Reversible adjustment coordination type rare earth nanocrystal-organic dye and application in detection

Abstract

The application discloses a reversible coordination type rare earth nanocrystal-organic dye, which comprises a rare earth nanocrystal and a chlorin dye, and the chlorin dye forms reversible coordination with the rare earth nanocrystal on the surface of the rare earth nanocrystal, wherein the chlorin dye has the structure shown in formula I: wherein R is halogen or a group * indicates a bonding position. The reversible coordination type rare earth nanocrystal-organic dye can form reversible coordination, the structure of the organic dye is not damaged, continuous detection can be carried out, the sensitivity of detection is high, and the anti-interference capability is strong. The application further discloses a preparation method of the reversible coordination type rare earth nanocrystal-organic dye, a luminescent probe, application of the luminescent probe in biological detection, and a method for detecting gastric juice pH by using the luminescent probe.

Description

Technical Field

[0001] This invention relates to the field of biological detection, and particularly to a reversible adjustable coordination type rare earth nanocrystal-organic dye and its application in detection. The invention includes the chemical structure of the organic dye in the complex composed of the rare earth nanocrystal-organic dye, the preparation method of the complex, and the application of the complex as a sensor in biological detection, especially in in vivo pH detection. Background Technology

[0002] Luminescence imaging technology has become a common tool in preclinical and biomedical research due to its non-invasiveness, high spatiotemporal resolution, and high sensitivity. Rare-earth nanocrystals, such as lanthanide nanocrystals, have become important luminescent probes due to their good luminescence properties and chemical stability, tunable emission wavelength, and luminescence lifetime. However, the application of rare-earth nanocrystals in luminescence imaging and in vivo sensing is currently limited, mainly because their small absorption cross-section makes it difficult to effectively absorb incident light, resulting in a weak signal and thus reducing the overall detection sensitivity. To obtain a stronger luminescence signal, a higher-power excitation source is required, but this may damage the sample, especially in biological samples. Due to the low absorption efficiency, more excitation light may be absorbed or scattered by other components in the sample, increasing the background signal and reducing the signal-to-noise ratio.

[0003] To overcome these problems, researchers have developed a variety of inorganic-organic interface interaction strategies, including using the large absorption cross-section of organic dyes to sensitize the luminescence of rare earth nanocrystals through energy transfer, and imaging probes based on energy transfer from rare earth nanocrystals to dyes, which expand the in vivo application of rare earth nanocrystals.

[0004] However, existing rare-earth nanocrystal-organic dye hybrid probes are primarily based on irreversible chemical reactions between the dye and the target analyte. These irreversible reactions destroy the organic dye on the nanocrystal surface, leading to an irreversible decrease in energy transfer efficiency. This prevents the hybrid probes from continuously and accurately detecting analyte concentrations. Therefore, these probes based on irreversible reaction mechanisms cannot achieve continuous real-time monitoring in dynamically changing biological environments, especially in applications requiring relatively long-term monitoring. They fail to meet the needs of biological detection. Furthermore, the irreversible chemical reaction means the probes cannot be reused after a single use, resulting in reagent waste and increased application costs.

[0005] Therefore, there is an urgent need to develop a novel rare-earth nanocrystalline-organic dye material that employs a new mechanism to achieve continuous monitoring and has high sensitivity to meet the requirements of accurate detection. Summary of the Invention

[0006] To address the aforementioned technical problems, embodiments of the present invention disclose a reversibly adjustable coordination type rare earth nanocrystal-organic dye, comprising rare earth nanocrystals and cyanine dye, wherein the cyanine dye forms reversible coordination with the rare earth nanocrystals on the surface of the rare earth nanocrystals, and wherein the cyanine dye has the structure shown in Formula I:

[0007]

[0008] Where R is a halogen or a group * indicates the bonding position; wherein, the rare earth nanocrystal-organic dye is reversibly responsive to pH, and the reversible pH response of the rare earth nanocrystal-organic dye leads to a change in the luminescence lifetime of the rare earth nanocrystal-organic dye.

[0009] By adopting the above technical solution, rare earth nanocrystal-organic dye materials with reversible adjustment mechanism can be obtained. In this reversible adjustment mechanism, the structure of the organic dye will not be destroyed, and continuous detection can be performed with high sensitivity and strong anti-interference ability.

[0010] Optionally, R is chlorine, preferably chlorine as it is easier to synthesize, and the cyanine dye has the structure shown in Formula II below:

[0011]

[0012] Optionally, R is In this case, R1 is a C1-C5 alkyl group.

[0013] Optionally, wherein, the group It forms coordination with the rare earth nanocrystals.

[0014] Optionally, the rare earth nanocrystals may be composed of Tm-doped nanocrystals. 3+ Eu 3+ and Yb 3+ One or more of NaYF4, wherein Tm 3+ Eu 3+ and Yb 3+ One or more of the substitution parts Y 3+ The location.

[0015] Optionally, the rare earth nanocrystals may be composed of Tm-doped nanocrystals. 3+ NaYF4, of which Tm 3+ The doping amount is 0.5% to 5%.

[0016] Optionally, the rare earth nanocrystals have a hexagonal phase structure, and the nanoparticle size of the rare earth nanocrystals is 5nm to 50nm.

[0017] According to another specific embodiment of the present invention, a method for preparing reversibly adjustable coordination type rare earth nanocrystal-organic dye is disclosed, comprising the following steps:

[0018] Rare earth nanocrystals are mixed with an organic solvent to form a solution. A deligation agent is added to the solution to remove the organic ligands on the surface of the rare earth nanocrystals, forming a precipitate.

[0019] The precipitate was collected by centrifugation and washing to obtain ligand-free rare earth nanocrystals.

[0020] Ligand-free rare earth nanocrystals are mixed with organic dyes and surfactants to form a uniformly dispersed mixture;

[0021] Rare earth nanocrystalline organic dyes were prepared by ultrasonic treatment and solvent evaporation.

[0022] By adopting the above technical solution, rare earth nanocrystal-organic dye materials with reversible adjustment mechanism can be obtained. In this reversible adjustment mechanism, the structure of the organic dye will not be destroyed, and continuous detection can be performed with high sensitivity and strong anti-interference ability.

[0023] Optionally, the rare earth nanocrystals are NaYF4:1%Tm, and the deliganding agent is a saturated NOBF4-CH2Cl2 solution.

[0024] Optionally, the surfactant is DSPE-PEG2000, and the organic solvent is cyclohexane.

[0025] Optionally, the weight ratio of the ligand-free rare earth nanocrystals, the organic dye, and the surfactant is 1:1:0.5.

[0026] According to another specific embodiment of the present invention, an embodiment of the present invention discloses a luminescent probe comprising the above-mentioned reversibly adjustable coordination type rare earth nanocrystal-organic dye.

[0027] By adopting the above technical solution, continuous luminescence lifetime imaging can be performed, with high detection accuracy, thus expanding the application range of luminescence probes.

[0028] According to another specific embodiment of the present invention, the present invention discloses the use of the above-mentioned luminescent probe in biological detection, wherein the biological detection is gastric juice pH detection.

[0029] Using the above technical solution, the detection of gastric juice pH is more continuous and more accurate.

[0030] According to another specific embodiment of the present invention, an embodiment of the present invention discloses a method for detecting gastric juice pH using the above-mentioned luminescent probe, comprising the following steps:

[0031] The luminescent probe is orally administered into the body to allow it to interact with the gastric environment;

[0032] H2 receptor antagonists or proton pump inhibitors were administered intravenously at a predetermined time.

[0033] Time-gated intensity imaging and lifetime imaging of the stomach of organisms were performed to monitor changes in the luminescence lifetime of the luminescent probe.

[0034] Single exponential fitting is performed on time-gated intensity images acquired under different time delays to generate lifetime images;

[0035] The luminescence lifetime of the luminescent probe is correlated with pH value, and changes in gastric juice pH are monitored by measuring changes in luminescence lifetime.

[0036] Using the above technical solution, the detection of gastric juice pH is more continuous and more accurate. Attached Figure Description

[0037] Figure 1 A schematic diagram of the reversible coordination mechanism according to an embodiment of the present invention is shown;

[0038] Figure 2 This diagram illustrates the protonation of cyanine dyes according to an embodiment of the present invention.

[0039] Figure 3 The following is a statistical chart showing the luminescence lifetime monitoring of Examples 1, 2 and Comparative Example 1 of the present invention during multiple cycles from pH=3 to pH=8;

[0040] Figure 4 The diagram shows the UV-Vis absorption spectroscopy test results of Embodiment 1, Embodiment 2 and Comparative Example 1 of the present invention;

[0041] Figure 5 This diagram illustrates the lifespan imaging of three groups of mice in Example 1 of the present invention.

[0042] Figure 6 This diagram illustrates the change in the average lifetime value of each group during a 4-hour monitoring period in Application Example 1 of the present invention.

[0043] Figure 7 This diagram illustrates the pH value changes of each group during a 4-hour monitoring period in Example 1 of the present invention. Detailed Implementation

[0044] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Although the description of the present invention is presented in conjunction with preferred embodiments, this does not mean that the features of the invention are limited to these embodiments. On the contrary, the purpose of describing the invention in conjunction with embodiments is to cover other options or modifications that may be derived based on the claims of the present invention. To provide a deep understanding of the invention, many specific details will be included in the following description. The invention may also be implemented without using these details. Furthermore, to avoid confusion or obscuring the focus of the invention, some specific details will be omitted in the description. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of the present invention can be combined with each other.

[0045] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0046] First, for sustainable detection, a rare-earth nanocrystal-organic dye probe with a reversible mechanism is needed to avoid irreversible chemical reactions that could damage the organic dye structure. When designing the reversible reaction, it is crucial to ensure that the structural stability of both the rare-earth nanocrystals (e.g., lanthanide nanocrystals) and the organic dye remains intact. This reversible mechanism should also maintain reaction efficiency, exhibiting rapid responsiveness and timely reflection of state changes. Furthermore, to guarantee effective luminescence performance and ensure the interaction at the inorganic-organic interface, the reversible mechanism must also consider the intermolecular interactions and energy transfer between the rare-earth nanocrystals and the organic dye.

[0047] Secondly, in terms of feedback of detection results, the reversible mechanism needs to improve detection sensitivity and anti-interference ability as much as possible, especially sensitivity to small changes, which can enhance the scope of practical application.

[0048] Furthermore, the setting of the triggering conditions for the reversible mechanism is also an issue that needs to be considered. These triggering conditions should be closely integrated with the reversible mechanism to ensure structural stability. Moreover, the triggering conditions should be applicable and widely applicable; otherwise, the reversible mechanism will be rendered ineffective.

[0049] Therefore, the first aspect of the present invention discloses a reversibly adjustable coordination type rare earth nanocrystal-organic dye, comprising rare earth nanocrystals and a cyanine dye capable of forming coordination with the rare earth nanocrystals on the surface of the rare earth nanocrystals, the cyanine dye having the structure shown in Formula I:

[0050]

[0051] Where R is a halogen or a group * indicates the bonding position. The luminescence lifetime of the coordination-type rare earth nanocrystal-organic dye changes with pH value.

[0052] Cyanide dyes with the above structure can coordinate with rare-earth nanocrystals. Furthermore, studies have shown that the coordination strength between cyanide dyes and rare-earth nanocrystals changes with variations in the pH of the external environment. This change does not disrupt the structure of the cyanide dye; reversibly adjustable coordination-type rare-earth nanocrystal-organic dyes can maintain structural integrity and continue to function. Specifically, refer to... Figure 1 Cyanide dyes are distributed on the surface of rare-earth nanocrystals. The benzoic acid groups of the cyanide dyes can provide lone pairs of electrons, and the rare-earth ions in the rare-earth nanocrystals can provide empty orbitals to participate in coordination. Further integration... Figure 1 At relatively low pH values ​​(e.g., pH 2–7), cyanine dyes have a weaker ability to donate lone pairs of electrons, weakening the coordination relationship between rare earth nanocrystals and cyanine dyes, and in some cases, causing coordination bond breakage. At relatively high pH values ​​(e.g., pH 8–12), cyanine dyes have a stronger ability to donate lone pairs of electrons, strengthening the coordination relationship between rare earth nanocrystals and cyanine dyes, reforming coordination bonds, and potentially further enhancing the coordination relationship. With changes in pH, the relationship between cyanine dyes and rare earth nanocrystals undergoes a continuous cycle of weakening coordination relationship – coordination bond breakage – coordination bond reformation – strengthening coordination relationship. Therefore, a reversible mechanism is constructed, ensuring that the structural stability of rare earth nanocrystals and organic dyes remains unaffected. While maintaining intermolecular interactions, it avoids introducing excessively strong reactions that could lead to structural damage. The structural design of cyanine dyes can incorporate protecting groups and steric effects to ensure the flexibility and compatibility of the reaction.

[0053] In particular, research has found that the cyanine dye structure of the embodiments of the present invention makes it hydrophobic. Even if the dye is removed from the nanocrystals after protonation, it can still remain within a certain distance range and will not migrate into the ambient aqueous phase, ensuring that it can be recoordinated and maintaining reversibility.

[0054] Furthermore, the luminescence lifetime of the reversibly modulated coordination-type rare-earth nanocrystal-organic dye also changes with variations in pH and coordination relationship. To improve detection accuracy, a reversible mechanism involving pH, coordination relationship, and luminescence lifetime was constructed.

[0055] pH can be considered a triggering condition, and one with wide-ranging applications. In practical applications, biological systems and their environments are complex and variable. In physiological processes, pH is a crucial indicator of many biochemical reactions, particularly important for understanding the complex metabolic states in comorbidities of the elderly (such as diabetes and cardiovascular diseases). For example, the function of certain immune cells (such as macrophages and T cells) is closely related to pH; a low pH environment may inhibit the activity of certain immune cells, affecting the immune response. In some tumor microenvironments, local pH changes may affect tumor immune escape or the effectiveness of immunotherapy. Furthermore, changes in pH may affect the concentration of some biomarkers, such as lactate, amino acids, and electrolytes, which are important detection indicators in biochemical experiments.

[0056] In existing technologies, there are schemes that establish a relationship between pH value and luminescence intensity. For example, Chinese patent application 202180085017.X discloses a pH-responsive cyanine dye and its conjugate, which measures pH by establishing a relationship between luminescence intensity and pH. However, luminescence intensity is affected by various uncertain factors, including luminescent group concentration, excitation conditions, and biological tissue shielding, which makes the precise extraction of in vivo luminescence signals complex and inaccurate. In practical applications, biological systems and their environments are complex and variable. Compared with luminescence intensity, luminescence lifetime is more adaptable to such changes. In addition, luminescence lifetime is generally not affected by biological tissue shielding and can resist interference from autofluorescence, absorption, and scattering in biological tissues. Therefore, establishing a relationship between lifetime and pH value is more accurate. Reflecting pH changes through changes in luminescence lifetime, and thus reflecting physiological changes, is a more reliable and accurate mechanism. In practical applications, individual differences in digestion rate, stomach position changes, etc., may lead to a decrease in detection accuracy. However, in this case, luminescence lifetime imaging can still provide relatively stable pH detection. Meanwhile, luminescent lifetime imaging is more sensitive to pH changes, and significant lifetime changes can still be observed even with small pH variations. Especially under acidic conditions, although the nonradiative energy transfer efficiency is lower, lifetime imaging can accurately reflect pH changes by capturing subtle variations in luminescent lifetime.

[0057] In summary, the technical solution provided in this invention achieves effective coordination between the dye and rare earth nanocrystals at higher pH values, thereby realizing efficient non-radiative energy transfer and shortening the luminescence lifetime. At lower pH values, the coordination between the dye and nanocrystals weakens, the non-radiative energy transfer efficiency decreases, and the luminescence lifetime is restored, realizing a reversible change mechanism. The change in pH value is reflected by the change in luminescence lifetime, enabling continuous monitoring. Furthermore, the feedback from the luminescence lifetime can improve detection sensitivity and anti-interference ability, reflect small changes, and is verifiable and repeatable.

[0058] In an embodiment of the present invention, R-base is... In this case, R1 is a C1-C5 alkyl group.

[0059] In embodiments of the present invention, the reversibly adjustable coordination type rare earth nanocrystal-organic dye has the structure shown in Formula II or Formula III:

[0060]

[0061] Preferably, the reversibly tunable coordination rare-earth nanocrystal-organic dye has the structure of Formula III. Specifically, the group... The (N-methylpiperazine group) can donate more electrons, enhancing the optical activity of the dye molecule. This electron-donating ability can enhance the stability of the excited state, thereby increasing luminescence efficiency. Its protonation state alters the electron distribution of the molecule, thus changing the absorption characteristics of the material and affecting the overall luminescence intensity and lifetime of the reversibly tunable coordination-type rare-earth nanocrystal-organic dye. (Reference) Figure 2 Under acidic conditions, the N-methylpiperazine group can accept hydrogen ions, weakening the electron-donating ability of its nitrogen atom and thus altering the energy level arrangement and conjugated system of the molecule. Through protonation and deprotonation, N-methylpiperazine modulates the energy levels within the conjugated system, resulting in more significant changes in the optical properties of the material at different pH values. For example, it can cause a redshift or blueshift of the absorption peak, improving the applicability of the probe in bioimaging and sensing applications.

[0062] In the cyanine dye portion of this invention, there are three units each with a specific function: a pH-responsive unit, a coordination unit, and a conjugated unit. The R group is the pH-responsive unit, which exhibits a certain pH responsiveness under different pH conditions, leading to changes in the luminescence properties of the rare-earth nanocrystal-organic dye. For example, the group... After protonation, the change in its energy level may lead to an increase or decrease in luminescence efficiency, which in turn affects the luminescence intensity. As a coordination unit, it changes the coordination relationship with rare earth nanocrystals; in cyanine dyes, removing the pH-responsive unit and the coordination unit part constitutes a conjugated unit.

[0063] In a specific embodiment of the present invention, the rare earth nanocrystals include Tm-doped components. 3+ Eu 3+ and Yb 3+ One or more of NaYF4, wherein Tm 3+ Eu 3+ and Yb 3+ One or more of the substitution parts Y 3+ The location. That is, rare earth nanocrystals are rare earth-doped nanocrystals.

[0064] Preferably, the rare earth nanocrystals are composed of Tm-doped nanocrystals. 3+ NaYF4, of which Tm 3+ The doping amount is 0.5% to 5%, that is, Tm 3+ The molar amount of Tm accounts for 1-3% of the total molar amount of rare earth ions. More preferably, Tm in NaYF4 3+ The doping amount is 1% (expressed as NaYF4:1%Tm), which can reduce energy transfer attenuation and self-absorption, further increasing luminescence efficiency. Specifically, the rare earth nanocrystals have a hexagonal phase structure, exhibiting better chemical stability and higher luminescence efficiency. In the embodiments of the present invention, the nanoparticle size of the rare earth nanocrystals is 5–50 nm. Within this particle size range, the coordination relationship between the rare earth nanocrystals and cyanine dyes is more stable, the energy transfer efficiency is higher, and it is easier to form a better interface effect. More specifically, the nanoparticle size of the rare earth nanocrystals is 19.7 ± 0.8 nm.

[0065] Specifically, in reversibly tunable coordination rare earth nanocrystals-organic dyes, on average, each rare earth nanocrystal molecule can form a coordination relationship with 10-20 cyanine dye molecules on its surface, that is, on average, each rare earth nanocrystal molecule can carry 10-20 cyanine dye molecules on its surface.

[0066] In a specific embodiment of the present invention, R is Cl. In an environment with pH=3, the luminescence lifetime of the reversibly adjustable coordination rare earth nanocrystal-organic dye is 150-180 μs. In an environment with pH=8, the luminescence lifetime of the reversibly adjustable coordination rare earth nanocrystal-organic dye is 60-80 μs.

[0067] In a specific embodiment of the present invention, R is a group. In an environment with pH=3, the luminescence lifetime of the reversibly tunable coordination rare earth nanocrystal-organic dye is 280–310 μs, while in an environment with pH=8, the luminescence lifetime of the pH-responsive nanomaterial is 160–190 μs.

[0068] At different pH values, the reversibly adjustable coordination-type rare earth nanocrystal-organic dye exhibits significant changes in luminescence lifetime, which facilitates accurate detection.

[0069] A second aspect of the present invention discloses a method for preparing a reversibly adjustable coordination type rare earth nanocrystal-organic dye, comprising the following steps:

[0070] Synthesis of reversibly adjustable coordination rare earth nanocrystal-organic dye: Rare earth nanocrystals and cyanine dyes were mixed in a solvent and then dispersed, desolventized, and centrifuged to obtain reversibly adjustable coordination rare earth nanocrystal-organic dye.

[0071] Specifically, the dispersion treatment is ultrasonic treatment for 1 to 2 minutes, the solvent removal treatment is solvent removal using a rotary evaporator at a pressure of 100 to 120 mbar, and the centrifugation treatment is centrifugation at 11,000 to 12,000 rpm.

[0072] More specifically, before synthesizing reversibly tunable coordination rare earth nanocrystals-organic dyes, the process also includes steps of synthesizing rare earth nanocrystal materials and synthesizing cyanine dyes.

[0073] A third aspect of the present invention discloses a luminescent probe comprising the reversibly adjustable coordination type rare-earth nanocrystal-organic dye described in the embodiments of the present invention. This luminescent probe is capable of continuous luminescence lifetime imaging for 1 to 8 hours, reflecting persistent changes in the target analyte.

[0074] A fourth aspect of this invention discloses the use of a luminescent probe in biodetection. The biodetection utilizes a luminescent probe comprising reversibly adjustable coordination rare-earth nanocrystals and an organic dye, enabling continuous luminescence lifetime imaging with high detection accuracy. Specifically, the biodetection is gastric juice pH detection.

[0075] The fifth aspect of the present invention discloses a method for detecting gastric juice pH using the above-mentioned luminescent probe, comprising the following steps:

[0076] The luminescent probe is orally administered into the body to allow it to interact with the gastric environment;

[0077] H2 receptor antagonists or proton pump inhibitors were administered intravenously at a predetermined time.

[0078] Time-gated intensity imaging and lifetime imaging of the stomach of organisms were performed to monitor changes in the luminescence lifetime of the luminescent probe.

[0079] Single exponential fitting is performed on time-gated intensity images acquired under different time delays to generate lifetime images;

[0080] The luminescence lifetime of the luminescent probe is correlated with pH value, and changes in gastric juice pH are monitored by measuring changes in luminescence lifetime.

[0081] The following will describe the implementation methods in more detail.

[0082] Example 1 (NaYF4:1%Tm@pH-820-b):

[0083] Example 1 is a reversibly adjustable coordination type rare earth nanocrystal-organic dye, where the rare earth nanocrystals are Tm 3+ The preparation method of 1% doped NaYF4 (NaYF4:1%Tm), in which R is Cl (represented as pH-820-b) in the cyanine dye is as follows.

[0084] (1) Synthesis of NaYF4: 1% Tm

[0085] YCl3 (0.99 mmol) and TmCl3 (0.01 mmol) were added to a three-necked flask containing oleic acid (6 mL) and octadecene (15 mL). The mixture was heated to 120 °C under an argon (Ar) atmosphere and maintained at that temperature while being magnetically stirred until a clear solution was formed. The solution was then cooled to 40 °C, remaining clear and transparent, at which point NH4F (0.4 mmol, 148.2 mg) and NaOH (2.5 mmol, 100 mg) dissolved in methanol (8 mL) were added dropwise to the mixture. The mixture was then heated to 120 °C and maintained for 30 minutes to remove methanol under an argon atmosphere, followed by evacuation for 10 minutes. The mixture was then heated to 300 °C under magnetic stirring and maintained for 60 minutes. The flask was then allowed to cool naturally to room temperature. Nanoparticles were collected by centrifugation (12000 rpm, 10 min), washed three times with cyclohexane / ethanol solution, and then dispersed in cyclohexane (10 mL).

[0086] (2) Synthesize dye pH-820-b (Formula II), and the specific synthetic route is shown below.

[0087]

[0088] (3) Synthesis of reversibly adjustable coordination rare earth nanocrystal-organic dyes

[0089] First, 20 mg of NaYF4:1%Tm dispersed in cyclohexane was added to 3 mL of saturated NOBF4-CH2Cl2 solution to remove the oleic acid ligands on the surface. A white precipitate was observed. The mixture was shaken for 2 minutes and then centrifuged at 11000 rpm for 10 minutes to collect the precipitate. 2 mL of methanol was added to disperse the ligand-free nanoparticles for later use. In a flask, 15 mg of DSPE-PEG2000 (a biomedical material composed of distearate phosphatidylethanolamine and polyethylene glycol with a molecular weight of 2000 Daltons covalently linked) was added to 4 mL of methanol. Then, 1 mL of ligand-free nanoparticles and pH-820-b dye (30 μL, 1 mg / mL, methanol solution) were added. The mixture was sonicated for 1 minute to ensure uniform dispersion. The solvent was removed using a rotary evaporator at 100 mbar. The resulting nanocomposite material was then redispersed in 1 mL of deionized water. Centrifuge at 12000 rpm and wash three times with deionized water to obtain reversible coordination-regulated rare earth nanocrystal-organic dye. Disperse the reversible coordination-regulated rare earth nanocrystal-organic dye in 1 mL of deionized water.

[0090] Example 2 (NaYF4: 1% Tm@pH-800-Nb)

[0091] Example 2 is a reversibly adjustable coordination type rare earth nanocrystal-organic dye, where the rare earth nanocrystals are Tm 3+ NaYF4 (NaYF4:1%Tm) with a doping concentration of 1%, where R is a functional group in the cyanine dye. (represented as pH-800-Nb), the specific preparation method in Example 2 is as follows.

[0092] (1) Synthesis of NaYF4: 1% Tm

[0093] YCl3 (0.99 mmol) and TmCl3 (0.01 mmol) were added to a three-necked flask containing oleic acid (6 mL) and octadecene (15 mL). The mixture was heated to 120 °C under an argon (Ar) atmosphere and maintained at that temperature while being magnetically stirred until a clear solution was formed. The solution was then cooled to 40 °C, remaining clear and transparent, at which point NH4F (0.4 mmol, 148.2 mg) and NaOH (2.5 mmol, 100 mg) dissolved in methanol (8 mL) were added dropwise to the mixture. The mixture was then heated to 120 °C and maintained for 30 minutes to remove methanol under an argon atmosphere, followed by evacuation for 10 minutes. The mixture was then heated to 300 °C under magnetic stirring and maintained for 60 minutes. The flask was then allowed to cool naturally to room temperature. Nanoparticles were collected by centrifugation (12000 rpm, 10 min), washed three times with cyclohexane / ethanol solution, and then dispersed in cyclohexane (10 mL).

[0094] (2) Synthetic dye pH-800-Nb (Formula III)

[0095] The synthesis route is shown below:

[0096]

[0097] pH-820-b (210 mg, 0.25 mmol) and N-methylpiperazine (0.1 mL, 0.76 mmol) were dissolved in 3 mL of anhydrous DMF (N,N-dimethylformamide), and then triethylamine (0.25 mL, 1.8 mmol) was added. The solution was stirred overnight in the dark. The solvent was evaporated to remove the residue, leaving a dark blue residue, which was purified by silica gel column chromatography (using a dichloromethane:methanol = 90:10 solvent system) to give the desired product as a blue solid (yield 15%).

[0098] 1H NMR (400MHz, MeOD-d4) δ (ppm) 8.16 (d, J = 8.8Hz, 2H), 7.92 (d, J = 8.0Hz, 4H), 7.85 (d, J = 8.8Hz, 3H), 7.70 (d, J = 13.6Hz, 2H), 7.53 (t, J = 7.4Hz, 2H),7.40-7.25(m,8H),5.85(d,J=13.6Hz,2H),5.35(s,4H),3.70(s,4 H),2.95(m,4H),2.62(m,4H),2.18(m,4H),1.94(s,12H),1.53(m,2H).

[0099] (3) Synthesize reversibly adjustable coordination type rare earth nanocrystal-organic dye.

[0100] First, NaYF4:1%Tm (20 mg) dispersed in cyclohexane was added to 3 mL of saturated NOBF4-CH2Cl2 solution to remove the oleic acid ligands on the surface. A white precipitate was observed. The mixture was shaken for 2 minutes and then centrifuged at 11000 rpm for 10 minutes to collect the precipitate. 2 mL of methanol was added to disperse the ligand-free nanoparticles for later use. In a flask, DSPE-PEG2000 (15 mg) was added to 4 mL of methanol. Then, 1 mL of ligand-free nanoparticles and pH-800-Nb dye (30 μL, 1 mg / mL, methanol solution) were added. The mixture was sonicated for 1 minute to ensure uniform dispersion. The solvent was removed using a rotary evaporator at 100 mbar. The resulting nanocomposite material was then redispersed in 1 mL of deionized water. Centrifuge at 12000 rpm and wash three times with deionized water to obtain reversible coordination-regulated rare earth nanocrystal-organic dye. Disperse the reversible coordination-regulated rare earth nanocrystal-organic dye in 1 mL of deionized water.

[0101] Comparative Example 1 (NaYF4:1%Tm@IR-820):

[0102] Comparative Example 1 is a rare earth nanocrystal-cyanine dye composite material, where the rare earth nanocrystals are Tm 3+ The cyanine dye used is commercially available IR-820, with a doping concentration of 1% NaYF4 (NaYF4:1%Tm). Coordination is achieved through sulfonic acid groups. The cyanine dye structure is shown below:

[0103]

[0104] The preparation method of rare earth nanocrystal-cyanine dye composite material is as described in Example 1.

[0105] Monitoring of luminescence lifetime across multiple cycles from pH 3 to pH 8:

[0106] The luminescence lifetime of Examples 1, 2, and Comparative Example 1 were monitored under different pH conditions, and multiple pH cycles from pH 3 to pH 8 were performed to observe changes in luminescence lifetime. Luminescence decay curves and time-gated intensity spectra were obtained using an FLS1000 fluorescence spectrophotometer (Edinburgh Instruments), equipped with an optical parametric oscillator (OPO) laser (10Hz). Luminescence lifetime and time-gated intensity images were collected using a self-built time-resolved imaging system equipped with a 785nm continuous wave (CW) diode laser, an 800±10nm filter (Croma), a digital delay generator (STC810, CISS Instruments), and an IsCMOS (2DSPC-GaAs-U, CISS Instruments) camera. All lifetime imaging data were processed using MATLAB. Lifetime imaging images were obtained by processing a series of time-gated intensity images acquired at different delay times. The luminescence lifetime of each pixel was obtained by fitting the decay curve of each pixel using a single exponential model. The luminescence decay curve follows Formula 1:

[0107]

[0108] Where I represents the luminescence intensity as a function of time, I0 represents the initial luminescence intensity, t represents time, and τ represents the luminescence lifetime. The photophysical meaning of τ is the time required for the luminescence intensity to decay to 1 / e of its initial value. C is a constant, resulting from unavoidable noise during lifetime imaging (such as dark current from the camera).

[0109] Test results are as follows Figure 3 As shown, according to Figure 3 The red and blue dashed lines in the figure show that Examples 1 and 2 exhibit excellent reversibility, maintaining a consistent response across five cycles and showing a significant, clearly distinguishable difference in luminescence lifetime between acidic and alkaline environments. In contrast, Comparative Example 1 showed a variable luminescence lifetime after repeated cycles, failing to exhibit a consistent cyclic response and exhibiting a smaller difference in luminescence lifetime between acidic and alkaline environments. Compared to the more hydrophobic pH-800-Nb and pH-820-b, the sulfonate groups of IR-820 impart greater water solubility. Therefore, IR-820 gradually separates from the nanocrystal surface into the aqueous phase. The dye is too far away to fully recoordinate with the nanocrystals, preventing stable reversible cycling. Due to the hydrophobicity of pH-800-Nb and pH-820-b, even after protonation and removal from the nanocrystals, they maintain a certain distance from the nanocrystals, ensuring complete reversibility.

[0110] UV-Vis absorption spectroscopy test:

[0111] The ultraviolet-visible absorption spectra of Examples 1, 2, and Comparative Example 1 were measured, and the results are as follows: Figure 4 As shown. Example 2 exhibits a certain degree of pH responsiveness, with a significant change in the absorption spectrum after protonation, resulting in a red shift of the absorption peak.

[0112] The properties of the different dyes in Examples 1, 2, and Comparative Example 1, and the pH response of the rare earth-dye hybrids are shown in Table 1 below:

[0113] Table 1:

[0114]

[0115] Therefore, as shown in Table 1, by rationally designing the coordinating groups on the organic dye, effective coordination between the dye and lanthanide nanocrystals was achieved at higher pH values, thereby realizing efficient nonradiative energy transfer and shortening the luminescence lifetime of the nanocrystals. At lower pH values, the coordination between the dye and nanocrystals weakened, reducing the nonradiative energy transfer efficiency and thus restoring the luminescence lifetime. It is noteworthy that this reversible coordination effect at the inorganic-organic interface can reversibly modulate the nonradiative energy transfer from the nanocrystals to the dye, resulting in a reversible change in the luminescence lifetime of the lanthanide nanocrystals. This reversible change is independent of whether the energy level of the dye itself is affected by pH. Furthermore, the results confirm that the energy levels of pH-820-b and IR-820 dyes are not affected by the solution pH. Moreover, when the pH value of the dye-free NaYF4:1%Tm@DSPE-PEG nanoparticles was changed, the fluorescence lifetime remained unchanged. Meanwhile, when pH-820-b and IR-820 coordinate with NaYF4:1%Tm to form lanthanide-dye hybrids, they exhibit significant pH responsiveness: longer lifetimes under acidic conditions and shorter lifetimes under alkaline conditions. These results indicate that the pH responsiveness of the rare-earth nanocrystal-organic dye composites does not originate from the pH sensitivity of the dyes themselves. Furthermore, since the nanocrystals themselves are not pH-responsive, the pH responsiveness of the rare-earth nanocrystal-organic dye composites stems from the reversible coordination between the nanocrystals and the dyes. Although pH-800-Nb itself is pH-sensitive, it exhibits the same pH-dependent lifetime behavior as pH-820-b and IR-820, with shorter lifetimes under alkaline conditions and longer lifetimes under acidic conditions. If the energy level of pH-800-Nb is the cause of its pH responsiveness, then the higher uptake rate of pH-800-Nb under acidic conditions would lead to more efficient energy transfer, resulting in a shorter lifetime under acidic conditions and a longer lifetime under alkaline conditions. However, the observed trend is exactly the opposite, further confirming that the response mechanism is unrelated to the pH sensitivity of the dye itself.

[0116] Example 3

[0117] Example 3 uses the reversible, adjustable coordination type rare earth nanocrystal-organic dye from Example 2 to continuously detect gastric acid in mice.

[0118] Both H2 receptor antagonists and proton pump inhibitors (PPIs) are effective in inhibiting gastric acid secretion, treating conditions such as gastroesophageal reflux disease (GERD), gastric ulcers, and Helicobacter pylori infection. Despite similar results, these two classes of drugs work through different mechanisms. H2 receptor antagonists competitively block the binding of histamine to H2 receptors on parietal cells, thereby inhibiting acid secretion. PPIs irreversibly bind to proton pumps within parietal cells, thus reducing gastric acid production. Traditionally, in vivo studies of antacids have relied on collecting gastric juice from multiple mice to obtain sufficient samples, which limits the ability to conduct long-term, non-invasive monitoring in a single mouse.

[0119] In Example 3, ranitidine hydrochloride was used as an H2 receptor antagonist (H2 receptor antagonist treatment group), esomeprazole was used as a PPI (PPI treatment group), and a control group was established by injecting saline. Gastric pH in mice was monitored for up to 4 hours after administration. Five minutes before intravenous injection, each mouse was orally administered the reversible regulated coordination rare-earth nanocrystal-organic dye from Example 2, allowing for 5 minutes of complete interaction between the dye and the gastric environment. The drugs were then administered intravenously (100 μL saline for the control group, 0.15 mg / 100 μL for each drug in the treatment groups). Each group consisted of three mice. Over the next 4 hours, time-gated intensity and lifetime imaging of the mouse stomach was performed. Lifetime images were generated in MATLAB using a single exponential fit of a series of time-gated intensity images acquired at different threshold delays, as shown below. Figure 5 As shown in the figure, in 4-hour continuous lifetime imaging, a general trend was observed in the H2 receptor antagonist group, where the gastric luminescence lifetime initially decreased and then increased, indicating an initial increase in gastric pH followed by a decrease. In contrast, the PPI group showed a decrease in gastric luminescence lifetime followed by stabilization, indicating a decrease in gastric pH followed by a period of stability. The control group showed no significant change in luminescence lifetime, indicating stable gastric pH under normal conditions.

[0120] like Figure 6 and Figure 7As shown, statistical analysis and linear fitting were performed on the lifetime imaging results. In the control group, corresponding to saline injection, the luminescence lifetime remained stable at approximately pH 4.0. In the ranitidine group, gastric pH began to rise 20 minutes after administration, peaked at 1 hour, and gradually decreased to baseline levels after 4 hours. Compared with the ranitidine group, mice in the esomeprazole group showed a delayed response, with significant pH changes beginning 80 minutes after administration, peaking at approximately 2 hours, and then maintaining an elevated pH. Notably, the peak gastric pH in the PPI group was higher than that in the ranitidine group. Overall, the PPI group had a longer onset time, but the pH maintenance time was longer than that in the H2 receptor antagonist group. This difference is attributed to the drug mechanism: H2 receptor antagonists reversibly bind to H2 receptors on parietal cells, reducing gastric acid by inhibiting cAMP production, while PPIs irreversibly inhibit the proton pump, leading to a sustained reduction in acid secretion.

[0121] Therefore, the reversibly adjustable coordination rare-earth nanocrystal-organic dye of Example 2, based on its reversible mechanism, allows for continuous pH monitoring, reflecting the true state of physiological indicators. When the pH changes, lifetime imaging provides a relatively linear response, making it easy to adjust and remeasure even with pH changes in subsequent measurements, obtaining new response data, thus demonstrating repeatability and verifiability. Furthermore, luminescence lifetime imaging provides relatively stable pH readings, accurately reflecting pH changes by capturing subtle variations in luminescence lifetime.

[0122] Furthermore, the rare-earth nanocrystal-organic dye complexes listed above also show potential applications in PCR detection. For example, Helicobacter pylori, a bacterium associated with gastric ulcers and gastric cancer, can survive in the acidic environment of the stomach. The pH of gastric juice can affect the activity and survival status of bacteria in a sample. By monitoring pH, it can be ensured that the acidic environment of gastric juice is suitable for Helicobacter pylori survival at the time of sample collection, thereby optimizing sampling conditions to improve the accuracy of PCR detection. In some cases, treatment of gastric diseases (such as the use of acid-suppressing drugs) may alter the pH of gastric juice. By monitoring changes in pH, the effectiveness of treatment can be assessed, and PCR detection can be used to determine whether the treatment has successfully eliminated the pathogen. More specifically, changes in gastric juice pH may affect the stability and extraction efficiency of DNA in a sample. By controlling pH, the conditions for DNA extraction and PCR amplification can be optimized, improving the sensitivity and specificity of detection.

[0123] While the present invention has been illustrated and described with reference to certain preferred embodiments, those skilled in the art should understand that the above description is a further detailed explanation of the invention in conjunction with specific embodiments, and should not be construed as limiting the specific implementation of the invention to these descriptions. Various changes in form and detail can be made by those skilled in the art, including several simple deductions or substitutions, without departing from the spirit and scope of the invention.

Claims

1. A reversibly modulated coordination-type rare earth nanocrystal-organic dye complex, characterized in that, Consisting of a rare earth nanocrystal and a cyanine dye, the composition of the rare earth nanocrystal includes NaYF4 doped with one or more of Tm 3+ , Eu 3+ , and Yb 3+ , one or more of Tm 3+ , Eu 3+ , and Yb 3+ replaces the position of part Y 3+ , and the cyanine dye forms a reversible coordination with the rare earth nanocrystal on the surface of the rare earth nanocrystal, wherein the cyanine dye has a structure shown in Formula I: Formula I; wherein R is halogen or a group , * indicates the position of bonding, R1is C1-C5 alkyl; The rare earth nanocrystal-organic dye pair is reversibly responsive to pH, and the reversibly responsive of the rare earth nanocrystal-organic dye pair to pH causes the luminescence lifetime of the rare earth nanocrystal-organic dye to change.

2. The reversible adjustable coordination type rare earth nanocrystal-organic dye composite as described in claim 1, characterized in that, The R is chlorine, and the chlorin dye has the following structure II: Formula II.

3. The reversible adjustable coordination type rare earth nanocrystal-organic dye composite as described in claim 1, characterized in that, The rare earth nanocrystal is a hexagonal phase structure, and the nanoparticle size of the rare earth nanocrystal is 5-50 nm. Group form a coordination with the rare earth nanocrystal.

4. The reversible adjustable coordination type rare earth nanocrystal-organic dye composite as described in claim 1, characterized in that, The composition of the rare earth nanocrystal includes Tm-doped NaYF4, wherein the doping amount of Tm is 0.5-5%. 3+ The composition of the rare earth nanocrystal includes Tm-doped NaYF4, wherein the doping amount of Tm is 0.5-5%. 3+ The composition of the rare earth nanocrystal includes Tm-doped NaYF4, wherein the doping amount of 5. The reversibly adjustable coordination type rare earth nanocrystal-organic dye composite as described in claim 4, characterized in that, The method comprises the following steps:

6. The method for preparing the reversibly adjustable coordination type rare earth nanocrystal-organic dye composite according to any one of claims 1 to 5, characterized in that, The rare earth nanocrystal is mixed with an organic solvent to form a solution, a de-ligating agent is added to the solution to remove organic ligands on the surface of the rare earth nanocrystal, and a precipitate is formed; The precipitate is collected by centrifugation and washing to obtain ligand-free rare earth nanocrystal; The ligand-free rare earth nanocrystal is mixed with an organic dye and a surfactant to form a uniformly dispersed mixture; The rare earth nanocrystal-organic dye composite is prepared by ultrasonic treatment and solvent evaporation. The rare earth nanocrystal is NaYF4:1%Tm, and the de-ligating agent is a saturated NOBF4-CH2Cl2 solution.

7. The production method according to claim 6, wherein The surfactant is DSPE-PEG2000, and the organic solvent is cyclohexane.

8. The production method according to claim 6, wherein The weight ratio of the ligand-free rare earth nanocrystal, the organic dye, and the surfactant is 1:1:0.

5.

9. The production method according to claim 6, wherein 11. Use of the luminescent probe of claim 10 in biological detection, which is stomach pH detection for non-disease diagnosis or treatment.

10. A luminescent probe, characterized by The method comprises the following steps: The luminescent probe is orally administered into a living body to interact with the stomach environment; 12. A method for detecting pH of gastric juice using the luminescent probe according to claim 10, characterized by, An H2 receptor antagonist or a proton pump inhibitor is administered by intravenous injection at a predetermined time; The stomach of the living body is subjected to time-gated intensity imaging and lifetime imaging to monitor the change in luminescence lifetime of the luminescent probe; The time-gated intensity images obtained at different time delays are subjected to single-exponential fitting processing to generate a lifetime image; The luminescence lifetime of the luminescent probe is correlated with the pH value, and the change in luminescence lifetime is used to monitor the change in stomach pH for non-disease diagnosis or treatment. ​ ​

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