Ratio-type photoacoustic imaging probe, preparation method and application

Abstract

According to the ratio-type photoacoustic imaging probe and the preparation method provided by the invention, the albumin is used as a modification reagent to prepare an albumin-zinc sulfide (ZnS) nano probe, the unreacted reagent is removed through ultrafiltration, then the indocyanine green is added for adsorption, the redundant ICG is removed through ultrafiltration, the albumin-zinc sulfide-indocyanine green (ZnS-ICG) probe is obtained, the reagent cost is low, the preparation method is simple, and the method is suitable for industrial production. According to the prepared ratio type photoacoustic imaging probe, a photoacoustic signal at 700nm of ICG can be used as a reference, a copper ion response type ratio photoacoustic imaging probe is constructed on the basis of cation exchange reaction and activation of a photoacoustic signal at 1064nm after copper ions and zinc ions are exchanged, and the copper ions are quantitatively measured through a ratio. And the probe has a near-infrared fluorescence signal and can be used for tracing distribution of the probe in vivo.

Description

Technical Field

[0001] The present application relates to the technical field of material detection, and in particular to a ratiometric photoacoustic imaging probe, a preparation method and an application thereof. Background Art

[0002] Alzheimer's disease (AD) is the most common neurodegenerative disease, characterized by extracellular amyloid plaques and intracellular neurofibrillary tangles. Common pathogenic factors include excitotoxicity, dysregulation of transition metal homeostasis, reduction of endogenous antioxidants, and neuroinflammation. Excessive accumulation of redox-active metal ions (especially copper) can lead to increased levels of oxidative stress in the brain, resulting in damage to cellular components and changes in signaling pathways associated with neurodegenerative diseases. Therefore, it is necessary to investigate the role of Cu in the brain of AD patients. 2+ Highly sensitive imaging / detection is essential for a comprehensive understanding of its pathological events in the brain. Currently, many fluorescent probes have been explored and applied to Cu in cells and organisms. 2+ However, due to its limited penetration depth (about 1 mm), difficulty in crossing the blood-brain barrier (BBB), and Cu 2+ The fluorescence quenching caused by Cu 2+ Intravital fluorescence imaging in the brain remains a great challenge.

[0003] Compared with fluorescence technology, photoacoustic (PA) imaging combines the advantages of optical imaging and ultrasound imaging, and exhibits multi-scale spatial resolution while penetrating deep tissues (centimeter depth). Due to these advantages, PA probes have been widely used in biomedical imaging, disease detection, and biosensing. However, PA signals are susceptible to interference from background signals, which in turn affects the detection accuracy of the analyte. Based on this, activatable PA probes and ratiometric PA probes have been developed. These probes can regulate the molecular structure and induce signal responses to the stimulation of the analyte, thereby achieving the detection of specific biomarkers and small molecules. Moreover, ratiometric PA imaging can effectively reduce the measurement error, making the measurement method have higher analytical sensitivity, specificity, and accuracy. Compared with small molecule PA probes, nanostructured probes have longer blood half-life and targeting performance, but copper ion ratiometric PA probes for Alzheimer's disease detection have rarely been studied.

[0004] Detecting copper ions through ratiometric photoacoustic imaging is the key to accurate AD detection. Therefore, it is of great significance to develop ratiometric photoacoustic imaging probes activated by copper ions. Currently, there are no reports on ratiometric photoacoustic imaging probes for copper ion detection. Summary of the invention

[0005] In view of this, it is necessary to provide a ratiometric photoacoustic imaging probe with high detection accuracy, a preparation method and an application thereof to address the problems of short probe metabolism time and low detection accuracy in the prior art.

[0006] To solve the above problems, this application adopts the following technical solutions:

[0007] One of the purposes of the present application is to provide a method for preparing a ratiometric photoacoustic imaging probe, comprising the following steps:

[0008] The pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution is adjusted to 3.0-6.0;

[0009] Adding the sodium sulfide solution to the albumin zinc ion mixed solution and stirring at room temperature for 0.5-1 hour to obtain the albumin-zinc sulfide nanoprobe;

[0010] Ultrafiltration treatment of the albumin-zinc sulfide nanoprobe;

[0011] Indocyanine green is added to the filtrate after the ultrafiltration treatment and stirred at room temperature, and then a second ultrafiltration treatment is performed to obtain the albumin-zinc sulfide-indocyanine green probe.

[0012] In some of the embodiments, in the step of adjusting the pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution to XXXX, the zinc ions include zinc acetate.

[0013] In some embodiments, the step of adjusting the pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution to XXXX specifically includes the following steps:

[0014] The zinc acetate is added dropwise to the albumin solution, and hydrochloric acid is added dropwise to adjust the pH value, and the mixture is stirred for 15-10 minutes.

[0015] In some of the embodiments, in the step of adjusting the pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution to 3.0-6.0, the molar ratio of the albumin solution to the zinc ion solution is 1:20-1:100.

[0016] In some embodiments, in the step of adding sodium sulfide solution to the albumin zinc ion mixed solution and stirring at room temperature for 0.5-1 hour to obtain the albumin-zinc sulfide nanoprobe, the amount of the sodium sulfide solution is 0.02-0.2 mol / L, the amount of the albumin zinc ion mixed solution is 0.02-0.2 mol / L, and the amount of Zn 2+ :S 2- The amount of substance is 0.5-2.

[0017] In some of the embodiments, in the step of adding indocyanine green to the filtrate after ultrafiltration and stirring at room temperature, and then performing a second ultrafiltration treatment to obtain an albumin-zinc sulfide-indocyanine green probe, the mass concentration of indocyanine green is 2 mg / mL and the volume is 0.02-0.15 mL.

[0018] The second object of the present application is to provide a ratiometric photoacoustic imaging probe prepared by any of the preparation methods described above.

[0019] The third purpose of the present application is to provide an application of the ratiometric photoacoustic imaging probe in detecting copper ions.

[0020] This application adopts the above technical solution, and its beneficial effects are as follows:

[0021] The ratio photoacoustic imaging probe and preparation method provided by the present application, albumin is used as a modification reagent to prepare an albumin-zinc sulfide (ZnS) nanoprobe, unreacted reagents are removed by ultrafiltration, and then indocyanine green is added for adsorption, and excess ICG is removed by ultrafiltration to obtain an albumin-zinc sulfide-indocyanine green (ZnS-ICG) probe, the reagent cost is low, the preparation method is simple, and the ratio photoacoustic imaging probe prepared can use the photoacoustic signal of ICG at 700nm as a reference, based on a cation exchange reaction, copper ions are exchanged with zinc ions and then activated at 1064nm. The copper ion response ratio photoacoustic imaging probe is constructed, and the copper ions are quantitatively determined by the ratio. And the probe has a near-infrared fluorescence signal, which can be used to trace the distribution of the probe in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments of the present application or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 This is a flow chart of the steps of the method for preparing a ratiometric photoacoustic imaging probe provided in an embodiment of the present application.

[0024] Figure 2 Schematic diagram of the synthesis and characterization of albumin-zinc sulfide-indocyanine green (ZnS-ICG) provided in Example 1 of the present application.

[0025] Figure 3 Schematic diagram of the photoacoustic signal of copper ion activation of ZnS-ICG provided in Example 1 of the present application. DETAILED DESCRIPTION

[0026] Embodiments of the present application are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present application, and should not be construed as limiting the present application.

[0027] In the description of the present application, it should be understood that the terms "upper", "lower", "horizontal", "inside", "outside", etc., indicating orientations or positional relationships, are based on the orientations or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present application and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be understood as a limitation on the present application.

[0028] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of this application, the meaning of "plurality" is two or more, unless otherwise clearly and specifically defined.

[0029] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0030] See also Figure 1 , a step flow chart of a method for preparing a ratiometric photoacoustic imaging probe is provided for the present application, including the following steps 110 to S140, and the implementation method of each step is described in detail below.

[0031] Step S110: adjusting the pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution to 3.0-6.0.

[0032] In this example, an appropriate amount of albumin (BSA) was weighed and dissolved in water to prepare a BSA solution.

[0033] In this embodiment, a certain amount of zinc acetate is weighed to prepare zinc ions (Zn 2+ ) solution.

[0034] Specifically, zinc acetate is added dropwise to the albumin solution, and then hydrochloric acid is added dropwise to adjust the pH value, and the mixture is stirred for 10-30 minutes to form an albumin-zinc ion mixed solution.

[0035] In this embodiment, the molar ratio of the albumin solution to the zinc ion solution is 1:20-1:100.

[0036] In this embodiment, the amount of the sodium sulfide solution is 0.02-0.2 mol / L, the amount of the albumin zinc ion mixed solution is 0.02-0.2 mol / L, and the amount of Zn 2+ :S 2- The amount of substance is 0.5-2.

[0037] Step S120: adding the sodium sulfide solution to the albumin zinc ion mixed solution and stirring at room temperature for 0.5-1 hour to obtain the albumin-zinc sulfide nanoprobe.

[0038] Specifically, an appropriate amount of sodium sulfide was weighed and prepared into a solution, and an albumin zinc acetate solution was added, and the mixture was stirred at room temperature for 30 minutes to obtain an albumin-zinc sulfide nanoprobe.

[0039] Step S130: ultrafiltration treatment of the albumin-zinc sulfide nanoprobe.

[0040] Specifically, the synthesized albumin-zinc sulfide is ultrafiltered through an ultrafiltration tube to remove albumin and various ions that have not participated in the reaction.

[0041] Step S140: adding indocyanine green to the filtrate after the ultrafiltration treatment and stirring at room temperature, and then performing a second ultrafiltration treatment to obtain an albumin-zinc sulfide-indocyanine green probe.

[0042] Specifically, indocyanine green is added to the filtrate after the ultrafiltration treatment and stirred at room temperature, and unreacted indocyanine green is removed by ultrafiltration.

[0043] In this embodiment, the mass concentration of indocyanine green is 2 mg / mL, and the volume is 0.02-0.15 mL.

[0044] The ratio photoacoustic imaging probe and preparation method provided by the present application, albumin is used as a modification reagent to prepare an albumin-zinc sulfide (ZnS) nanoprobe, unreacted reagents are removed by ultrafiltration, and then indocyanine green is added for adsorption, and excess ICG is removed by ultrafiltration to obtain an albumin-zinc sulfide-indocyanine green (ZnS-ICG) probe, the reagent cost is low, the preparation method is simple, and the ratio photoacoustic imaging probe prepared can use the photoacoustic signal of ICG at 700nm as a reference, based on a cation exchange reaction, copper ions are exchanged with zinc ions and then activated at 1064nm. The copper ion response ratio photoacoustic imaging probe is constructed, and the copper ions are quantitatively determined by the ratio. And the probe has a near-infrared fluorescence signal, which can be used to trace the distribution of the probe in the body.

[0045] The above technical solution of the present application is described in detail below in conjunction with specific embodiments.

[0046] Example 1

[0047] 1) Weigh a certain amount of albumin (BSA) and dissolve it in water to prepare a 3-5 mg / mL BSA solution;

[0048] 2) Weigh a certain amount of zinc acetate and prepare 0.02-0.2 mol / L zinc ion (Zn 2+ ) solution;

[0049] 3) Add zinc acetate dropwise to the albumin solution, then add hydrochloric acid dropwise to adjust the pH to 3.0-6.0, and stir for 15-30 minutes;

[0050] 4) Weigh a certain amount of sodium sulfide to make a 0.02-0.2 mol / L solution, add albumin zinc acetate (Zn2+:S2- molar ratio 0.5-2) solution, and stir at room temperature for 30-60 minutes;

[0051] 5) The synthesized albumin-zinc sulfide was passed through a 100KD ultrafiltration tube (6500 rpm, 15 minutes) to remove the albumin and various ions that did not participate in the reaction;

[0052] 6) Add 0.1-1 mL of indocyanine green (0.2 mg / mL) and stir at room temperature, and remove unreacted indocyanine green by ultrafiltration.

[0053] 7) Determine the fluorescence imaging and ratiometric photoacoustic imaging performance.

[0054] Example 2

[0055] 1) Weigh 12 mg of albumin (BSA) and dissolve it in water to prepare a 3 mg / mL BSA solution;

[0056] 2) Weigh 1.84 mg of zinc acetate and prepare a 0.02 mol / L zinc ion (Zn2+) solution;

[0057] 3) Add zinc acetate dropwise to the albumin solution, then add hydrochloric acid dropwise to adjust the pH to 4.0, and stir for 15 minutes;

[0058] 4) Weigh 1.21 mg of sodium sulfide, prepare a 0.02 mol / L solution, add the albumin zinc acetate solution, and stir at room temperature for 30 minutes;

[0059] 5) The synthesized albumin-zinc sulfide was passed through a 100KD ultrafiltration tube (6500 rpm, 15 minutes) to remove the albumin and various ions that did not participate in the reaction;

[0060] 6) Add 1 mL of indocyanine green (0.2 mg / mL) and stir at room temperature, and remove unreacted indocyanine green by ultrafiltration.

[0061] 7) Determine hemolysis, cytotoxicity, and evaluate the biosafety of materials.

[0062] See also Figure 2 , which is the synthesis and characterization of albumin-zinc sulfide-indocyanine green (ZnS-ICG) provided in Example 1 of the present application. Including a) synthesis schematic diagram; b) transmission electron microscope image of ZnS-ICG, c) ultraviolet absorption spectra of ZnS, ICG and ZnS-ICG, d) fluorescence spectra and fluorescence photos of ICG and ZnS-ICG.

[0063] In this embodiment, albumin was used as a modification reagent to prepare albumin-zinc sulfide (ZnS) nanoprobes, unreacted reagents were removed by ultrafiltration, and then indocyanine green was added for adsorption, and excess ICG was removed by ultrafiltration to obtain albumin-zinc sulfide-indocyanine green (ZnS-ICG). Transmission electron microscopy results showed that ZnS-ICG was about 3nm, and after adsorbing ICG, the characteristic absorption peak and fluorescence emission properties of ICG were retained.

[0064] See also Figure 3 , which is the photoacoustic signal of copper ion activation of ZnS-ICG provided in Example 1 of the present application. a) Schematic diagram of copper ion exchange; b) Effect of different copper ion concentrations on the absorption of ZnS-ICG at 1064nm; c) Effect of different copper ion concentrations on the absorption of ZnS-ICG at 700nm; d) Transmission electron microscopy image after copper ion exchange, e) Effect of copper ion concentration on probe photoacoustic signal, f) Relationship between copper ion concentration and PA1064nm / PA700nm.

[0065] Furthermore, the experiment tested the changes in the absorption signal and photoacoustic signal of the material after adding different concentrations of copper ions. With the increase of copper ion concentration, the absorption at 1064nm gradually increased, indicating that copper ions and zinc ions produced a cation exchange reaction, but the absorption intensity at 700nm remained unchanged, laying the foundation for the construction of a ratiometric photoacoustic probe.

[0066] It can be understood that the technical features of the above-described embodiments can be arbitrarily combined. In order to make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0067] The above are only preferred embodiments of the present application, and only specifically describe the technical principles of the present application. These descriptions are only for explaining the principles of the present application and cannot be interpreted as limiting the scope of protection of the present application in any way. Based on the explanation here, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application, and other specific implementation methods of the present application that can be associated with the technicians in this field without creative work, should be included in the scope of protection of the present application.

Claims

1. A method for preparing a ratiometric photoacoustic imaging probe, It is characterized in that The steps include: The pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution is adjusted to 3.0-6.0; Adding the sodium sulfide solution to the albumin zinc ion mixed solution and stirring at room temperature for 0.5-1 hour to obtain the albumin-zinc sulfide nanoprobe; Ultrafiltration treatment of the albumin-zinc sulfide nanoprobe; Indocyanine green is added to the filtrate after the ultrafiltration treatment and stirred at room temperature, and then a second ultrafiltration treatment is performed to obtain the albumin-zinc sulfide-indocyanine green probe.

2. The method for preparing a ratiometric photoacoustic imaging probe according to claim 1, It is characterized in that In the step of adjusting the pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution to 3.0-6.0, the zinc ions include zinc acetate.

3. The method for preparing a ratiometric photoacoustic imaging probe according to claim 2, It is characterized in that The step of adjusting the pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution to 3.0-6.0 specifically includes the following steps: The zinc acetate is added dropwise to the albumin solution, and hydrochloric acid is added dropwise to adjust the pH value, and the mixture is stirred for 10-30 minutes.

4. The method for preparing a ratiometric photoacoustic imaging probe according to claim 1, It is characterized in that In the step of adjusting the pH value of the albumin-zinc ion mixed solution formed by mixing the albumin solution and the zinc ion solution to 3.0-6.0, the molar ratio of the albumin solution to the zinc ion solution is 1:20-1:

100.

5. The method for preparing a ratiometric photoacoustic imaging probe according to claim 1, It is characterized in that In the step of adding the sodium sulfide solution to the albumin zinc ion mixed solution and stirring at room temperature for 0.5-1 hour to obtain the albumin-zinc sulfide nanoprobe, the amount of the sodium sulfide solution is 0.02-0.2 mol / L, the amount of the albumin zinc ion mixed solution is 0.02-0.2 mol / L, and the amount of Zn 2+ :S 2- The amount of substance is 0.5-2.

6. The method for preparing a ratiometric photoacoustic imaging probe according to claim 1, It is characterized in that In the step of adding indocyanine green to the filtrate after ultrafiltration and stirring at room temperature, and then performing a second ultrafiltration to obtain an albumin-zinc sulfide-indocyanine green probe, the mass concentration of indocyanine green is 2 mg / mL and the volume is 0.02-0.15 mL.

7. A ratiometric photoacoustic imaging probe, It is characterized in that The compound is prepared by the preparation method according to any one of claims 1 to 2.

8. Use of the ratiometric photoacoustic imaging probe as claimed in claim 4 in detecting copper ions.

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