PH-responsive self-assembled nanoparticles based on metal coordination as well as preparation method and application of pH-responsive self-assembled nanoparticles

Abstract

The invention relates to the technical field of self-assembly nano-particles, in particular to pH response type self-assembly nano-particles based on metal coordination and a preparation method and application of the pH response type self-assembly nano-particles. The pH response type self-assembly nanoparticles based on the metal coordination effect are prepared from the following raw materials: DOTA-Yb, DOTA-Er and a metal ion aqueous solution. The pH response type self-assembled nano particles provided by the invention respectively have different fluorescence intensities at 1550 nm and 1050 nm under different pH conditions and under 980 nm exciting light, and have pH responsiveness; after being disassembled under different pH conditions, the fluorescent probe respectively has fluorescence emission peaks at 1550nm and 1050nm under 980nm exciting light, and has dual-wavelength emission property, and the pH value can be detected by utilizing the ratio of the fluorescence intensity of two emission wavebands.

Description

Technical Field

[0001] The present invention relates to the technical field of self - assembled nanoparticles, and particularly to a pH - responsive self - assembled nanoparticle based on metal coordination, a preparation method thereof, and an application thereof. Background Art

[0002] In the field of biological labeling, fluorescence labeling is the most concerned labeling method. The detection sensitivity of fluorescence labeling mainly depends on the fluorescence intensity of the label. At present, the commonly used fluorescence labeling materials mainly include organic fluorescent dyes and quantum dot nanoprobes, but they have obvious limitations: the fluorescence intensity of organic fluorescent dyes is low, and they are extremely prone to photobleaching, resulting in the attenuation of fluorescence signals; the color purity of quantum dot nanoprobes is low, the fluorescence intensity is unstable, and the fluorescence signal is easily interfered in a complex biological environment. Therefore, there is an urgent need to develop a new type of fluorescence labeling material.

[0003] In recent years, fluorescent nanoparticles have received extensive attention in the fields of luminescence, display, biological labeling, etc. due to their high brightness, good stability, low diffusibility, low deformability, and non - fading at high temperatures. At present, most of the fluorescent nanoparticles prepared in the prior art are nanoparticles containing cadmium (Cd). However, cadmium is a heavy metal with potential biological toxicity and environmental pollution risks.

[0004] In addition, most of the existing fluorescent nanoparticles lack the ability to respond to micro - environmental changes (such as pH value changes), and it is difficult to achieve precise detection and imaging for specific physiological or pathological conditions. Summary of the Invention

[0005] The present invention provides a pH - responsive self - assembled nanoparticle based on metal coordination, and the pH - responsive self - assembled nanoparticle based on metal coordination has pH responsiveness and dual - wavelength emission properties.

[0006] The present invention also provides a preparation method of a pH - responsive self - assembled nanoparticle based on metal coordination. Through this preparation method, the above - mentioned pH - responsive self - assembled nanoparticle based on metal coordination can be prepared. Therefore, the prepared pH - responsive self - assembled nanoparticle based on metal coordination has pH responsiveness and dual - wavelength emission properties.

[0007] The present invention also provides a dual - emission ratio - type fluorescence probe, which includes the above - mentioned pH - responsive self - assembled nanoparticle based on metal coordination. Therefore, the dual - emission ratio - type fluorescence probe has pH responsiveness and dual - wavelength emission properties; the dual - emission ratio - type fluorescence probe can be used to detect the pH value, and it uses the ratio of the fluorescence intensities of two emission bands (fluorescence ratio value) to detect the pH value, which can effectively reduce the interference caused by the probe concentration and environmental factors and improve the detection accuracy.

[0008] The first aspect of the present invention provides a pH-responsive self-assembled nanoparticle based on metal coordination. The raw materials for preparing the pH-responsive self-assembled nanoparticle based on metal coordination include DOTA-Yb, DOTA-Er, and an iron source;

[0009] The DOTA-Yb is prepared from a first raw material system including 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and a ytterbium source;

[0010] The DOTA-Er is prepared from a second raw material system including 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and an erbium source.

[0011] For the pH-responsive self-assembled nanoparticle based on metal coordination as described above, in the pH-responsive self-assembled nanoparticle based on metal coordination, the mass ratio of DOTA-Yb, DOTA-Er, and the iron source is 10:10:(0.1-2).

[0012] For the pH-responsive self-assembled nanoparticle based on metal coordination as described above, the iron source includes ferric chloride.

[0013] For the pH-responsive self-assembled nanoparticle based on metal coordination as described above, in the first raw material system, the ytterbium source is ytterbium chloride, and the mass ratio of the ytterbium chloride to the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is (1-2):1.

[0014] For the pH-responsive self-assembled nanoparticle based on metal coordination as described above, in the second raw material system, the erbium source is erbium chloride, and the mass ratio of the erbium chloride to the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is (1-2):1.

[0015] The second aspect of the present invention provides a preparation method for the pH-responsive self-assembled nanoparticle based on metal coordination as described above, including the following steps:

[0016] Dissolve 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and the ytterbium source in the first raw material system in water, carry out a first stirring reaction under the condition of a pH value of 6.4-6.6, and then obtain DOTA-Yb through a first drying treatment and a first purification treatment;

[0017] Dissolve 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and the erbium source in the second raw material system in water, carry out a second stirring reaction under the condition of a pH value of 6.4-6.6, and then obtain DOTA-Er through a second drying treatment and a second purification treatment;

[0018] Dissolve the DOTA-Yb and the DOTA-Er in water respectively to obtain a DOTA-Yb solution and a DOTA-Er solution;

[0019] Mix the DOTA-Yb solution and the DOTA-Er solution to obtain a mixed solution;

[0020] Add an aqueous solution of an iron source to the mixed solution, carry out a third stirring reaction under the condition that the pH value is 7.0 - 8.5, and then carry out centrifugation treatment and washing treatment to obtain the pH-responsive self-assembled nanoparticles based on metal coordination.

[0021] For the preparation method of the pH-responsive self-assembled nanoparticles based on metal coordination as described above, the concentration of iron ions in the aqueous solution of the iron source is 2 mg / mL - 15 mg / mL.

[0022] For the preparation method of the pH-responsive self-assembled nanoparticles based on metal coordination as described above, the time of the first stirring reaction is 10 h - 20 h;

[0023] And / or, the time of the second stirring reaction is 10 h - 20 h.

[0024] For the preparation method of the pH-responsive self-assembled nanoparticles based on metal coordination as described above, the time of the third stirring reaction is 5 min - 240 min.

[0025] The third aspect of the present invention provides a dual-emission ratio fluorescent probe, which comprises the pH-responsive self-assembled nanoparticles based on metal coordination as described above;

[0026] The dual-emission ratio fluorescent probe is applied to detect the pH value.

[0027] In summary, the solution of the present invention has at least the following effects:

[0028] The pH-responsive self-assembled nanoparticles based on metal coordination provided by the present invention have pH responsiveness and dual-wavelength emission properties: (1) Under different pH conditions, the pH-responsive self-assembled nanoparticles have different fluorescence intensities at 1550 nm and 1050 nm under 980 nm excitation light, showing pH responsiveness; (2) After disassembling under different pH conditions, the pH-responsive self-assembled nanoparticles based on metal coordination have fluorescence emission peaks at 1550 nm and 1050 nm under 980 nm excitation light, showing dual-wavelength emission properties. The pH value can be detected by using the ratio of fluorescence intensities in two emission bands (fluorescence ratio value), effectively reducing the interference caused by concentration and environmental factors and improving the detection accuracy, and having broad application prospects in the fields of detection and fluorescence imaging, etc. Description of the Drawings

[0029] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0030] Figure 1 It is the TEM image of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention;

[0031] Figure 2 It is the transmission electron microscopy energy spectrum test result diagram of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention, where Figure 2 a is the dark field image of the pH-responsive self-assembled nanoparticles based on metal coordination in TEM, Figure 2 b is the superimposed distribution diagram of different elements in the pH-responsive self-assembled nanoparticles based on metal coordination, Figure 2 c is the distribution diagram of Fe element in the pH-responsive self-assembled nanoparticles based on metal coordination, Figure 2 d is the distribution diagram of Er element in the pH-responsive self-assembled nanoparticles based on metal coordination, Figure 2 e is the distribution diagram of Yb element in the pH-responsive self-assembled nanoparticles based on metal coordination;

[0032] Figure 3 It is the histogram of the hydrodynamic diameter distribution of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention;

[0033] Figure 4UV-Vis absorption spectrum of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention;

[0034] Figure 5 Fluorescence intensities of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention in buffer solutions with different pH values (pH = 4.5, pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 6.8, pH = 7.4) in the emission wavelength range of 1000 - 1150 nm under the excitation light with a wavelength of 980 nm;

[0035] Figure 6 Fluorescence intensities of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention in buffer solutions with different pH values (pH = 4.5, pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 6.8, pH = 7.4) in the emission wavelength range of 1450 - 1650 nm under the excitation light with a wavelength of 980 nm;

[0036] Figure 7 Working curve of FL1050 / FL1550 vs. pH value of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention at different pH values;

[0037] Figure 8 Fluorescence at 1050 nm and 1550 nm of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention in buffer solutions with different pH values (pH = 4.5, pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 6.8, pH = 7.4) under the excitation light with a wavelength of 980 nm;

[0038] Figure 9 Fluorescence at 1550 nm of the pH-responsive self-assembled nanoparticles based on metal coordination in Examples 1 - 3 of the present invention under the excitation light with a wavelength of 980 nm.

[0039] Figure 10 Fluorescence at 1550 nm of the pH-responsive self-assembled nanoparticles based on metal coordination in Examples 1 and 4 of the present invention under the excitation light with a wavelength of 980 nm.

[0040] Figure 11 Fluorescence at 1550 nm of the pH-responsive self-assembled nanoparticles based on metal coordination in Examples 1 and 5 - 7 of the present invention under the excitation light with a wavelength of 980 nm. Detailed implementation manners

[0041] To make the objectives, technical solutions, and advantages of the present invention clearer, the following will, in combination with the embodiments of the present invention, clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in this field or according to the product specifications. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchases.

[0042] It should be noted that the descriptions involving "first", "second", "third", etc. in the present invention are used to distinguish similar objects, rather than to describe a specific order or sequence, and thus should not be construed as a limitation to the present invention.

[0043] The first aspect of the present invention provides a pH-responsive self-assembled nanoparticle based on metal coordination. The raw materials for preparing the pH-responsive self-assembled nanoparticle based on metal coordination include DOTA-Yb, DOTA-Er, and an aqueous metal ion solution;

[0044] DOTA-Yb is made from a first raw material system including 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and a ytterbium source;

[0045] DOTA-Er is made from a second raw material system including 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and an erbium source.

[0046] The present invention does not particularly limit the specific sources of the raw materials for preparing the above-mentioned pH-responsive self-assembled nanoparticles based on metal coordination, which can be obtained through commercial channels or prepared by methods well-known in the art.

[0047] In the present invention, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA) is a twelve-membered tetraaza macrocyclic ligand, which can be used as a chelating agent to form stable complexes with lanthanide ions (such as ytterbium ions and erbium ions).

[0048] The present invention prepares pH-responsive self-assembled nanoparticles based on metal coordination through raw materials including DOTA-Yb, DOTA-Er, and an aqueous metal ion solution. The pH-responsive self-assembled nanoparticles based on metal coordination have pH responsiveness and dual-wavelength emission properties: (1) Under different pH conditions, the pH-responsive self-assembled nanoparticles have different fluorescence intensities at 1550 nm and 1050 nm under 980 nm excitation light, showing pH responsiveness; (2) After the pH-responsive self-assembled nanoparticles based on metal coordination disassemble under different pH conditions, they have fluorescence emission peaks at 1550 nm and 1050 nm under 980 nm excitation light, showing dual-wavelength emission properties. The pH value can be detected by using the ratio of the fluorescence intensities of the two emission bands (fluorescence ratio value), and it has broad application prospects in the fields of detection and fluorescence imaging, etc.

[0049] An explanation of the principle of the present invention: Due to the fluorescence resonance energy transfer effect (FRET effect) of the pH-responsive self-assembled nanoparticles based on metal coordination of the present invention, the energy of Yb is transferred to Er, sensitizing the fluorescence intensity of Er at 1550 nm under 980 nm excitation light (FL1550); when the pH-responsive self-assembled nanoparticles based on metal coordination disassemble in an acidic environment, the FRET effect between Yb and Er is interrupted, restoring the fluorescence intensity of Yb at 1050 nm under 980 nm excitation light (FL1050). By calculating the ratio of these two fluorescence intensities (FL1050 / FL1550), high-precision pH value detection is achieved.

[0050] In a specific embodiment, in the above-mentioned pH-responsive self-assembled nanoparticles based on metal coordination, the mass ratio of DOTA-Yb, DOTA-Er, and the iron source is 10:10:(0.1 - 2).

[0051] When the mass-volume ratio of DOTA-Yb, DOTA-Er, and the aqueous metal ion solution is within the above range, it is beneficial to prepare the above-mentioned pH-responsive self-assembled nanoparticles based on metal coordination with pH responsiveness and dual-wavelength emission properties.

[0052] In a specific embodiment, the iron source includes ferric chloride.

[0053] In the present invention, ferric ions (Fe 3+ ) are metal ions that can form coordination bonds with DOTA.

[0054] In a specific embodiment, in the above-mentioned first raw material system, the ytterbium source is ytterbium chloride, and the mass ratio of ytterbium chloride to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is (1 - 2):1.

[0055] When in the first raw material system, the ytterbium source is ytterbium chloride and the parameter of the mass ratio of ytterbium chloride to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is within the above range, ytterbium ions can fully chelate with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid.

[0056] In a specific embodiment, in the above second raw material system, the erbium source is erbium chloride, and the mass ratio of erbium chloride to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is (1-2):1.

[0057] When in the second raw material system, the erbium source is erbium chloride and the parameter of the mass ratio of erbium chloride to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is within the above range, erbium ions can fully chelate with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid.

[0058] The second aspect of the present invention provides a method for preparing the above pH-responsive self-assembled nanoparticles based on metal coordination, comprising the following steps:

[0059] Dissolve 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and the ytterbium source in the first raw material system in water, carry out a first stirring reaction under the condition that the pH value is 6.4-6.6, and after the first drying treatment and the first purification treatment, obtain DOTA-Yb;

[0060] Dissolve 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and the erbium source in the second raw material system in water, carry out a second stirring reaction under the condition that the pH value is 6.4-6.6, and after the second drying treatment and the second purification treatment, obtain DOTA-Er;

[0061] Dissolve DOTA-Yb and DOTA-Er in water respectively to obtain a DOTA-Yb solution and a DOTA-Er solution;

[0062] Mix the DOTA-Yb solution and the DOTA-Er solution to obtain a mixed solution;

[0063] Add an aqueous solution of an iron source to the mixed solution, carry out a third stirring reaction under the condition that the pH value is 7.0-8.5, and after centrifugation and washing, the pH-responsive self-assembled nanoparticles based on metal coordination are obtained.

[0064] The present invention does not particularly limit the specific type of the above water, and it can be selected according to actual needs. In some embodiments, the above water can be deionized water.

[0065] The present invention does not particularly limit the temperature and time of the first drying treatment and the second drying treatment, and can be selected according to actual needs.

[0066] The present invention also does not particularly limit the specific operations of the above-mentioned centrifugation treatment and washing treatment, and methods well-known in the art can be adopted.

[0067] Through the above preparation, the present invention can prepare the above-mentioned metal coordination-based pH-responsive self-assembled nanoparticles with pH responsiveness and dual-wavelength emission properties.

[0068] In a specific embodiment, the concentration of iron ions in the above-mentioned aqueous iron source solution is 2 mg / mL to 15 mg / mL.

[0069] When the concentration of iron ions in the aqueous iron source solution is within the above range, sufficient binding sites can be provided, enabling the iron ions to coordinate with the carboxyl groups on DOTA-Yb and DOTA-Er, thereby self-assembling to form metal coordination-based pH-responsive self-assembled nanoparticles with pH responsiveness and dual-wavelength emission properties.

[0070] In a specific embodiment, the time of the above-mentioned first stirring reaction is 10 h to 20 h.

[0071] When the parameter of the time of the first stirring reaction is within the above range, the reaction between 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and the ytterbium source in the first raw material system proceeds sufficiently, thereby obtaining DOTA-Yb.

[0072] In a specific embodiment, the time of the above-mentioned second stirring reaction is 10 h to 20 h.

[0073] When the parameter of the time of the second stirring reaction is within the above range, the reaction between 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and the erbium source in the second raw material system proceeds sufficiently, thereby obtaining DOTA-Er.

[0074] In a specific embodiment, the time of the above-mentioned third stirring reaction is 5 min to 240 min.

[0075] When the parameter of the time of the third stirring reaction is within the above range, it is beneficial for the reaction between metal ions (iron ions) and DOTA-Yb and DOTA-Er to proceed sufficiently. At the same time, it avoids the aggregation or precipitation of nanoparticles caused by too long reaction time, which is beneficial for preparing uniform metal coordination-based pH-responsive self-assembled nanoparticles.

[0076] In a specific embodiment, the above first purification treatment includes: dissolving the product after the first drying treatment in chloroform, adding water, and then performing extraction.

[0077] The first purification treatment of the product after the first drying treatment in the present invention is to remove excess Yb ions.

[0078] In a specific embodiment, the above second purification treatment includes: dissolving the product after the second drying treatment in chloroform, adding water, and then performing extraction.

[0079] The second purification treatment of the product after the second drying treatment in the present invention is to remove excess Er ions.

[0080] In a specific embodiment, it further includes performing rotary evaporation and drying on the above DOTA-Yb and the above DOTA-Er respectively. The specific operation of performing rotary evaporation and drying in the present invention is not particularly limited and can be carried out according to the methods well known in the art.

[0081] The third aspect of the present invention provides a dual-emission ratio fluorescent probe, which includes the above pH-responsive self-assembled nanoparticles based on metal coordination; the dual-emission ratio fluorescent probe is applied to detect the pH value. Since the dual-emission ratio fluorescent probe includes the above pH-responsive self-assembled nanoparticles based on metal coordination, the dual-emission ratio fluorescent probe also has pH responsiveness and dual-wavelength emission properties; the dual-emission ratio fluorescent probe uses the ratio of the fluorescence intensities of two emission bands (fluorescence ratio value) to detect the pH value, which can effectively reduce the interference caused by the probe concentration and environmental factors and improve the detection accuracy.

[0082] Hereinafter, the present invention will be further introduced through specific examples.

[0083] Example 1

[0084] In this example, the pH-responsive self-assembled nanoparticles based on metal coordination are prepared through the following process:

[0085] Step 1: Dissolve 5 mg of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA) and 6 mg of ytterbium chloride (YbCl3) in water, stir and react at a pH value of 6.5 for 12 h to obtain product A. Perform a drying treatment on product A, then dissolve the dried product A in chloroform, add water, and perform extraction to remove excess Yb ions. After rotary evaporation and drying, DOTA-Yb is obtained;

[0086] Step 2: Dissolve 5 mg of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid (DOTA) and 6 mg of erbium chloride (ErCl3) in water, stir and react for 12 h under the condition of pH 6.5 to obtain product B. Dry product B, then dissolve the dried product B in chloroform, add water for extraction to remove excess Er ions, and obtain DOTA-Er after rotary evaporation and drying;

[0087] Step 3: Respectively take 10 mg of DOTA-Yb in Step 1 and 10 mg of DOTA-Er in Step 2 and dissolve them in 1 mL of water to obtain a DOTA-Yb solution and a DOTA-Er solution;

[0088] Step 4: Mix the DOTA-Yb solution and the DOTA-Er solution in Step 3 to obtain a mixed solution;

[0089] Step 5: Dissolve 0.5 mg of ferric chloride (FeCl3) in 50 μL of water to obtain an aqueous FeCl3 solution;

[0090] Step 6: Add the aqueous FeCl3 solution in Step 5 to the mixed solution in Step 4, stir and react for 240 min under the condition of pH 8.5 to obtain product C. Centrifuge and wash product C to obtain pH-responsive self-assembled nanoparticles based on metal coordination;

[0091] Example 2

[0092] The preparation of the pH-responsive self-assembled nanoparticles based on metal coordination provided in this example is basically the same as that in Example 1, except that:

[0093] Step 5: Replace 0.5 mg of ferric chloride (FeCl3) with 0.1 mg of ferric chloride (FeCl3).

[0094] Example 3

[0095] The preparation of the pH-responsive self-assembled nanoparticles based on metal coordination provided in this example is basically the same as that in Example 1, except that:

[0096] Step 5: Replace 0.5 mg of ferric chloride (FeCl3) with 0.2 mg of ferric chloride (FeCl3).

[0097] Example 4

[0098] The preparation of the pH-responsive self-assembled nanoparticles based on metal coordination provided in this example is basically the same as that in Example 1, except that:

[0099] Step 6: Replace the stirring reaction for 240 min at pH 8.5 with a stirring reaction for 240 min at pH 7.0.

[0100] Example 5

[0101] The preparation of the pH-responsive self-assembled nanoparticles based on metal coordination provided in this example is basically the same as that in Example 1, except that:

[0102] Step 6: Replace the stirring reaction for 240 min at pH 8.5 with a stirring reaction for 5 min at pH 8.5.

[0103] Example 6

[0104] The preparation of the pH-responsive self-assembled nanoparticles based on metal coordination provided in this example is basically the same as that in Example 1, except that:

[0105] Step 6: Replace the stirring reaction for 240 min at pH 8.5 with a stirring reaction for 60 min at pH 8.5.

[0106] Example 7

[0107] The preparation of the pH-responsive self-assembled nanoparticles based on metal coordination provided in this example is basically the same as that in Example 1, except that:

[0108] Step 6: Replace the stirring reaction for 240 min at pH 8.5 with a stirring reaction for 120 min at pH 8.5.

[0109] Performance Test

[0110] 1. Morphology Characterization

[0111] The pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention were tested by transmission electron microscopy (TEM), Figure 1 which is the TEM image of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention; the energy spectrum test (EDS) of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 was carried out, Figure 2 which is the result diagram of the energy spectrum test of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention. Among them, Figure 2 a in is the dark field image of the pH-responsive self-assembled nanoparticles based on metal coordination in TEM, Figure 2 b in is the superposition distribution diagram of different elements in the pH-responsive self-assembled nanoparticles based on metal coordination,Figure 2 c is the distribution map of Fe element in the pH-responsive self-assembled nanoparticles based on metal coordination. Figure 2 d is the distribution map of Er element in the pH-responsive self-assembled nanoparticles based on metal coordination. Figure 2 e is the distribution map of Yb element in the pH-responsive self-assembled nanoparticles based on metal coordination.

[0112] It can be seen from Figure 1 that the pH-responsive self-assembled nanoparticles based on metal coordination provided by the present invention are spherical in shape.

[0113] It can be seen from Figure 2 that self-assembly chelates Yb and Er in the pH-responsive self-assembled nanoparticles based on metal coordination, providing conditions for the occurrence of the FRET effect.

[0114] 2. Particle size and particle size distribution (PDI) test

[0115] The pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention were tested for particle size and particle size distribution (PDI). Figure 3 is the histogram of the hydrodynamic diameter distribution of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention.

[0116] It can be seen from Figure 3 that the pH-responsive self-assembled nanoparticles based on metal coordination provided by the present invention have good dispersibility and uniformity.

[0117] 3. Ultraviolet-visible absorption spectroscopy test

[0118] The pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention were tested for ultraviolet-visible absorption spectroscopy. Figure 4 is the ultraviolet-visible absorption spectrum of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention.

[0119] It can be seen from Figure 4 that the pH-responsive self-assembled nanoparticles based on metal coordination provided by the present invention have a broad absorption peak.

[0120] 4. Ratio fluorescence imaging test

[0121] (1) The pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention were respectively placed in buffer solutions with different pH values (pH = 4.5, pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 6.8, pH = 7.4). The excitation light wavelength was selected as 980 nm, and the fluorescence intensity of the pH-responsive self-assembled nanoparticles based on metal coordination in the emission wavelength range of 1000 - 1150 nm was detected, and the fluorescence intensity of the pH-responsive self-assembled nanoparticles based on metal coordination in the emission wavelength range of 1450 - 1650 nm was detected. Figure 5 is the fluorescence intensity of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention in buffer solutions with different pH values (pH = 4.5, pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 6.8, pH = 7.4) in the emission wavelength range of 1000 - 1150 nm under the excitation light with a wavelength of 980 nm. Figure 6 is the fluorescence intensity of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention in buffer solutions with different pH values (pH = 4.5, pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 6.8, pH = 7.4) in the emission wavelength range of 1450 - 1650 nm under the excitation light with a wavelength of 980 nm. Figure 7 is the working curve of FL1050 / FL1550 of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention versus the pH value at different pH values.

[0122] As Figures 5 - 7 can be seen, the pH-responsive self-assembled nanoparticles based on metal coordination provided by the present invention have dual emission peaks in the emission wavelength ranges of 1000 - 1150 nm and 1450 - 1650 nm, and the fluorescence intensity is correlated with the change of the pH value.

[0123] For the fluorescence detection of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention at different pH values, fluorescence emission peaks are respectively present at 1550 nm and 1050 nm under the excitation light of 980 nm, showing dual-wavelength emission properties. The pH value can be detected by using the ratio of the fluorescence intensities of the two emission bands (fluorescence ratio value). The working curve of FL1050 / FL1550 of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention versus the pH value at different pH values is as Figure 7As shown, its linear relationship satisfies y = 0.2694x - 0.9869. FL1050 / FL1550 refers to the ratio of the fluorescence intensity at 1050 nm to the fluorescence intensity at 1550 nm (fluorescence ratio value) under the excitation light of 980 nm wavelength.

[0124] (2) The pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 of the present invention were respectively placed in buffer solutions with different pH values (pH = 4.5, pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, pH = 6.8, pH = 7.4). The excitation light wavelength was selected as 980 nm. The fluorescence at 1050 nm (fluorescence 1050) of the pH-responsive self-assembled nanoparticles based on metal coordination at different pH values was recorded, the fluorescence at 1550 nm (fluorescence 1550) of the pH-responsive self-assembled nanoparticles based on metal coordination at different pH values was recorded, and the ratio of the fluorescence at 1050 nm to the fluorescence at 1550 nm of the pH-responsive self-assembled nanoparticles based on metal coordination at different pH values (ratio) was recorded. Using the color scale as a control, the results are as Figure 8 shown.

[0125] It can be seen from Figure 8 that as the pH value decreases, the fluorescence intensity of the pH-responsive self-assembled nanoparticles based on metal coordination provided by the present invention at 1050 nm increases, and the fluorescence intensity at 1550 nm weakens.

[0126] 5. Fluorescence resonance energy transfer effect (FRET effect)

[0127] (1) The FRET effect generated by the self-assembly of the pH-responsive self-assembled nanoparticles based on metal coordination in Examples 1-3 of the present invention was tested. The specific method was: the excitation light wavelength was selected as 980 nm, and the fluorescence of the pH-responsive self-assembled nanoparticles based on metal coordination at 1550 nm was recorded. The results are as Figure 9 shown.

[0128] It can be seen from Figure 9 that when the addition amount of ferric chloride is 0.5 mg, the near-infrared second-region fluorescence of Er at 1550 nm is the strongest, indicating that the FRET effect of the pH-responsive self-assembled nanoparticles based on metal coordination provided in Example 1 of the present invention is the best.

[0129] (2) The FRET effect generated by the self-assembly of the pH-responsive self-assembled nanoparticles based on metal coordination in Examples 1 and 4 of the present invention was tested. The specific method was: the excitation light wavelength was selected as 980 nm, and the fluorescence of the pH-responsive self-assembled nanoparticles based on metal coordination at 1550 nm was recorded. The results are asFigure 10 as shown

[0130] As Figure 10 can be seen, when the pH value is 8.5, the near-infrared fluorescence in the second near-infrared region of Er at 1550 nm is the strongest, indicating that the FRET effect of the pH-responsive self-assembled nanoparticles based on metal coordination provided in Example 1 of the present invention is the best.

[0131] (3) Test the FRET effect generated by the self-assembly of the pH-responsive self-assembled nanoparticles based on metal coordination in Example 1 and Examples 5-7 of the present invention. The specific method is as follows: Select the excitation light wavelength to be 980 nm, and record the fluorescence of the pH-responsive self-assembled nanoparticles based on metal coordination at 1550 nm. The results are as Figure 11 shown

[0132] As Figure 11 can be seen, when the reaction time is 240 min, the near-infrared fluorescence of Er at 1550 nm is the strongest, indicating that the FRET effect of the pH-responsive self-assembled nanoparticles based on metal coordination provided in Example 1 of the present invention is the best.

[0133] Finally, it should be noted that: the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pH-responsive self-assembled nanoparticle based on metal coordination, characterized in that: The raw materials for preparing the pH-responsive self-assembled nanoparticles based on metal coordination include DOTA-Yb, DOTA-Er and an iron source; The DOTA-Yb is prepared from a first raw material system including 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and an ytterbium source; The DOTA-Er is prepared from a second raw material system including 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and an erbium source.

2. The pH-responsive self-assembled nanoparticles based on metal coordination according to claim 1, characterized in that: In the pH-responsive self-assembled nanoparticles based on metal coordination, the mass ratio of DOTA-Yb, DOTA-Er and the iron source is 10: 10：(0.1～2)。 3. The pH-responsive self-assembled nanoparticles based on metal coordination according to claim 2, characterized in that: The iron source includes ferric chloride.

4. The pH-responsive self-assembled nanoparticles based on metal coordination according to claim 1, characterized in that: In the first raw material system, the ytterbium source is ytterbium chloride, and the mass ratio of the ytterbium chloride to the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is (1-2):

1.

5. The pH-responsive self-assembled nanoparticles based on metal coordination according to claim 1, characterized in that: In the second raw material system, the erbium source is erbium chloride, and the mass ratio of the erbium chloride to the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid is (1-2):

1.

6. A method for preparing pH-responsive self-assembled nanoparticles based on metal coordination according to any one of claims 1 to 5, characterized in that: The following steps are involved: Dissolving 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and ytterbium source in the first raw material system in water, performing a first stirring reaction at a pH value of 6.4 to 6.6, and then performing a first drying treatment and a first purification treatment to obtain DOTA-Yb; Dissolving 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracarboxylic acid and an erbium source in the second raw material system in water, performing a second stirring reaction at a pH value of 6.4 to 6.6, and then performing a second drying treatment and a second purification treatment to obtain DOTA-Er; Dissolving the DOTA-Yb and the DOTA-Er in water respectively to obtain a DOTA-Yb solution and a DOTA-Er solution; Mixing the DOTA-Yb solution and the DOTA-Er solution to obtain a mixed solution; The iron source aqueous solution is added to the mixed solution, and a third stirring reaction is carried out under the condition of pH value of 7.0 to 8.5, followed by centrifugation and washing to obtain the pH-responsive self-assembled nanoparticles based on metal coordination.

7. The method for preparing pH-responsive self-assembled nanoparticles based on metal coordination according to claim 6, characterized in that: The concentration of iron ions in the iron source aqueous solution is 2 mg / mL to 15 mg / mL.

8. The method for preparing pH-responsive self-assembled nanoparticles based on metal coordination according to claim 6, characterized in that: The first stirring reaction time is 10h to 20h; And / or, the second stirring reaction time is 10 h to 20 h.

9. The method for preparing pH-responsive self-assembled nanoparticles based on metal coordination according to claim 6, characterized in that: The third stirring reaction time is 5 min to 240 min.

10. A dual-emission ratiometric fluorescent probe, characterized in that: It comprises the pH-responsive self-assembled nanoparticles based on metal coordination as described in any one of claims 1 to 5; The dual-emission ratiometric fluorescent probe is used to detect pH value.

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