Dye complex, preparation method and application in detection

Abstract

The present application discloses a dye complex, a preparation method and its application in detection, and relates to the technical field of luminescent materials, wherein the dye complex comprises a cyanine dye and a lanthanide nanocrystal that undergoes reversible controlled non-radiative energy transfer with the cyanine dye; wherein the dye complex reversibly responds to pH, and the reversible response of the dye complex to pH causes the luminescence lifetime of the dye complex to change; the present application utilizes lanthanide nanocrystals as energy donors, and designs the cyanine dye of the above-mentioned specific structure as an energy acceptor, and by regulating the non-radiative energy transfer pathway, induces a reversible change in the luminescence lifetime of the lanthanide nanocrystal, thereby enabling in-situ reversible detection of pH in a living body. The structural formula of the cyanine dye is shown in Formula I: Formula I.

Description

Technical Field

[0001] The present application relates to the technical field of luminescent materials, and in particular to a dye complex, a preparation method and application thereof in detection. Background Art

[0002] In physiological processes, pH is an important indicator of many biochemical reactions and is of great significance for understanding the complex metabolic states in elderly comorbidities such as diabetes and cardiovascular disease. The functions of certain immune cells (such as macrophages and T cells) are closely related to pH, and a low pH environment may inhibit their activity, thereby affecting the immune response. In the tumor microenvironment, local pH changes may affect the immune escape of the tumor or the effect of immunotherapy. Therefore, detecting pH can provide background information for immune detection to evaluate the functional status of the immune system. In addition, changes in pH may also affect the concentrations of biomarkers such as lactate, amino acids, and electrolytes, which are important detection indicators in biochemical experiments.

[0003] To achieve in vivo imaging and detection of biomarkers, researchers have used nanocrystals as energy donors and designed energy receptors to detect the concentration of specific biomarkers such as HClO, ONOO-, and glutathione (GSH). However, the core of this detection method is the irreversible reaction between the biomarker and the energy receptor. In these reactions, the chemical structure of the energy receptor is irreversibly changed or new compounds are formed, such as through redox reactions or the formation of covalent bonds, causing the energy receptor to be permanently changed. As the reaction proceeds, the structure of the energy receptor may be destroyed and lose its original function, thereby destroying the energy transfer pathway between the nanocrystal and the energy receptor. This means that after the reaction, even if a new energy receptor is introduced, the original energy transfer efficiency cannot be restored. Therefore, in order to continuously perform detection, new energy receptors need to be continuously replenished. This method has certain advantages in one-time detection, but due to its irreversibility, it is impossible to dynamically monitor the changes in pH in the body.

[0004] Therefore, there is an urgent need to develop reversible energy transfer systems or introduce other technologies to compensate for this limitation. Summary of the invention

[0005] The purpose of the present application is to provide a dye complex, a preparation method and its application in detection to overcome at least one of the above-mentioned defects. The present application creatively designs the structure of cyanine dyes so that the absorption characteristics of cyanine dyes change with changes in pH, and this change is reversible, that is, under different pH conditions, the dye can be restored to its original state and will not be destroyed. In the dye complex formed by cyanine dyes and lanthanide nanocrystals, there is a reversible and controlled non-radiative energy transfer (NET) mechanism between lanthanide nanocrystals and the cyanine dyes, which is realized by the reversible absorption changes of the dyes at different pH values, which means that the energy transfer pathway between lanthanide nanocrystals and the cyanine dyes designed by us can be adjusted when the pH changes. This way of detecting pH is because the change in luminescence lifetime is independent of its aggregation state, thereby achieving concentration-independent detection, which makes the consistency of in vivo and in vitro experiments higher.

[0006] To achieve the above objectives, the present application provides the following technical solutions:

[0007] The first aspect of the present application provides a dye complex, comprising a cyanine dye and a lanthanide nanocrystal that undergoes reversible controlled non-radiative energy transfer with the cyanine dye; wherein the structural formula of the cyanine dye is as shown in Formula I:

[0008] Formula I;

[0009] Wherein, R1 is one of hydrogen or halogen, R2 is one of hydrogen or C1-C5 straight-chain alkyl, R3, R4, R5, R6 are the same or different and are independently selected from C1-C5 alkyl and alkoxy;

[0010] Wherein, the group in Formula I Connecting to the lanthanide in the lanthanide nanocrystal via a non-covalent bond;

[0011] The dye complex responds reversibly to pH, and the reversible response of the dye complex to pH causes the luminescence lifetime of the dye complex to change.

[0012] Specifically, for example, R1 is preferably one of hydrogen, fluorine, chlorine, bromine, and iodine, and R2 is hydrogen or methyl, ethyl, propyl, butyl, or pentyl. R3, R4, R5, and R6 can be independently selected from C1 to C5 straight-chain alkyl or alkoxy groups, that is, each substituent can be, for example, methyl (-CH3), ethyl (-C2H5), propyl (-C3H7), butyl (-C4H9), pentyl (-C5H 11) or their corresponding alkoxy forms, such as methoxy (-OCH3), ethoxy (-OC2H5), etc. When an alkyl group is selected, the substituent is in the form of a hydrocarbon chain, while when an alkoxy group is selected, there will be an oxygen atom attached to the hydrocarbon chain. In addition, R3, R4, R5, and R6 can be the same or different, which means that in the same molecule, these four sites can use exactly the same substituent, such as all ethyl, or they can be different, such as R3 is methyl, R4 is ethoxy, R5 is propyl, and R6 is butoxy.

[0013] In the dye complex provided above, the inventor creatively designed and synthesized the structure described in Formula I for the first time. In this structure, the structure of the benzoic acid connected to R2 and the position and group of the amino group on the five-membered ring connected to R5 and R6 are crucial to achieve pH reversible response. This type of structure has not yet appeared in the current structural reports related to cyanine dyes. This is because the structure of the benzoic acid connected to R2 is mainly connected to the lanthanide elements in the lanthanide nanocrystals through non-covalent bonds, and the benzoic acid group is used to enhance its non-radiative energy transfer with the surface of the nanoparticles, such as hydrogen bonds, electrostatic interactions or van der Waals forces. This connection mode can not only enhance the non-radiative energy transfer between the benzoic acid group and the surface of the nanoparticles, but also provide unique advantages for the application of dyes. A significant advantage of non-covalent bond connection is its dynamic adjustability, which enables the dye to achieve reversible in-situ detection when the environment changes (such as pH changes). This property is very critical for real-time monitoring of changes in environmental conditions, especially in complex biological or chemical systems. The pH response mechanism of cyanine dyes is based on the protonation of the nitrogen atom in the benzyl indole group (the nitrogen atom on the five-membered ring connected to R5 and R6). This protonation and deprotonation process will respond to pH, but pH response alone is not enough. This pH response can also cause the luminescence lifetime of the dye complex to change, thereby establishing a pH reversible in situ detection method based on luminescence lifetime. Therefore, only hydrogen can be connected to the nitrogen atom on the five-membered ring connected to R5 and R6, and the one connected to the tail end of R2 must be a benzoic acid group to achieve this chain response mechanism.

[0014] In addition, in order to achieve in vivo luminescence lifetime imaging, the embodiments of the present application use lanthanide nanocrystals with a long luminescence lifetime. The different energy levels of lanthanide ions in the lanthanide nanocrystals allow adjustment of the excitation and emission wavelengths, and due to the 4f-4f transition and the shielding of external electrons, the lanthanide nanocrystals exhibit a controllable long luminescence lifetime (microseconds to milliseconds) that is not affected by the external environment. In addition, they have excellent photostability, which helps prevent photodamage.

[0015] Based on these advantages, nanocrystals of different sizes, structures and lanthanide ion doping can be designed to obtain different luminescence lifetimes for multiple in vivo imaging. However, this stable luminescence also brings challenges to the detection of pH. In order to solve this problem, the present application uses lanthanide nanocrystals as energy donors and designs the above-mentioned cyanine dyes with specific structures as energy acceptors. By regulating the non-radiative energy transfer pathway, the reversible change of the luminescence lifetime of lanthanide nanocrystals is induced, thereby enabling in situ reversible detection of pH in vivo.

[0016] Furthermore, the structural formula of the cyanine dye is preferably as shown in Formula II:

[0017] Formula II;

[0018] That is, R1 in Formula I is selected from chlorine, R2 is selected from methyl, and R3, R4, R5, and R6 are selected from methyl. In this preferred structure, the chlorine atom is a common halogen substituent, and the introduction of chlorine usually does not require harsh reaction conditions, making the entire synthesis process more gentle and efficient. In addition, the methyl group is a small and simple alkyl group. The introduction of the methyl group will not significantly increase the complexity of the synthesis steps, nor will it introduce a large steric hindrance, which can reduce the selectivity problem in the reaction step and improve the yield and purity.

[0019] Furthermore, the lanthanide nanocrystals are preferably a matrix material and a doping element comprising a lanthanide fluoride; wherein the lanthanide fluoride is preferably one of NaYF4 or NaGdF4, and the doping element is preferably at least one of Tm, Yb, Er, and Eu. As for the selection of matrix materials, both NaYF4 and NaGdF4 are highly stable fluoride materials with good chemical inertness and thermal stability. In addition, both also have a suitable crystal structure that can accommodate doping ions of different types and concentrations. As for the selection of doping elements, at least one of Tm, Yb, Er, and Eu is preferred. These doping ions have good energy transfer efficiency, which can improve the luminescence efficiency of the nanocrystals, so that they can still achieve brighter luminescence at low excitation power.

[0020] Furthermore, in the lanthanide nanocrystals, the molar percentage of the doping element is preferably 0.1%-2%. Within this range, the luminescence efficiency can be maximized without significantly introducing a quenching effect.

[0021] Furthermore, the dye complex further comprises: a surfactant connected to the surface of the lanthanide nanocrystal by non-covalent bonds.

[0022] Furthermore, the surfactant is preferably 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000 (DSPE-PEG2000). The surfactant DSPE-PEG2000 is connected to the surface of the nanocrystals through non-covalent bonds, which can effectively improve the dispersibility of the nanocrystals in the aqueous phase and other solvents. This is because the hydrophilicity of the PEG chain can prevent the aggregation and precipitation between the nanocrystals and maintain their stable dispersion state. In addition, the protective layer formed by the surfactant can reduce the impact of the environment on the nanocrystals, protect their optical properties from the influence of the external environment such as changes in pH and ionic strength, thereby maintaining their luminous efficiency.

[0023] In a second aspect of the embodiments of the present application, a method for preparing a dye complex is provided, the method comprising: allowing the cyanine dye and the lanthanide nanocrystal to self-assemble. The self-assembly process generally does not require complex chemical reaction steps or condition control, and can simplify the preparation process of the complex. This method generally relies on non-covalent interactions, such as electrostatic effects, van der Waals forces, or π-π interactions, thereby reducing the need for harsh reaction conditions.

[0024] Furthermore, the cyanine dye is prepared by chemically reacting the following compound 1, compound 2 and compound 3:

[0025] , , ;

[0026] Among them, R1 is one of hydrogen or halogen, R2 is one of hydrogen or C1-C5 straight-chain alkyl, R3, R4, R5, R6 are the same or different and are independently selected from C1-C5 alkyl and alkoxy.

[0027] Furthermore, the formula II is prepared by chemical reaction of the following compound 4, compound 5 and compound 6:

[0028] , , .

[0029] Furthermore, the molar ratio of compound 1, compound 2 and compound 3 is (0.5-1.5):(0.5-1.5):(0.5-1.5); the chemical reaction is carried out under an inert gas atmosphere, and the reaction temperature is 40°C-60°C, and the reaction time is 4 hours-6 hours.

[0030] Furthermore, the molar ratio of compound 1, compound 2 and compound 3 is 1:1:1, the reaction temperature is 50° C., and the reaction time is 5 hours.

[0031] In some preferred embodiments, the preparation method of the lanthanide nanocrystals includes: adding YCl3 and TmCl3 to a mixture of liquid unsaturated fatty acids and liquid olefins to obtain a first mixture; stirring the first mixture at a first temperature under an inert gas atmosphere until it becomes transparent; cooling the first mixture to a second temperature and keeping it transparent, and then dropping a first solution dissolved with NH4F and NaOH into the first mixture to obtain a second mixture; heating the second mixture to a third temperature and keeping it for a first target time to remove the solvent, and then degassing for a second target time; heating the second mixture from which the solvent has been removed to a fourth temperature while stirring and keeping it for a third target time, then cooling it to room temperature, and collecting the lanthanide nanocrystals by centrifugation.

[0032] Preferably, the method for preparing lanthanide nanocrystals satisfies at least one of the following conditions: (1) the molar ratio of YCl3 to TmCl3 is (90-100):1; (2) the liquid unsaturated fatty acid includes oleic acid, and the liquid olefin includes octadecene; (3) the first temperature is 110°C-130°C, the second temperature is 30°C-50°C, and the first solution is a methanol solution of NH4F and NaOH; (4) the third temperature is 110°C-130°C, the first target time is 20 minutes-40 minutes, and the second target time is 5 minutes-15 minutes; (5) the fourth temperature is 250°C-350°C, and the third target time is 30 minutes-90 minutes.

[0033] Furthermore, in some preferred embodiments, the method for causing the cyanine dye and the lanthanide nanocrystals to self-assemble comprises: removing impurity ligands on the surface of the lanthanide nanocrystals and dispersing them to obtain a dispersion of ligand-free lanthanide nanocrystals; adding the dispersion of ligand-free lanthanide nanocrystals and the cyanine dye to a second solution dispersed with a surfactant and performing ultrasound, and then removing the solvent to obtain the dye complex.

[0034] Preferably, the dispersion liquid of the ligand-free lanthanide nanocrystals uses methanol as a dispersion medium, and the second solution dispersed with a surfactant is a methanol solution of DSPE-PEG2000.

[0035] More preferably, the method for removing impurity ligands on the surface of the lanthanide nanocrystals and dispersing them includes: adding a cyclohexane solution of the lanthanide nanocrystals to a saturated NOBF4-CH2Cl2 solution, and obtaining a ligand-free lanthanide nanocrystal precipitate by centrifugation; and dispersing the ligand-free lanthanide nanocrystal precipitate with methanol to obtain a dispersion of the ligand-free lanthanide nanocrystals.

[0036] In a third aspect of the embodiments of the present application, an application of the aforementioned dye complex in pH detection is provided. Preferably, an application suitable for in situ pH detection in vivo is provided. The dye complex can exhibit a reversible change in luminescence lifetime in response to changes in pH, and when applied to pH detection, quantitative, in situ and long-term monitoring of pH in vivo can be achieved.

[0037] The principle of using the dye complex provided in the examples of the present application to detect pH is as follows:

[0038]

[0039] In the above process, first of all, from the perspective of energy transfer mechanism, lanthanide nanocrystals and cyanine dyes form a composite system through coordination, so that energy transfer (for example, fluorescence resonance energy transfer, FRET) can be carried out effectively. Through coordination, energy is transferred from lanthanide nanocrystals (energy donors) to cyanine dyes (energy acceptors), and the pH dependence of cyanine dyes can significantly affect this energy transfer process. Secondly, from the perspective of chemical environmental sensitivity, the chemical properties of cyanine dyes change due to pH changes, and this change usually affects the optical properties of the coordination structure, including changes in absorption and emission spectra. After lanthanide nanocrystals are coordinated with cyanine dyes, the pH response performance of cyanine dyes will directly affect the overall luminescence behavior of the composite material. Under different pH conditions, cyanine dyes may experience fluorescence quenching or enhancement effects, thereby changing the energy transfer efficiency with lanthanide nanocrystals. This change is usually reflected in the length of the luminescence lifetime, and the effect of pH on cyanine dyes indirectly provides information through the luminescence lifetime.

[0040] Furthermore, the application of dye complexes in pH detection mainly relies on the aforementioned dye complexes as pH nanosensors for detection.

[0041] By adjusting the reversible absorption changes of cyanine dyes at different pH values, the reversible and controlled non-radiative energy transfer (NET) efficiency between lanthanide nanocrystals and cyanine dyes is achieved, so that the luminescence lifetime of the dye complex can be reversibly changed when used as a nanosensor without causing damage to the cyanine dye. This aggregation-independent mechanism ensures that the process is independent of concentration, and the high-fidelity signal extraction of lifetime imaging can achieve accurate lifetime imaging and monitoring of pH in vivo.

[0042] In a fourth aspect of an embodiment of the present application, a method suitable for in situ pH detection in a living body is provided, the detection method comprising: obtaining the luminescence lifetime of the part to be tested where the dye complex is located, and obtaining the pH value of the part to be tested based on the relationship between the pH value and the luminescence lifetime. Select the luminescence lifetime, and establish a relationship with the pH. The luminescence lifetime is an inherent property of the luminophore and is not affected by the luminophore concentration or test conditions, such as laser duration, laser power density, device configuration, and optical path length. Therefore, the luminescence lifetime is considered to be a high-fidelity signal, which makes luminescence lifetime imaging a theoretical quantitative imaging technique.

[0043] Compared with the prior art, the technical solution of this application has the following beneficial effects:

[0044] The present application designs a cyanine dye with a specific structure that undergoes a significant energy level change when protonated, and uses an effective inorganic-organic interface assembly to construct a reversible energy transfer pathway, which allows the protonation state of the dye to adjust the energy transfer efficiency, thereby changing the luminescence lifetime of the dye complex, so that the dye complex can respond to changes in pH and show reversible changes in luminescence lifetime without damaging the cyanine dye. Using high-fidelity signal extraction of lifetime imaging, reversible in-situ long-term monitoring of pH in vivo can be achieved. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The following figures describe in detail the exemplary embodiments disclosed in this application. The chemical structures introduced in the study follow the Markush principle, and the described embodiments are only used as demonstrations to prove the effect of this technology. This means that the listed compounds and their structures represent a class of compounds with similar properties and functions. Professionals in the field of chemistry will understand that these embodiments are not restrictive, but are used to demonstrate the potential application scope and effectiveness of the technology. Other compounds that conform to the Markush structure can also achieve similar technical effects. Those of ordinary skill in the art will understand that these embodiments are non-limiting, exemplary embodiments, and the drawings are only for the purpose of illustration and description, and are not intended to limit the scope of this application. Other embodiments may also accomplish the invention intent in this application. It should be understood that the drawings are only for illustrating the principles of the present invention and cannot be used to limit the present invention. Among them:

[0046] Figure 1 This is a schematic diagram of the preparation principle of the dye complex of Example 7 of the present application;

[0047] Figure 2 UV-visible absorption spectra of the dye complex prepared in Example 7 of the present application at different pH values;

[0048] Figure 3A schematic diagram of the reversible change of the luminescence decay curve of the dye complex prepared in Example 7 of the present application during the acid-base cycle;

[0049] Figure 4 The luminescence decay curve of the dye complex prepared in Example 7 of the present application under acid-base conditions;

[0050] Figure 5 This is the luminescence decay curve of the dye complex prepared in Example 7 of the present application at different pH values;

[0051] Figure 6 The relationship between the lifetime and pH value of the dye complex prepared in Example 7 of the present application is a titration fitting curve at different penetration depths;

[0052] Figure 7 This is a schematic diagram of time-gated intensity imaging of Mouse 1 in Example 9 of the present application 1 hour after PPI administration;

[0053] Figure 8 This is a schematic diagram of the time-gated luminescence intensity in the stomach of Mouse 1, Mouse 2 and Mouse 3 after PPI administration in Example 9 of the present application;

[0054] Fig. 9 The time-dependent luminescence lifetime curves of the stomachs of Mouse 1, Mouse 2, and Mouse 3 in Example 9 of the present application;

[0055] Fig.10 This is a curve showing the change of gastric pH value over time of Mouse 1, Mouse 2 and Mouse 3 in Example 9 of the present application. DETAILED DESCRIPTION

[0056] The technical scheme of the present application will be clearly and completely described below in conjunction with the accompanying drawings and specific embodiments. It should be noted that the present application is not limited to the structures listed herein, and is also changeable. Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as those generally understood by technicians in the technical field of the present invention. The terms used in this specification in the specification of the present application are only for the purpose of describing specific embodiments and are not intended to limit the present application. Unless otherwise specified, the reagents and raw materials used can be purchased through commercial channels. The experimental methods for which specific conditions are not indicated in the following examples are selected according to conventional methods and conditions, or according to the product specifications.

[0057] Some of the reagents used in the examples of the present invention: Y2O3 and Tm2O3 were purchased from Adama, and NOBF4 was purchased from Alfa Aesar. Oleic acid, octadecene and NH4F were purchased from Sigma-Aldrich, DSPE-PEG2000 and rabeprazole (sodium) were purchased from MCE®. 10% Intralipid was purchased from Sigma-Aldrich. Methanol, ethanol, cyclohexane, sodium hydroxide and hydrochloric acid were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai).

[0058] The equipment and instruments used in the embodiments of the present invention are as follows: 1 HNMR and 13 CNMR spectra were performed on a Bruker AVANCE III HD at 500 MHz and 100 MHz, respectively. High-resolution mass spectra (HR-MS) were measured on a Bruker Micro TOF II instrument. Transmission electron microscopy (TEM) tests were performed using a Talos F200X G2 (Thermo Fisher Scientific) field emission transmission electron microscope with an accelerating voltage of 200 kV. UV-visible absorption spectra were performed on a UV2600i UV-visible spectrophotometer. All luminescence decay curves and time-gated intensity spectra were performed on a FLS1000 fluorescence spectrometer equipped with a laser. Luminescence lifetime and time-gated intensity images were collected on a laboratory-built time-resolved imaging system.

[0059] Example 1

[0060] Preparation of cyanine dye FD-822 (Formula Ⅱ)

[0061] The preparation route of cyanine dye FD-822 of the present embodiment is as follows:

[0062] ;

[0063] This embodiment provides a method for preparing a cyanine dye FD-822, which specifically comprises the following steps:

[0064] Compound 5 (2 mmol) and compound 6 (2 mmol) were added to an ethanol solution of compound 4 (2 mmol), and the mixture was stirred at 50 ° C under an argon atmosphere for 5 hours. After the mixture was cooled to room temperature, the reaction solution was added to saturated brine and extracted with dichloromethane. The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and then concentrated to obtain a crude product. The crude product was purified by gradient elution flash chromatography (dichloromethane / methanol is 100 / 0 to 100 / 1 to 100 / 5, v / v) to obtain FD-822 as a dark green solid, and its nuclear magnetic data are as follows:

[0065] 1H NMR (500 MHz, Chloroform-d) δ = 8.29 (d, J = 16.1 Hz, 1H), 8.11 (dd,J = 8.1, 4.2 Hz, 2H), 8.07 (t, J = 7.6 Hz, 2H), 7.95 (t, J = 7.8 Hz, 2H),7.87 (d, J = 8.6 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 8.7 Hz, 1H),7.66 (d, J = 12.5 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.47 (dt, J = 18.8, 7.5Hz, 3H), 7.35 (d, J = 8.0 Hz, 2H), 7.30 (d, J = 7.5 Hz, 1H), 7.01 (d, J = 8.7Hz, 1H), 6.74 (d, J = 16.1 Hz, 1H), 5.47 (d, J = 12.6 Hz, 1H), 5.03 (s, 2H),2.58 (t, J = 6.1 Hz, 2H), 2.41 (t, J = 6.2 Hz, 2H), 2.05 (s, 6H), 1.81 (t, J= 6.1 Hz, 2H), 1.74 (s, 6H)。

[0066] 13 C NMR (126 MHz, CDCl3) δ 170.08, 155.84, 141.77, 132.61, 130.85,130.31, 130.16, 129.92, 129.83, 129.36, 129.15, 128.89, 128.40, 127.21,126.99, 126.43, 126.32, 125.69, 124.62, 122.67, 121.77, 109.01, 94.06, 72.08,66.17, 54.06, 48.23, 46.23, 27.35, 26.83, 26.28, 24.33, 21.33, 15.21. HR-MS(ESI Positive) calc. for C 46 H 42 ClN2O2 + , 689.29[M + ], found 689.287。

[0067] In addition, the UV-visible absorption and steady-state emission spectra of protonated FD-822 in different solvents showed that the absorption peak was mainly located at 822 nm, while the emission peak was located at 850 nm.

[0068] Example 2

[0069] The preparation of another cyanine dye is the same as in Example 1, except that the raw material in the synthesis of compound 4 is replaced with POBr3, and the product with the following structure is finally obtained:

[0070] ;

[0071] Its NMR data are as follows:

[0072] 13C NMR: δ 19.0 (1C, s), 25.1-25.2 (4C, 25.1 (s), 25.1 (s)), 26.1-26.3(2C, 26.1 (s), 26.2 (s)), 39.0 (1C, s), 50.3-50.4 (2C, 50.4 (s), 50.4 (s)),109.9 (1C, s), 110.8 (1C, s), 111.9 (1C, s), 119.6 (1C, s), 123.6-123.7 (2C,123.6 (s), 123.6 (s)), 123.8-123.9 (2C, 123.9 (s), 123.9 (s)), 125.0-125.1(3C, 125.0 (s), 125.0 (s), 125.1 (s)), 125.1-125.2 (2C, 125.2 (s), 125.2(s)), 126.1 (1C, s), 126.7-126.8 (2C, 126.7 (s), 126.7 (s)), 126.9-127.0 (2C,126.9 (s), 126.9 (s)), 128.1 (1C, s), 128.3-128.4 (2C, 128.4 (s), 128.4 (s)),128.9 (1C, s), 129.4 (2C, s), 129.6 (2C, s), 129.8 (1C, s), 130.1 (1C, s),130.7-130.8 (2C, 130.7 (s), 130.7 (s)), 135.3 (1C, s), 136.9 (1C, s), 138.1(1C, s), 151.1 (1C, s), 152.0 (1C, s), 167.1 (1C, s)。

[0073] 1H NMR(500 MHz, Chloroform-d): δ 1.54 (6H, s), 1.77-1.97 (8H, 1.85(dtt, J = 13.8, 6.6, 2.8 Hz), 1.92 (s)), 2.56-2.73 (4H, 2.64 (ddd, J = 14.2,6.6, 2.8 Hz), 2.65 (ddd, J = 14.0, 6.6, 2.8 Hz)), 5.20 (2H, s), 6.63 (1H, d,J = 10.2 Hz), 6.97 (1H, d, J = 15.9 Hz), 7.19-8.11 (18H, 7.25 (dd, J = 8.9,0.5 Hz), 7.32 (dddd, J = 8.0, 6.9, 1.9, 0.5 Hz), 7.33 (dddd, J = 8.5, 1.6, 0.5Hz), 7.37 (dddd, J = 7.9, 7.5, 1.7, 0.5 Hz), 7.41 (d, J = 10.2 Hz), 7.52(ddd, J = 8.5, 6.9, 1.9 Hz), 7.53 (dd, J = 8.9, 0.5 Hz), 7.61 (ddd, J = 8.6,7.5, 1.5 Hz), 7.74 (ddt, J = 8.9, 1.6, 0.5 Hz), 7.82 (d, J = 15.9 Hz), 7.91(ddt, J = 8.6, 1.7, 0.5 Hz), 7.91 (ddd, J = 8.5, 1.7, 0.5 Hz), 7.92 (dtt, J =7.9, 1.5, 0.5 Hz), 7.94 (ddt, J = 8.9, 1.9, 0.5 Hz), 8.00 (dtt, J = 8.0, 1.9, 0.5 Hz), 8.05 (ddt, J = 8.5, 1.9, 0.5 Hz)).

[0074] Example 3

[0075] The preparation of another cyanine dye is the same as in Example 1, except that the raw material in the synthesis of compound 4 is replaced with hypophosphorous acid, and the product with the following structure is finally obtained:

[0076] ;

[0077] Its NMR data are as follows:

[0078] 1H NMR: δ 1.06-1.17 (6H, 1.11 (s), 1.11 (s)), 1.27-1.37 (6H, 1.32(s), 1.32 (s)), 1.53 (1H, dtt, J = 13.5, 10.2, 3.1 Hz), 2.05-2.53 (4H, 2.14(ddd, J = 14.2, 10.3, 3.0 Hz), 2.14 (dtt, J = 13.5, 3.1, 2.5 Hz), 2.30 (ddd,J = 14.4, 10.2, 3.1 Hz), 2.45 (ddd, J = 14.2, 3.0, 2.5 Hz)), 2.61 (1H, ddd, J= 14.4, 3.1, 2.5 Hz), 4.43 (1H, d, J = 5.2 Hz), 4.63-4.73 (2H, 4.68 (s), 4.68(s)), 5.41 (1H, dd, J = 17.4, 5.2 Hz), 5.77-6.00 (2H, 5.84 (d, J = 17.4 Hz),5.94 (d, J = 10.4 Hz)), 6.00-6.18 (2H, 6.05 (s), 6.12 (d, J = 10.4 Hz)), 6.48(1H, dd, J = 8.8, 0.5 Hz), 6.83 (1H, dd, J = 8.9, 0.5 Hz), 7.26-7.45 (4H,7.33 (dddd, J = 7.9, 7.5, 1.6, 0.5 Hz), 7.38 (dddd, J = 7.9, 7.5, 1.7, 0.5Hz), 7.38 (ddd, J = 8.5, 1.4, 0.5 Hz)), 7.51-7.98 (10H, 7.58 (ddd, J = 8.6,7.5, 1.4 Hz), 7.61 (ddd, J = 8.6, 7.5, 1.5 Hz), 7.73 (dddt, J = 7.9, 1.6,1.4, 0.5 Hz), 7.73 (ddt, J = 8.9, 1.8, 0.5 Hz), 7.76 (ddt, J = 8.8, 1.6, 0.5Hz), 7.80 (ddt, J = 8.6, 1.6, 0.5 Hz), 7.90 (ddt, J = 8.6, 1.7, 0.5 Hz), 7.90(ddd, J = 8.5, 1.7, 0.5 Hz), 7.92 (dddt, J = 7.9, 1.8, 1.5, 0.5 Hz)). .

[0079] 13 C NMR: δ 19.0 (1C, s), 24.7 (1C, s), 25.1-25.2 (2C, 25.1 (s), 25.1(s)), 26.3-26.4 (2C, 26.3 (s), 26.3 (s)), 29.1 (1C, s), 44.7 (1C, s), 50.4(1C, s), 57.2 (1C, s), 69.4 (1C, s), 109.9 (1C, s), 111.9 (1C, s), 113.0 (1C,s), 117.0 (1C, s), 123.6-123.7 (2C, 123.6 (s), 123.6 (s)), 123.8-123.9 (2C,123.9 (s), 123.9 (s)), 125.0-125.1 (3C, 125.0 (s), 125.0 (s), 125.1 (s)),125.1-125.2 (2C, 125.2 (s), 125.2 (s)), 126.7-126.8 (2C, 126.7 (s), 126.7(s)), 126.9-127.0 (2C, 126.9 (s), 126.9 (s)), 127.7 (2C, s), 128.3-128.4 (2C,128.4 (s), 128.4 (s)), 128.9 (1C, s), 129.6 (2C, s), 129.8 (1C, s), 130.1(1C, s), 130.7-130.8 (2C, 130.7 (s), 130.7 (s)), 131.9 (1C, s), 136.3 (1C,s), 137.6 (1C, s), 142.0 (1C, s), 152.0 (1C, s), 153.2 (1C, s), 167.1 (1C, s).

[0080] Example 4

[0081] The preparation of another cyanine dye is the same as in Example 1, except that compound 5 in the synthesis step is replaced by the following structure:

[0082] ;

[0083] The structure of the target cyanine dye is shown below:

[0084] ;

[0085] Its NMR data are as follows:

[0086] 1H NMR: δ 0.81-0.93 (12H, 0.87 (t, J = 6.5 Hz), 0.87 (t, J = 6.5Hz)), 1.19-1.35 (24H, 1.25 (tt, J = 6.9, 6.5 Hz), 1.25 (tt, J = 6.9, 6.5 Hz),1.25 (quint, J = 6.5 Hz), 1.25 (quint, J = 6.5 Hz), 1.27 (tt, J = 6.8, 6.5Hz), 1.27 (tt, J = 6.8, 6.5 Hz), 1.28 (tt, J = 6.9, 6.5 Hz), 1.28 (tt, J =6.9, 6.5 Hz), 1.29 (h, J = 6.5 Hz), 1.29 (h, J = 6.5 Hz), 1.30 (h, J = 6.5Hz), 1.30 (h, J = 6.5 Hz)), 1.87 (2H, dtt, J = 13.8, 6.6, 2.8 Hz), 2.19-2.31(4H, 2.25 (t, J = 6.8 Hz), 2.25 (t, J = 6.8 Hz)), 2.34-2.46 (4H, 2.40 (t, J =6.5 Hz), 2.40 (t, J = 6.5 Hz)), 2.58-2.76 (4H, 2.66 (ddd, J = 14.0, 6.6, 2.8Hz), 2.68 (ddd, J = 14.3, 6.6, 2.8 Hz)), 5.22 (2H, s), 6.67 (1H, d, J = 10.3Hz), 7.04 (1H, d, J = 15.9 Hz), 7.21-7.68 (9H, 7.27 (dd, J = 8.9, 0.5 Hz),7.31 (dddd, J = 8.0, 6.9, 1.9, 0.5 Hz), 7.33 (ddd, J = 8.5, 1.6, 0.5 Hz),7.37 (dddd, J = 7.9, 7.5, 1.7, 0.5 Hz), 7.43 (d, J = 10.3 Hz), 7.52 (ddd, J =8.5, 6.9, 1.9 Hz), 7.54 (dd, J = 8.9, 0.5 Hz), 7.61 (ddd, J = 8.6, 7.5, 1.5Hz)), 7.75 (1H, ddt, J = 8.9, 1.6, 0.5 Hz), 7.84-8.11 (8H, 7.91 (d, J = 15.9Hz), 7.91 (ddd, J = 8.5, 1.7, 0.5 Hz), 7.92 (ddt, J = 8.6, 1.7, 0.5 Hz), 7.92(dtt, J = 7.9, 1.5, 0.5 Hz), 7.94 (ddt, J = 8.9, 1.9, 0.5 Hz), 8.00 (dtt, J =8.0, 1.9, 0.5 Hz), 8.05 (ddt, J = 8.5, 1.9, 0.5 Hz))。.

[0087] 13C NMR: δ 14.0-14.1 (4C, 14.1 (s), 14.1 (s)), 19.0 (1C, s), 22.4-22.6 (4C, 22.5 (s), 22.5 (s)), 25.8-25.9 (4C, 25.9 (s), 25.9 (s)), 26.1-26.3 (2C,26.1 (s), 26.2 (s)), 31.4-31.6 (4C, 31.5 (s), 31.5 (s)), 32.4-32.4 (4C, 32.4(s), 32.4 (s)), 39.0 (1C, s), 57.6-57.6 (2C, 57.6 (s), 57.6 (s)), 109.9 (1C,s), 111.9 (1C, s), 119.6 (1C, s), 123.6-123.7 (2C, 123.6 (s), 123.6 (s)), 123.8-123.9 (2C, 123.9 (s), 123.9 (s)), 125.0-125.1 (3C, 125.0 (s), 125.0(s), 125.1 (s)), 125.1-125.2 (2C, 125.2 (s), 125.2 (s)), 126.1 (1C, s),126.7-126.8 (2C, 126.7 (s), 126.7 (s)), 126.9-127.0 (2C, 126.9 (s), 126.9(s)), 128.1 (1C, s), 128.3-128.4 (2C, 128.4 (s), 128.4 (s)), 128.9 (1C, s), 129.4 (2C, s), 129.6 (2C, s), 129.8 (1C, s), 130.1 (1C, s), 130.7-130.8 (2C,130.7 (s), 130.7 (s)), 131.0 (1C, s), 135.3 (1C, s), 136.9 (1C, s), 138.1(1C, s), 151.1 (1C, s), 152.0 (1C, s), 167.1 (1C, s).

[0088] Example 5

[0089] Another cyanine dye was prepared in the same manner as in Example 1, except that the reactants in the synthesis step Replace it with the following structure: ;

[0090] The structure of the target cyanine dye is shown below:

[0091] ;

[0092] Its NMR data are as follows:

[0093] 1H NMR: δ 1.37 (2H, tt, J = 7.4, 7.0 Hz), 1.49-1.65 (8H, 1.54 (s),1.58 (tt, J = 7.5, 7.4 Hz)), 1.78-2.05 (10H, 1.86 (dtt, J = 13.8, 6.6, 2.8Hz), 1.92 (s), 1.98 (tt, J = 8.4, 7.0 Hz)), 2.58-2.74 (6H, 2.66 (ddd, J =14.0, 6.6, 2.8 Hz), 2.66 (ddd, J = 14.3, 6.6, 2.8 Hz), 2.64 (t, J = 7.5 Hz)),3.64 (2H, t, J = 8.4 Hz), 6.67 (1H, d, J = 10.2 Hz), 6.97 (1H, d, J = 15.9Hz), 7.13 (2H, ddd, J = 8.5, 1.7, 0.5 Hz), 7.20-7.68 (7H, 7.26 (dd, J = 8.9,0.5 Hz), 7.32 (dddd, J = 8.0, 6.9, 1.9, 0.5 Hz), 7.37 (dddd, J = 7.9, 7.5,1.7, 0.5 Hz), 7.42 (d, J = 10.2 Hz), 7.52 (ddd, J = 8.5, 6.9, 1.9 Hz), 7.51(dd, J = 8.9, 0.5 Hz), 7.61 (ddd, J = 8.6, 7.5, 1.5 Hz)), 7.75 (1H, ddt, J =8.9, 1.6, 0.5 Hz), 7.83-8.11 (8H, 7.90 (d, J = 15.9 Hz), 7.91 (ddt, J = 8.6,1.7, 0.5 Hz), 7.92 (dtt, J = 7.9, 1.5, 0.5 Hz), 7.92 (ddd, J = 8.5, 1.4, 0.5Hz), 7.94 (ddt, J = 8.9, 1.9, 0.5 Hz), 8.00 (dtt, J = 8.0, 1.9, 0.5 Hz), 8.05(ddt, J = 8.5, 1.9, 0.5 Hz))。

[0094] 13C NMR: δ 19.0 (1C, s), 25.1-25.2 (4C, 25.1 (s), 25.1 (s)), 26.1-26.3 (2C, 26.1 (s), 26.2 (s)), 29.3-29.4 (2C, 29.4 (s), 29.4 (s)), 31.6 (1C, s), 36.1 (1C, s), 39.0 (1C, s), 50.3-50.4 (2C, 50.4 (s), 50.4 (s)), 109.9 (1C,s), 111.9 (1C, s), 119.6 (1C, s), 123.6-123.7 (2C, 123.6 (s), 123.6 (s)),123.8-123.9 (2C, 123.9 (s), 123.9 (s)), 125.0-125.1 (3C, 125.0 (s), 125.0(s), 125.1 (s)), 125.1-125.2 (2C, 125.2 (s), 125.2 (s)), 126.1 (1C, s),126.7-126.8 (2C, 126.7 (s), 126.7 (s)), 126.9-127.0 (2C, 126.9 (s), 126.9(s)), 128.1 (1C, s), 128.3-128.4 (2C, 128.4 (s), 128.4 (s)), 128.8-129.0 (3C,128.9 (s), 128.9 (s)), 129.6 (2C, s), 129.8 (1C, s), 130.1 (1C, s), 130.7-130.8 (2C, 130.7 (s), 130.7 (s)), 131.0 (1C, s), 135.3 (1C, s), 138.1 (1C,s), 148.6 (1C, s), 151.1 (1C, s), 152.0 (1C, s), 167.1 (1C, s).

[0095] Example 6

[0096] Preparation of Lanthanide Nanocrystals NaYF4:1%Tm

[0097] This embodiment provides a method for preparing lanthanide nanocrystals, comprising the following steps:

[0098] (1) YCl3 (0.99 mmol) and TmCl3 (0.01 mmol) were added to a three-necked flask containing 7.13 g of oleic acid and 11.84 g of octadecene to obtain a first mixed solution.

[0099] (2) The first mixture was heated to 120° C. and maintained at 120° C. with magnetic stirring under an argon atmosphere until a transparent solution was formed.

[0100] (3) The transparent solution is cooled to 40° C. to keep it transparent, and then a methanol solution containing NH 4 F and NaOH is added dropwise to the first mixture to obtain a second mixed solution.

[0101] (4) The second mixture was heated to 120°C and maintained for 30 minutes to remove methanol under an argon atmosphere and then degassed for an additional 10 minutes.

[0102] (5) The second mixture from which methanol was removed was heated to 300°C under magnetic stirring and maintained for 60 minutes. Subsequently, the culture flask was naturally cooled to room temperature and lanthanide nanocrystals NaYF4:1%Tm were collected by centrifugation.

[0103] The prepared NaYF4:1%Tm was washed three times with cyclohexane / ethanol solution and dispersed in cyclohexane. The results showed that the prepared NaYF4:1%Tm showed a uniform ellipsoidal morphology with an average minor axis diameter of 21.6±1.0nm. Using 785nm laser excitation, Tm was observed at 800nm. 3+ The characteristic emission peak and the luminescence decay curve obtained by transient fluorescence spectroscopy showed a single exponential decay, and the luminescence lifetime in cyclohexane was determined to be 660 μs.

[0104] Example 7

[0105] Preparation of dye complex NaYF4:1%Tm@FD-822@DSPE-PEG2000

[0106] The preparation principle of the dye complex NaYF4:1%Tm@FD-822@DSPE-PEG2000 can be found in Figure 1 .

[0107] This embodiment provides a method for preparing a dye complex NaYF4:1%Tm@FD-822@DSPE-PEG2000, which specifically includes the following steps:

[0108] (1) The cyclohexane solution of NaYF4:1%Tm prepared in Example 6 was added to a saturated NOBF4-CH2Cl2 solution to remove the surface oleic acid ligands. The solution was then shaken and centrifuged to obtain a ligand-free NaYF4:1%Tm precipitate. Methanol was added to disperse the ligand-free NaYF4:1%Tm precipitate to obtain a ligand-free NaYF4:1%Tm dispersion.

[0109] (2) A methanol solution of DSPE-PEG2000 was added to the culture flask, and then a ligand-free NaYF4:1%Tm dispersion and FD-822 (1 mg / mL methanol solution) were added and ultrasonicated. The solvent was removed using a rotary evaporator to obtain the dye complex NaYF4:1%Tm@FD-822@DSPE-PEG2000.

[0110] The dye complex NaYF4:1%Tm@FD-822@DSPE-PEG2000 prepared above was redispersed in deionized water, centrifuged and washed with deionized water, and finally dispersed in deionized water for storage.

[0111] The results showed that the size and morphology of NaYF4:1%Tm@FD-822@DSPE-PEG2000 were uniform in water. The particle size was measured by nanoflow cytometry to be 61.4±6.1nm. According to the molar extinction coefficient of FD-822 at pH=8, which is 5.05×10 4 L·mol -1 cm -1 , it can be calculated that approximately 13 FD-822 molecules are bound to the surface of each lanthanide nanocrystal.

[0112] refer to Figure 2 , under alkaline conditions, the dye complex shows a peak at 542 nm and a minimum absorption at 822 nm. As the pH decreases, the absorption at 542 nm decreases, while the peak at 822 nm appears and increases, similar to the response observed in FD-822 nanomicelles. This suggests that the coordination of FD-822 with NaYF4:1%Tm effectively maintains its pH-responsive behavior.

[0113] Figure 3 The reversibility of the luminescence lifetime of NaYF4:1%Tm@FD-822@DSPE-PEG2000 is shown in acid-base cycles. Figure 4 The luminescence decay curve of NaYF4:1%Tm@FD-822@DSPE-PEG2000 under acid-base conditions is shown, where base means the solution pH is adjusted to 8, and acid means the solution pH is adjusted to 3.

[0114] Example 8

[0115] The luminescence signal of NaYF4:1%Tm@FD-822@DSPE-PEG2000 prepared in Example 7 was measured at different pH levels and different lipid thicknesses. Figure 5 The luminescence decay curves at different pH values ​​are shown. Figure 6 The titration fitting curve relationship between lifetime and pH value at different penetration depths is shown. The luminescence intensity increases with increasing pH. Due to the uneven spatial distribution of the excitation light, changes in luminescence intensity are observed at different positions within the quartz tube, as shown in the intensity imaging without 1% lipid emulsion shielding. As the pH value increases, the luminescence lifetime gradually increases, and the range of change is consistent with the results of the luminescence decay curve.

[0116] Compared with the time-gated intensity imaging results, the lifetime imaging results show a uniform spatial distribution and a smaller standard deviation. Although increasing the thickness of the lipid emulsion causes a sharp decrease in the luminescence intensity, resulting in a slightly higher standard deviation of the lifetime fitting, the overall photoluminescence lifetime remains unchanged. These results indicate that lifetime imaging is not affected by the excitation intensity and spatial distribution.

[0117] In addition, the inventors also found that the lifetime imaging fitting curves overlapped well after 0mm, 3mm and 6mm tissue shielding. Due to tissue shielding, intensity imaging is difficult to accurately extract luminescent signals from the body. In contrast, lifetime imaging can reliably capture in situ luminescent signals, thereby accurately determining the pH value in the body.

[0118] Comparative Example 1

[0119] The difference from Example 7 is that FD-822 is replaced by the following dye 1:

[0120] .

[0121] Comparative Example 2

[0122] The difference from Example 7 is that FD-822 is replaced by the following dye 2:

[0123] .

[0124] Comparative Example 3

[0125] The difference from Example 7 is that FD-822 is replaced by the following dye 3:

[0126] .

[0127] Comparative Example 4

[0128] The difference from Example 7 is that FD-822 is replaced by the following dye 4:

[0129] .

[0130] The dye complexes prepared in Example 7 and Comparative Examples 1-4 were used to detect microenvironment markers. The detection results are shown in Table 1.

[0131] Table 1 Microenvironmental marker detection results

[0132]

[0133] The test results show that the dye and NaYF4:1%Tm are compounded to detect microenvironmental markers, among which the complexes of Comparative Examples 1, 2, and 4 have a lifespan response, but the pH cannot be detected because the organic molecules are destroyed after the reaction, so the acid-base detection cannot be achieved. The complex of Comparative Example 3 is used for pH detection, but the molecule does not respond to the lifespan and can only be detected by the ratiometric method. The ratiometric method is based on the intensity of the light signal, and the intensity signal will always be affected by the tissue. The present application uses a specific dye structure so that there is energy transfer between the dye and the lanthanide compound, so there is a response to the lifespan, the pH can be detected by the lifespan, and the molecular structure of the dye will not be destroyed. The lifespan is equivalent to the average of multiple light signal intensities, which can reduce errors and achieve longer in-situ detection.

[0134] Example 9

[0135] The NaYF4:1%Tm@FD-822@DSPE-PEG2000 prepared in Example 7 was used as a pH nanosensor for in vivo biokinetic studies.

[0136] As a proof of concept, NaYF4:1%Tm@FD-822@DSPE-PEG2000 prepared in Example 7 was first injected into the mouse stomach by gavage, and then the effects and duration of different administration routes and doses of PPI (rabeprazole (sodium)) on the gastric pH of mice were compared for in vivo biokinetic monitoring.

[0137] Mouse 1 and mouse 2 were intravenously injected with 200 μL and 100 μL PPI (at a concentration of 1.5 mg / mL), respectively, while mouse 3 received 200 μL PPI by gastric gavage. Control mice were intravenously injected with 200 μL saline. Subsequently, we performed time-resolved imaging (including time-gated intensity and lifetime) and monitoring at different time points after drug administration (0, 0.5, 1, 2, and 4 hours).

[0138] From time-gated intensity imaging, it was observed that the excitation light was completely filtered out. When time-gated intensity imaging was performed on mouse 1 1 hour after PPI administration, see Figure 7The signal-to-noise ratio (SBR) reached 2106, thanks to the ability of lanthanide nanocrystals to absorb and emit at the same energy level, combined with the time-resolved imaging system built in the laboratory, which produced high-brightness, high signal-to-background ratio (SBR) images, allowing us to clearly observe the time-gated luminescence intensity in the mouse stomach after PPI administration, reference Figure 8 . These images show that some pH nanosensors move from the stomach to the intestines, making the stomach outline less clear. Therefore, the luminescence intensity information cannot accurately reflect changes in gastric pH. Overall, the time-gated luminescence intensity in the mouse stomach initially increased significantly after PPI administration and then decreased over time. This is because the increase in gastric pH after PPI administration causes a blue shift in FD-822 absorption, which reduces NETs on lanthanide nanocrystals and enhances luminescence intensity. However, the intensity signal is affected by the penetration depth and excitation conditions, so factors such as mouse posture, individual differences, and slight changes in the position of the excitation light may cause deviations. For example, the decrease in luminescence intensity in mouse 2 at 2 h is due to changes in body posture. Therefore, it is difficult to accurately assess differences in gastric pH between mice based solely on luminescence intensity.

[0139] The above demonstrates the accuracy of lifetime imaging, which shows that the luminescence lifetime in the stomach of experimental mice initially increases and then decreases. Lifetime imaging clearly captures the signal from the stomach without interference from the intestine, which may be due to continuous peristalsis causing intestinal signal fluctuations. These fluctuations are filtered out during the lifetime fitting process, leaving only the stomach signal. From the time-dependent luminescence lifetime curve of the mouse stomach, refer to Fig. 9 , almost no change was observed in the control group, while the luminescence lifetime of the experimental group increased significantly after PPI administration. It is worth noting that, see Fig.10 , Mouse 1, which received the higher dose, maintained a relatively high gastric pH even 4 h after administration, whereas Mouse 3, which received PPI by gastric gavage, showed a delayed response compared with Mouse 1 and Mouse 2.

[0140] The results showed that before PPI administration, the gastric pH of all mice was low, approximately 3.2, with little change. After PPI administration, mice 1 and 2 that received intravenous injections showed a significant increase in pH within 0.5 hours, reaching a peak at 2 hours. Mouse 3 received PPI by gastric gavage, and the pH increased significantly after 1 hour, also reaching a peak at 2 hours. Both mice 2 and 3 returned to baseline pH at 4 hours, while mouse 1 maintained the pH at 4.5 at 4 hours, indicating that the pH value continued to be high. It is worth noting that lifetime imaging is not affected by excitation conditions or tissue obstruction. It can accurately track luminescence signals, thereby determining gastric pH and key kinetic parameters, such as the onset and duration of the PPI effect. In contrast, intensity imaging cannot reliably capture these patterns.

[0141] Furthermore, in control mice, some nanosensors in the mouse stomach were still present after 4 hours, allowing their luminescence lifetime signals to be captured. Lifetime imaging and distribution showed that the luminescence lifetime in the stomach was significantly lower than in the intestine, indicating that the pH of the stomach is much lower compared to the intestine. This difference was also visible in intensity imaging.

[0142] Example 10

[0143] The process of establishing the relationship between pH and life span

[0144] In order to derive the mathematical expression of the luminescence lifetime pH response curve, it is assumed that: (1) the energy transfer between the nanoparticles and the dye follows the Förster resonance energy transfer (FRET) mechanism; (2) the single wavelength case is considered, where only the emission of lanthanide nanocrystals at 800 nm and the absorption of FD-822 at 800 nm are considered.

[0145] ;

[0146] Here, ε is the molar extinction coefficient, c is the total concentration of the dye, and L is the optical path length. Considering a single wavelength, the overlap integral J can be simplified to:

[0147] ;

[0148] Here, J represents the overlap integral which is directly related to the pH value of the solution. Substituting the overlap integral J into the Förster distance equation yields:

[0149] ;

[0150] Here, K 2 is the orientation factor, φ D is the quantum yield of the rare earth nanoparticles, n is the refractive index of the medium, N Ais Avogadro's constant. By substituting the simplified J, we get:

[0151] ;

[0152] Let K be the constant term, this simplifies to:

[0153] ;

[0154] therefore:

[0155] ;

[0156] Next, the energy transfer efficiency η can be expressed as:

[0157] ;

[0158] Let M = R 6 / K, we get:

[0159] ;

[0160] Finally, the relationship between the luminescence lifetime τ of the nanosensor and the energy transfer efficiency η is:

[0161] ;

[0162] Here, represents the luminescence lifetime of the nanosensor, Represents the intrinsic luminescence lifetime of lanthanide nanocrystals.

[0163] Embodiment 11

[0164] This example demonstrates the potential for pH detection in PCR reactions. Hepatitis B virus (HBV) is an important cause of liver disease worldwide. Effective HBV detection is essential for early diagnosis and treatment. However, due to the complexity of the liver environment, the performance of traditional PCR detection in the liver microenvironment may be affected by pH changes. Therefore, it is particularly important to combine in situ pH long-term in vivo monitoring to optimize PCR detection.

[0165] The in situ pH quantitative lifespan imaging system, equipped with a highly sensitive pH sensor, is suitable for monitoring pH changes in the liver microenvironment in vivo. Patients with hepatitis B virus were selected to ensure that the study was conducted without antiviral treatment. The pH value of the liver microenvironment was recorded in real time when the patient underwent liver biopsy or percutaneous liver puncture sampling. The pH sensor was implanted in the liver area to monitor pH fluctuations for up to 4 hours. During the monitoring period, the pH changes at different time points were recorded. The association between pH and HBV viral load was analyzed, with special attention to the changes in pH at different stages of infection. According to the real-time monitored pH data, a suitable buffer was selected to adjust the pH of the removed liver tissue samples to ensure that it was within the optimal range of PCR reaction (pH=7-8). Subsequently, PCR reaction was performed to amplify specific gene regions of HBV. Fluorescence quantitative PCR (qPCR) technology was used to evaluate the relationship between amplification efficiency and viral load under different pH conditions, and the amplification results of samples without pH adjustment and samples with pH optimization were compared. This study found that the pH value in the liver microenvironment fluctuated significantly over different time periods, especially in the stage of high HBV viral load, when the pH value showed an acidic trend. The PCR amplification efficiency of samples after pH adjustment increased by about 60%, and both sensitivity and specificity were significantly improved. The detected HBV viral load was higher than that of samples without pH adjustment.

[0166] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not used as additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features continue to be combined with each other to form various embodiments not listed above, which are all regarded as the scope of the present invention; further, for ordinary technicians in this field, they can be improved or transformed according to the above description, and all these improvements and transformations should belong to the protection scope of the claims attached to the present invention.

Claims

1. A dye composite, characterized in that The dye complex comprises a cyanine dye and a lanthanide element nanocrystal that undergoes reversible controlled non-radiative energy transfer with the cyanine dye; wherein the structural formula of the cyanine dye is as shown in Formula I: Formula I; Wherein, R1 is one of hydrogen or halogen, R2 is one of C1-C5 straight chain alkyl, R3, R4, R5, R6 are the same or different and are independently selected from C1-C5 alkyl and alkoxy; Wherein, the group in Formula I Connected to the lanthanide in the lanthanide nanocrystal via a non-covalent bond; The dye complex responds reversibly to pH, and the reversible response of the dye complex to pH causes the luminescence lifetime of the dye complex to change.

2. The dye composite according to claim 1, characterized in that The structural formula of the cyanine dye is shown in Formula II: Formula II.

3. The dye composite according to claim 1, characterized in that The lanthanide nanocrystals include a base material of lanthanide fluoride and a doping element; Wherein, the lanthanide fluoride is one of NaYF4 and NaGdF4, the doping element is at least one of Tm, Yb, Er and Eu, and the molar percentage of the doping element is 0.5%-5%.

4. The dye composite according to any one of claims 1 to 3, characterized in that The dye complex further includes a surfactant that is non-covalently bonded to the surface of the lanthanide nanocrystal.

5. The dye composite according to claim 4, characterized in that The surfactant is 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-polyethylene glycol 2000.

6. A method for preparing the dye composite according to any one of claims 1 to 5, characterized in that: The preparation method comprises: allowing the cyanine dye and the lanthanide element nanocrystal to self-assemble.

7. The method for preparing the dye composite according to claim 6, characterized in that: The cyanine dye is prepared by chemically reacting the following compound 1, compound 2 and compound 3: 、 、 ； Among them, the selection of R1, R2, R3, R4, R5 and R6 is the same as claim 1.

8. The method for preparing the dye composite according to claim 7, characterized in that: The molar ratio of compound 1, compound 2 and compound 3 is (0.5-1.5):(0.5-1.5):(0.5-1.5); the chemical reaction is carried out under an inert gas atmosphere, and the reaction temperature is 40°C-60°C, and the reaction time is 4 hours-6 hours.

9. The method for preparing the dye composite according to claim 6, characterized in that: The method for preparing the lanthanide nanocrystals comprises: Adding YCl3 and TmCl3 to a mixture of liquid unsaturated fatty acid and liquid olefin to obtain a first mixture; Stirring the first mixture under an inert gas atmosphere at a first temperature until it becomes transparent; Cooling the first mixture to a second temperature and keeping it transparent, and then dropping a first solution containing NH4F and NaOH dissolved therein into the first mixture to obtain a second mixture; heating the second mixture to a third temperature and maintaining it for a first target time to remove solvent, and then degassing for a second target time; The second mixture from which the solvent has been removed is heated to a fourth temperature and maintained for a third target time while being stirred, and then cooled to room temperature, and the lanthanide nanocrystals are collected by centrifugation.

10. The method for preparing the dye composite according to claim 9, characterized in that: The method for preparing the lanthanide nanocrystals satisfies at least one of the following conditions: (1) The molar ratio of YCl3 to TmCl3 is (90-100):1; (2) The liquid unsaturated fatty acid includes oleic acid, and the liquid olefin includes octadecene; (3) The first temperature is 110°C-130°C, the second temperature is 30°C-50°C, and the first solution is a methanol solution of NH4F and NaOH; (4) The third temperature is 110°C-130°C, the first target time is 20 minutes-40 minutes, and the second target time is 5 minutes-15 minutes; (5) The fourth temperature is 250° C. to 350° C., and the third target time is 30 minutes to 90 minutes.

11. The method for preparing the dye composite according to any one of claims 6 to 10, characterized in that: The method for causing the cyanine dye and the lanthanide nanocrystal to self-assemble comprises: removing impurity ligands on the surface of the lanthanide element nanocrystals and dispersing them to obtain a dispersion of ligand-free lanthanide element nanocrystals; The dispersion of the ligand-free lanthanide element nanocrystals and the cyanine dye are added to a second solution dispersed with a surfactant and ultrasonicated, and then the solvent is removed to obtain the dye complex.

12. The method for preparing the dye composite according to claim 11, characterized in that: The method for removing and dispersing the impurity ligands on the surface of the lanthanide nanocrystals comprises: adding the cyclohexane solution of the lanthanide element nanocrystals to a saturated NOBF4-CH2Cl2 solution, and obtaining a ligand-free lanthanide element nanocrystal precipitate by centrifugation; Methanol is used to disperse the ligand-free lanthanide element nanocrystal precipitate to obtain a dispersion of the ligand-free lanthanide element nanocrystal.

13. Use of the dye complex according to any one of claims 1 to 5 in pH detection.

14. A method for detecting pH in vivo, characterized in that: The dye complex described in any one of claims 1 to 5 is used as a pH nanosensor for detection, and the detection method comprises: obtaining the luminescence lifetime of the test site where the dye complex is located, and obtaining the pH value of the test site based on the relationship between the pH value and the luminescence lifetime, for the purpose of non-disease diagnosis or treatment.

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