of the gold core particles of L-AuNP@TMT was statistically analyzed.

[0073] Figure 3 The TEM image of L-AuNP@TMT according to an embodiment of the present invention is shown.

[0074] Figure 4 The statistical histogram of the particle distribution of L-AuNP@TMT according to an embodiment of the present invention is shown.

[0075] As Figure 3 and Figure 4 shown, the size of the gold core of L-AuNP@TMT is 2.42 ± 0.53 nm (N = 100), and the particle morphology distribution is uniform and the crystal structure is clear and complete.

[0076] Example 4.

[0077] Measure the temperature sensitivity of the luminescence intensity of L-AuNP@TMT:

[0078] Figure 5 The schematic diagram of the change in the full-wavelength luminescence intensity of L-AuNP@TMT according to an embodiment of the present invention is shown. The abscissa is the wavelength, with the unit of nanometer (nm), and the ordinate is the photoluminescence intensity (dimensionless). Different curves represent the changes in the photoluminescence intensity of p-L-AuNP@TMT at different temperatures in the range from 0 °C to 60 °C, and the temperature gradient is 5 °C.

[0079] As Figure 5 shown, the change in the luminescence intensity of L-AuNP@TMT (excitation wavelength: 405 nm, slit width Ex: 5 nm, Em: 2.5 nm). When the temperature is increased from 0 °C to 60 °C, the luminescence peak of the temperature-sensitive probe L-AuNP@TMT does not shift significantly, but its luminescence intensity decreases correspondingly with the temperature increase process.

[0080] Figure 6 The schematic diagram of the fitting curve of the relative luminescence intensity at the emission peak of L-AuNP@TMT according to an embodiment of the present invention is shown. The fitting function is y = -1.26x + 100.25, and the correlation coefficient between the relative luminescence intensity and the temperature is R 2 = 0.99.

[0081] As Figure 6As shown, the fitting curve of the luminescence intensity of L-AuNP@TMT versus temperature. As the temperature increases from 5 °C to 60 °C, the luminescence intensity of L-AuNP@TMT gradually decreases and shows a linear relationship with the temperature change: for every 1 °C increase in temperature, the luminescence intensity decreases by 1.26%. It can be seen that the luminescence intensity of L-AuNP@TMT has good temperature sensitivity.

[0082] Figure 7 The figure shows the schematic diagram of the relative luminescence intensity of L-AuNP@TMT versus temperature under the delay (100 ns) acquisition condition according to an embodiment of the present invention. The abscissa is the ambient temperature (the temperature of the solution where the probe is located), with the unit of °C, and the ordinate is the relative luminescence intensity (a dimensionless relative quantity).

[0083] 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 L-AuNP@TMT gradually decreases with the increase of the ambient temperature and still shows a good linear relationship: the fitting function is y = -0.9x + 119, and the correlation coefficient between the relative luminescence intensity and the temperature is R 2 = 0.99. For every 1 °C increase in temperature, the luminescence intensity decreases by 0.9%. Therefore, L-AuNP@TMT is suitable for use in time-gated imaging for temperature detection.

[0084] Example 6.

[0085] Measure the luminescence lifetime of L-AuNP@TMT:

[0086] Figure 8 The figure shows the schematic diagram of the luminescence lifetime of L-AuNP@TMT according to an embodiment of the present invention.

[0087] As Figure 8 shown, at 25 °C, after triple-exponential fitting, the luminescence lifetime of L-AuNP@TMT is τ1 = 99.83 ns (9.12%); τ2 = 622.28 ns (42.52%); τ3 = 2386.83 ns (48.36%), and the average fluorescence lifetime is 1427.96 ns. It can be seen that the luminescence lifetime of L-AuNP@TMT is much longer than the lifetime of plant autofluorescence, and the interference of plant autofluorescence can be removed by time-gated imaging.

[0088] Example 7.

[0089] Measure the pH stability of L-AuNP@TMT:

[0090] To explore the effect of pH on the luminescence intensity of L-AuNP@TMT, the pH of the L-AuNP@TMT solution was adjusted to five gradients of 6-10 with a buffer solution, and the luminescence intensity of L-AuNP@TMT under different pH conditions was measured.

[0091] Figure 9 The schematic diagram of the stability of the luminescence intensity of L-AuNP@TMT according to the embodiment of the present invention in the pH range of 6-10 is shown. The abscissa is pH, and the ordinate is the relative luminescence intensity (relative fluorescence intensity, dimensionless).

[0092] As Figure 9 shown, in the range of pH 6.0-10.0, as the pH increases, the luminescence intensity of L-AuNP@TMT is relatively stable. Therefore, the influence of pH change on the luminescence intensity of L-AuNP@TMT within this pH range can be excluded, and the accuracy of the acquisition result can be improved. Example 8.

[0093] Measure the Ca 2+ stability of L-AuNP@TMT:

[0094] 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 change on the luminescence intensity of L-AuNP@TMT is crucial for the accuracy of the acquisition result. Measure the change of the luminescence intensity of L-AuNP@TMT with the Ca 2+ concentration (10 -5 -10 -9 mol / L).

[0095] Figure 10 The schematic diagram of the change of the luminescence intensity of L-AuNP@TMT according to the embodiment of the present invention with the Ca 2+ concentration is shown. The abscissa is the Ca 2+ concentration mol / L, and the ordinate is the relative luminescence intensity (relative fluorescence intensity, dimensionless).

[0096] As Figure 10 shown, in the range of calcium ion concentration of 10 -5 -10 -9 mol / L, the luminescence intensity of L-AuNP@TMT remains stable. Therefore, the influence of Ca 2+ on the luminescence intensity of L-AuNP@TMT within this concentration range can be excluded, and the accuracy of the acquisition result can be improved.

[0097] Example 9.

[0098] Measure the internal temperature of tobacco leaves with the L-AuNP@TMT thermosensitive probe:

[0099] Inject 50 μl of L-AuNP@TMT with a concentration of 100 μg / ml into the tobacco leaves using a syringe. After 8 hours, place the tobacco in a temperature-controlled box. Using a PI-MAX4 time-gated imaging system, while changing the ambient temperature and completely filtering out the autofluorescence background of the plant with a 100 ns delay, simultaneously collect and record the luminescence intensity of L-AuNP@TMT in the tobacco leaves and the change in ambient temperature. After the collection is completed, analyze the correspondence between the internal temperature change of the tobacco leaves and the ambient temperature change.

[0100] Figure 11 Shows a schematic diagram of the luminescence intensity of the L-AuNP@TMT thermosensitive probe inside tobacco changing with the ambient temperature under high-temperature stress according to an embodiment of the present invention.

[0101] Figure 12 Shows a schematic diagram of the correspondence between the internal temperature change amount of tobacco and the ambient temperature change amount under high-temperature stress according to an embodiment of the present invention.

[0102] Figure 13 Shows a schematic diagram of the luminescence intensity of the L-AuNP@TMT thermosensitive probe inside tobacco changing with the ambient temperature under low-temperature stress according to an embodiment of the present invention.

[0103] Figure 14 Shows a schematic diagram of the correspondence between the internal temperature change amount of tobacco and the ambient temperature change amount under low-temperature stress according to an embodiment of the present invention.

[0104] As Figure 11 shown, the luminescence intensity of the L-AuNP@TMT thermosensitive probe inside tobacco changes with the ambient temperature under high-temperature stress: when 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 - 750 s, the luminescence intensity of the probe gradually decreases as the ambient temperature increases; in the range of 750 - 1200 s, the luminescence intensity of the probe gradually increases as the ambient temperature decreases. The change in the luminescence intensity of the probe is synchronized with the change in the ambient temperature, indicating that the L-AuNP@TMT thermosensitive probe has a fast time response to temperature changes and can be used for detecting rapid temperature change events / phenomena.

[0105] As Figure 12 shown, the amplitude of the ambient temperature change is higher than that of the internal temperature change of the plant, reflecting that there may be a temperature self-adaptive mechanism in the tobacco plant to "resist" the ambient temperature change, which may be related to the plant's self-protection mechanism.

[0106] Figure 13 and Figure 14A schematic diagram showing the correspondence between the change in the temperature inside tobacco and the change in the ambient temperature under low-temperature stress is shown. Similar to the results of high-temperature stress, there may be a low-temperature stress adaptation mechanism in tobacco that can "resist" the change in ambient low temperature.

[0107] In the embodiments of the present invention, a method for obtaining the internal temperature by detecting the luminescence signal by inputting a temperature-sensitive probe into a plant body 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 L-AuNP@TMT 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.

[0108] 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 approaches may occur to 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 L-AuNP@TMT, characterized in that, Comprising: Preparing a temperature-sensitive probe L-AuNP@TMT with a particle size range of 2.42 ± 0.53 nm; Injecting the temperature-sensitive probe L-AuNP@TMT into a plant to obtain a plant body containing the temperature-sensitive probe L-AuNP@TMT; Exciting the temperature-sensitive probe L-AuNP@TMT to make the temperature-sensitive probe L-AuNP@TMT emit a luminescence signal; Delayed acquisition of the luminescence signal to obtain a sample signal; Determining the temperature inside the plant according to the sample signal; Wherein, the delay is less than the luminescence lifetime of the temperature-sensitive probe L-AuNP@TMT and greater than the luminescence lifetime of the autofluorescence of the plant body.

2. The method according to claim 1, characterized in that The luminescence signal is the luminescence intensity.

3. The method according to claim 1, characterized in that The luminescence lifetime of the temperature-sensitive probe L-AuNP@TMT > 1000 ns.

4. The method according to claim 1, characterized in that The delay is set to ≥ 10 ns.

5. The method according to claim 4, characterized in that The delay is set to 100 ns.

6. The method according to claim 1, characterized in that Preparing a temperature-sensitive probe L-AuNP@TMT with a particle size range of 2.42 ± 0.53 nm includes: Dropping an aqueous solution of TMT into distilled water to obtain a first mixture; Dropping an aqueous solution of sodium hydroxide into the first mixture, continuously stirring during the dropping process, to obtain a second mixture; Dropping an aqueous solution of chloroauric acid into the second mixture, continuously stirring during the dropping process, to obtain a third mixture; Irradiating the third mixture with an ultraviolet lamp to obtain a fourth mixture; Separating the precipitate in the fourth mixture to obtain the temperature-sensitive probe L-AuNP@TMT.

7. The method according to claim 6, characterized in that The wavelength of the ultraviolet lamp is 390 nm, the power is 8 w, and the irradiation time is 10 minutes.

8. The method according to claim 6, characterized in that During the dropping of sodium hydroxide and the dropping of chloroauric acid, the temperature is controlled at 25 °C.

9. The method according to claim 6, characterized in that The molar ratio of TMT, sodium hydroxide, and chloroauric acid is 10:1:

5.

10. A temperature-sensitive probe for detecting the internal temperature of a plant body, characterized in that The particle size range of the temperature-sensitive probe is 2.42 ± 0.53 nm, and it is prepared by the following method: Dropping an aqueous solution of TMT into distilled water to obtain a first mixture, Dropping an aqueous solution of sodium hydroxide into the first mixture, continuously stirring during the dropping process, to obtain a second mixture; dropping an aqueous solution of chloroauric acid into the second mixture, continuously stirring during the dropping process, to obtain a third mixture; irradiating the third mixture with an ultraviolet lamp to obtain a fourth mixture; Separating the precipitate in the fourth mixture to obtain the temperature-sensitive probe L-AuNP@TMT.

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