A zwitterionic chemiluminescent reactive oxygen species probe cleared by kidneys and its preparation method and application

Abstract

The present invention discloses a zwitterionic chemiluminescent reactive oxygen species probe that is cleared by the kidneys, and a preparation method and application thereof, and relates to the technical field of preparation of ROS-responsive fluorescent nanoprobes. The preparation method comprises the following steps: S1, using NaBH 3 CN is used as a reducing agent, and MSA-AuNPs protected by the thiol ligand MSA are prepared by chemical reduction; S2, ABEI is chemically coupled to the surface of MSA-AuNPs prepared in step S1 by covalent bonds to prepare nanoprobe ABEI-MSA-AuNPs. The present invention discloses a zwitterionic chemiluminescent reactive oxygen species probe that is cleared by the kidneys, and a preparation method and application thereof. The chemiluminescent molecule ABEI is chemically coupled to the surface of MSA-AuNPs with near-infrared fluorescence properties by covalent bonds to prepare nanoprobe ABEI-MSA-AuNPs. The zwitterionic effect induced by charge pairing effectively inhibits the protein adsorption of the nanoprobe in the body, increases the clearance rate of the nanoprobe by the kidneys, and can achieve rapid and sensitive detection of ROS levels in disease sites in the body by dual-channel ratio urine analysis.

Description

Technical Field

[0001] The present invention relates to the technical field of ROS responsive fluorescent nanoprobe preparation, and in particular to a zwitterionic chemiluminescent reactive oxygen species probe cleared by kidneys, and a preparation method and application thereof. Background Art

[0002] Reactive oxygen species (ROS) are signal molecules that regulate the life activities of organisms and have an important impact on various physiological processes. However, excessive ROS produced in the body will oxidize various biological macromolecules such as DNA, protein and cholesterol, and then cause various serious diseases such as cancer and hepatitis. Therefore, ROS has become an important detection indicator for various diseases. So far, many technologies have been developed to detect ROS, such as chromatography, fluorescence imaging, electrochemical analysis, chemiluminescence, etc.

[0003] Chemiluminescence (CL) is a means and strategy with unique advantages and broad application prospects in the detection of various biomarkers such as ROS and enzyme activity, and has attracted widespread attention in recent years. For example, polyethylene glycol (PEG) and photosensitizers are coupled with luminol, a chemiluminescent agent with high sensitivity and high selectivity to ROS, and self-assembled to form nanoparticles. This chemiluminescent probe can show excellent inflammation detection effects in a variety of animal disease models. Chemiluminescence is produced by chemical reactions, generally oxidation reactions, which generate photons by generating electronic excited states. These photons release energy in the form of light during the process of electron relaxation back to the ground state. Unlike photoluminescence, since the chemiluminescence process is driven by chemical reactions to supply energy and does not require an excitation light source, chemiluminescence usually has the advantages of minimal light interference (such as autofluorescence), high signal-to-noise ratio, high detection sensitivity, and simple operation.

[0004] Commonly used chemiluminescent compounds include luminol and its derivatives, oxalates, dioxetane, etc. However, they usually have problems such as short emission wavelength (usually 400-500nm) and poor water solubility, which limits their application in in vivo detection. In order to solve these problems, many studies have coupled nanoparticles with luminescent compounds based on the chemiluminescence resonance energy transfer mechanism (CRET) to enhance the hydrophilicity of the probe and extend its emission wavelength. For example, studies have shown that self-luminescent semiconductor polymer nanoparticles can effectively bind hydrophobic oxalates and other near-infrared fluorophores. The nanoparticles have been successfully used for non-invasive in vivo chemiluminescence imaging of various animal disease models.

[0005] However, nanoparticles constructed based on this strategy are often large in size and have large hydrophobic parts, which leads to the constructed nanoparticles being phagocytosed by the mononuclear phagocyte system (MPS, such as the liver) after entering the systemic circulation, and the body is slowly cleared, which in turn causes irreversible toxic side effects to the body. In addition, the limited tissue light penetration depth also makes it difficult for non-invasive imaging based on chemiluminescence to achieve clinical transformation. In recent years, related studies have reported separable nanoparticles that can be triggered to decompose into renal-clearable fragments under certain pathological conditions, which can bypass the light penetration limitation and achieve rapid detection of diseases through urine analysis. Although this method can promote the renal clearance of materials to a certain extent, nanoparticles that are not targeted to the disease site may still eventually accumulate in the liver. Summary of the invention

[0006] The present invention provides a zwitterionic chemiluminescent reactive oxygen species probe that is cleared by the kidneys, as well as a preparation method and application thereof. The probe has extremely low biological adsorption and can realize simple and accurate detection of pathological ROS through near-infrared window ratio urine analysis, thus providing a novel and transformable strategy for solving the early detection of ROS-related diseases.

[0007] To achieve the above object, the present invention provides a method for preparing a zwitterionic chemiluminescent reactive oxygen species probe that is cleared by the kidney, comprising the following steps:

[0008] S1. Using NaBH3CN as a reducing agent, MSA-AuNPs protected by thiol ligand MSA were prepared by chemical reduction method;

[0009] S2. ABEI is chemically coupled to the surface of MSA-AuNPs prepared in step S1 via covalent bonds to prepare nanoprobes ABEI-MSA-AuNPs.

[0010] Preferably, in step S1, the chemical reduction method specifically includes the following steps: adding 100mM HAuCl4 solution dropwise to 60-80mM MSA aqueous solution, stirring and reacting at room temperature until the color of the solution completely changes from golden yellow to colorless and transparent; continuing to add 120mM NaBH3CN aqueous solution, stirring and reacting at room temperature for 2-4 hours; after the reaction is completed, centrifuging at 15000rpm to obtain MSA-AuNPs.

[0011] Preferably, the volume ratio of MSA aqueous solution:HAuCl4 solution:NaBH3CN aqueous solution is 3:0.01:1.

[0012] Preferably, in step 2, the specific steps of the coupling reaction include: mixing MSA-AuNPs, carbodiimide hydrochloride EDCI, 1-hydroxybenzotriazole HOBT, and N,N-diisopropylethylamine DIPEA, and stirring the reaction for 1 hour; continuing to add ABEI and stirring the reaction for 3-5 hours; after the reaction, purifying the solution with a 1500Da dialysis bag, and the purified solution is centrifuged at 15000rpm to obtain ABEI-MSA-AuNPs.

[0013] Preferably, MSA-AuNPs:EDCI:HOBT:DIPEA:ABEI=(1-2)eq:(1-2)eq:(1-2)eq:(1-2)eq:(3-4)eq.

[0014] The present invention discloses a zwitterionic chemiluminescent reactive oxygen species probe that is cleared by the kidneys, and a preparation method and application thereof, which have the following beneficial effects:

[0015] (1) The thiol ligand (mercaptosuccinic acid, MSA)-protected MSA-AuNPs were prepared by chemical reduction, and the common chemiluminescent molecule N-(4-aminobutyl)-N-ethylisoluminol (ABEI) was covalently coupled to the surface of MSA-AuNPs with near-infrared fluorescence properties to prepare the nanoprobe ABEI-MSA-AuNPs.

[0016] (2) Based on the chemiluminescence resonance energy transfer effect and the large luminescence Stokes shift of MSA-AuNPs, the energy generated by the chemical reaction between ABEI and ROS can be effectively transferred to MSA-AuNPs, and then MSA-AuNPs emit near-infrared light with a wavelength greater than 800 nm;

[0017] (3) ABEI alone cannot be effectively excreted through urine after injection through the tail vein, and MSA-AuNPs alone are also mainly enriched in the liver after injection through the tail vein. However, after ABEI and MSA-AuNPs are chemically coupled through covalent bonds, the zwitterionic effect induced by charge pairing (ABEI is positively charged and MSA-AuNPs are negatively charged, and the surface of ABEI-MSA-AuNPs contains both positive and negative groups, that is, it exhibits a zwitterionic effect) can effectively inhibit the protein adsorption of the nanoprobe in the body, and significantly reduce the liver enrichment of the conjugate ABEI-MSA-AuNPs (the liver enrichment of ABEI-MSA-AuNPs 24 hours after intravenous injection is about 100 times lower than that of MSA-AuNPs), which greatly increases the renal clearance rate of the nanoprobe;

[0018] (4) By constructing a mouse acute kidney injury model, we further demonstrated that ABEI-MSA-AuNPs can achieve rapid and sensitive detection of ROS levels in disease sites in vivo through dual-channel ratiometric urine analysis.

[0019] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The ultraviolet absorption spectra of the ultrasmall gold nanoparticles MSA-AuNPs, ABEI-MSA-AuNPs and ABEI synthesized in Example 1;

[0021] Figure 2 is the Zeta potential of gold nanoparticles before and after the reaction of MSA-AuNPs with ABEI in Example 1;

[0022] Figure 3 The agarose gel electrophoresis diagram of the ultra-small gold nanoparticles MSA-AuNPs and ABEI-MSA-AuNPs synthesized in Example 1;

[0023] Figure 4 The transmission electron micrographs of the ultra-small gold nanoparticles MSA-AuNPs and ABEI-MSA-AuNPs synthesized in Example 1; wherein (a) is a transmission electron micrograph of MSA-AuNPs, and (b) is a transmission electron micrograph of ABEI-MSA-AuNPs;

[0024] Figure 5 Gel electrophoresis patterns of the interaction test between ABEI-MSA and GS-AuNPs and serum proteins;

[0025] Figure 6 The distribution of MSA-AuNPs and ABEI-MSA-AuNPs in the liver and urine, where (a) is the distribution of MSA-AuNPs and ABEI-MSA-AuNPs in the liver, and (b) is the distribution of MSA-AuNPs and ABEI-MSA-AuNPs in the urine;

[0026] Figure 7 The distribution of ABEI-MSA-AuNPs in the kidney; (a) is an image of a kidney section of an ICR mouse after silver staining observed under an optical microscope, and (b) is a partial magnified image of (a);

[0027] Figure 8The results of the five ABEI-MSA-AuNPs tests for detecting acute kidney injury are shown in Figure 1; (a) shows the near-infrared fluorescence signal of ABEI-MSA-AuNPs in the urine of normal mice and diseased mice and the luminescence signal after adding luminol; (b) shows the level of reactive oxygen species in normal mice and diseased mice;

[0028] Fig. 9 The DCFH reactive oxygen species staining results of six kidney sections were tested in the experiment, where (a) is the DCF fluorescence signal of the kidneys of normal mice and diseased mice; (b) is the semi-quantitative analysis statistical graph of the DCF fluorescence signal of normal mice and diseased mice;

[0029] Fig.10 The luminescence of urine after local injection of ABEI-MSA-AuNPs in seven normal ICR mice and peritonitis ICR mice was tested. (a) is the near-infrared fluorescence signal of ABEI-MSA-AuNPs in the urine of normal ICR mice and peritonitis ICR mice and the luminescence signal after adding luminol, and (b) is the level of reactive oxygen in normal ICR mice and peritonitis ICR mice. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages disclosed in the embodiments of the present invention clearer, the embodiments of the present invention are further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the embodiments of the present invention and are not intended to limit the embodiments of the present invention. Based on the embodiments in this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. Examples of the embodiments are shown in the accompanying drawings, where the same or similar reference numerals throughout represent the same or similar elements or elements with the same or similar functions.

[0031] It should be noted that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product or server that includes a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units that are not explicitly listed or inherent to these processes, methods, products or devices.

[0032] Like reference numerals and letters denote similar items in the following drawings, and thus, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0033] Embodiment 1

[0034] The synthesis of ultrasmall gold nanoparticles (MSA-AuNPs) protected by mercaptosuccinic acid (MSA) and the functionalization modification process of their surface active oxygen responsive chemiluminescent material N-(4-aminobutyl)-N-ethylisoluminol (ABEI) are as follows:

[0035] (1) Preparation of MSA-AuNPs: 25 μL of 100 mM HAuCl4 solution was added dropwise to 7.5 mL of 70 mM MSA aqueous solution, and the mixture was stirred at room temperature for 20 minutes. The color of the solution completely changed from golden yellow to colorless and transparent. Then, 2.5 mL of 120 mM NaBH3CN aqueous solution was added, and the mixture was stirred at room temperature for 3 hours. After the reaction was completed, ultra-small gold nanoparticles MSA-AuNPs were obtained by centrifugation at 15,000 rpm.

[0036] (2) Preparation of ABEI-MSA-AuNPs: 2 eq of MSA-AuNPs, 2 eq of EDCI, 2 eq of 1-hydroxybenzotriazole (HOBT), and 2 eq of N,N-diisopropylethylamine (DIPEA) were mixed and stirred for reaction for 1 hour.

[0037] Then, 4 eq of ABEI was added and stirred for 5 hours. After the reaction, a 1500 Da dialysis bag was used for purification, and then centrifuged at 15000 rpm to obtain ABEI-MSA-AuNPs, an ultra-small gold nanoparticle functionalized with ABEI.

[0038] Experimental test 1 demonstrated the successful synthesis of nanoparticles functionalized with reactive oxygen species-responsive chemiluminescent substances.

[0039] like Figure 1 Shown are the ultraviolet absorption spectra of the ultrasmall gold nanoparticles MSA-AuNPs, ABEI-MSA-AuNPs and ABEI synthesized in Example 1.

[0040] Neither the mercaptosuccinic acid protected gold nanoparticles (MSA-AuNPs) nor the luminol functional modified gold nanoparticles (ABEI-MSA-AuNPs) showed a surface plasmon resonance absorption peak at a wavelength of 500-550nm, indicating that both are ultra-small gold nanoparticles. In addition, the ultraviolet absorption of ABEI-MSA-AuNPs showed the characteristic absorption peak of ABEI, indicating that ABEI was successfully coupled to MSA-AuNPs.

[0041] like Figure 2 , which shows the Zeta potential of gold nanoparticles before and after the reaction of MSA-AuNPs with ABEI in Example 1.

[0042] The results show that the potential of AuNPs increases by 3 times after the reaction. This is because the coupling of MSA-AuNPs to ABEI is through the formation of amide bonds, so the number of ionizable carboxyl groups of the protective ligand (MSA) is reduced, and the N atom on ABEI is positively charged, and the combined effect of the two leads to an increase in potential.

[0043] like Figure 3 The figure shows the agarose gel electrophoresis of the ultra-small gold nanoparticles MSA-AuNPs and ABEI-MSA-AuNPs synthesized in Example 1. Since the molecular weight of ABEI-MSA-AuNPs is larger than that of MSA-AuNPs, the electric potential is also increased, resulting in a slower electrophoresis rate of ABEI-MSA-AuNPs.

[0044] like Figure 4 The following are transmission electron micrographs of the ultra-small gold nanoparticles MSA-AuNPs and ABEI-MSA-AuNPs synthesized in Example 1. (a) is a transmission electron micrograph of MSA-AuNPs, and (b) is a transmission electron micrograph of ABEI-MSA-AuNPs.

[0045] It can be clearly observed that both the synthesized MSA-AuNPs and ABEI-MSA-AuNPs are monodisperse nanoparticles with ultrasmall core sizes, and the size and monodispersity of gold nanoparticles are not changed after coupling with ABEI.

[0046] Experimental test 2 proves that ABEI-MSA-AuNPs are zwitterionic gold nanoparticles with anti-serum protein binding ability

[0047] Agarose gel electrophoresis was used to investigate the interaction between ABEI-MSA-AuNPs and serum proteins. ABEI-MSA-AuNPs and classic amphiphilic ionic glutathione-protected gold nanoparticles (GS-AuNPs) were incubated with fetal bovine serum (FBS) at 37°C for 30 minutes, and then the protein staining solution CBB was added to mark the electrophoresis of the protein during the electrophoresis. CBB solution and a mixed solution of CBB and FBS were used as blank groups, and ABEI-MSA-AuNPs and GS-AuNPs were used as control groups. The electrophoresis results showed that, like GS-AuNPs, ABEI-MSA-AuNPs had excellent anti-protein adsorption ability (the black box is the position of the gold nanoparticles, and the blue band is the position of the protein), as shown in Figure 2. Figure 5 shown.

[0048] Experimental test three demonstrated that ABEI-MSA-AuNPs accumulated very little in MPS organs and could be eliminated from the body through urine.

[0049] The results of test one and two show that ABEI-MSA-AuNPs have high anti-serum protein binding ability and their core size is less than 3nm, which allows ABEI-MSA-AuNPs nanoparticles to effectively evade the uptake of MPS, be cleared from the blood to the kidneys, and then excreted from the body through urine.

[0050] like Figure 6 As shown in (a), 24 hours after tail vein injection of MSA-AuNPs and ABEI-MSA-AuNPs, respectively, the liver enrichment of ABEI-MSA-AuNPs was about 100 times lower than that of MSA-AuNPs (0.58%ID / g vs 55.4%ID / g), which indicates that ABEI-MSA-AuNPs are rarely enriched in MPS organs in vivo and have a low potential biosafety risk.

[0051] In addition, if Figure 6 As shown in (b), the urinary clearance rate of ABEI-MSA-AuNPs is as high as 40% ID, while only 3% ID of MSA-AuNPs can be excreted through urine. The above results show that after ABEI functionalization, the liver accumulation of AuNPs can be significantly reduced and its urinary clearance ability can be improved.

[0052] Experimental testing of four kidney tissue sections further demonstrated that ABEI-MSA-AuNPs can be filtered through the glomeruli and accumulated in the kidneys.

[0053] The location of ABEI-MSA-AuNPs gold nanoparticles in kidney tissue sections was observed under an optical microscope using the silver staining method. Figure 7 As shown in (a) and (b), the ICR mouse kidney sections treated with silver staining clearly show that ABEI-MSA-AuNPs are mainly distributed in the proximal tubule (PT) cells after being filtered by the glomerulus (G) (as shown by the white arrows in the figure). This indicates that the proximal tubule can effectively absorb ABEI-MSA-AuNPs, which is consistent with the experimental results of the effective accumulation of ABEI-MSA-AuNPs in the kidney in test three.

[0054] The experiment tested five normal ICR mice and ICR mice with acute kidney injury by injecting ABEI-MSA-AuNPs through the tail vein, collecting the mouse urine and detecting the luminescence. The results proved that the chemiluminescent reactive oxygen species probe can be excreted through urine and can reflect the differences in the content of reactive oxygen species in the body.

[0055] Different doses of glycerol (5 mL / kg and 8 mL / kg) were injected into the leg muscles on both sides for 2 hours to construct acute kidney injury disease models of different severity. Then, normal mice and disease mice were injected with ABEI-MSA-AuNPs through the tail vein, their urine was collected, and the luminescence of the nanoparticles in the urine was detected.

[0056] like Figure 8 As shown in (a), the near-infrared fluorescence signal of ABEI-MSA-AuNPs can be detected in the urine of normal mice and diseased mice, which once again verifies that the nanoparticles can be used as nanoprobes that can be cleared by the kidneys. The addition of luminol as a reaction reagent to urine can detect the near-infrared luminescence signal of gold nanoparticles after CRET, and the intensity of the luminescence signal is related to the level of reactive oxygen in the body.

[0057] The detection index of the active oxygen level in the body using this probe was introduced, and the results were as follows Figure 8 As shown in (b). This indicates that there is a significant difference in the content of reactive oxygen species in normal mice and diseased mice, and the nanoprobe can also distinguish diseased mice into mild and severe cases (5mL / kg and 8mL / kg) based on the principle that the content of reactive oxygen species varies with the severity of the disease damage.

[0058] Experimental test six analyzed the differences in reactive oxygen levels between normal mice and diseased mice through DCFH-DA-stained kidney sections, verifying that the chemiluminescent reactive oxygen probe ABEI-MSA-AuNPs can indeed respond to and distinguish different levels of reactive oxygen in the body.

[0059] Using the fluorescent probe DCFH-DA for reactive oxygen detection is a common method for quantitatively detecting the level of reactive oxygen in cells based on changes in fluorescence intensity. Reactive oxygen in cells can oxidize DCFH to generate fluorescent DCF, so a fluorescence microscope can be used to detect the fluorescence signal of DCF in kidney slices, and the level of reactive oxygen can be analyzed based on the strength of the fluorescence signal.

[0060] like Fig. 9 As shown in (a), the DCF fluorescence signals in the kidneys of mice with acute renal injury induced by low-dose (5 mL / kg) and high-dose (8 mL / kg) glycerol were significantly stronger than those in healthy mice, indicating that the level of reactive oxygen species in the kidneys of diseased mice was significantly higher than that in healthy mice.

[0061] The DCF fluorescence intensity was semi-quantitatively analyzed. Fig. 9As shown in (b), the average gray value of the normal group is ~36, while that of the mild group is ~61, and that of the severe group is ~84. Further analysis shows that there are statistically significant differences between the normal group and the disease group, and between the disease groups. The above experimental results are consistent with the experimental results of the fifth test that the nanoprobe ABEI-MSA-AuNPs can detect diseases by responding to different levels of reactive oxygen in the body.

[0062] The experiment tested seven normal ICR mice and peritonitis ICR mice by local injection of ABEI-MSA-AuNPs, collected the mouse urine, and detected the luminescence, proving that the chemiluminescent reactive oxygen species probe can detect different levels of reactive oxygen in the body through urine.

[0063] Different doses of zymosan (250 mg / kg and 500 mg / kg) were injected intraperitoneally for 5 h to construct peritonitis disease models with different degrees of inflammation, i.e., different ROS levels. Then, ABEI-MSA-AuNPs were locally injected (intraperitoneal injection) into normal mice and diseased mice, and their urine was collected.

[0064] like Fig.10 As shown in (a), the near-infrared fluorescence signal of ABEI-MSA-AuNPs was detected in the urine of both normal mice and diseased mice, indicating that the nanoparticles can be excreted from the body through urine through the kidneys by local injection. Then, the reaction reagent luminol was added to the urine, and the near-infrared luminescence signal of MSA-AuNPs after CRET was detected in accordance with the fifth test.

[0065] The probe was introduced to detect the level of active oxygen in the body. Fig.10 As shown in (b). The results showed that there were significant differences in the levels of reactive oxygen species between normal mice and peritonitis mice, and between mice with peritonitis induced by different doses of drugs. This shows that ABEI-MSA-AuNPs can be administered not only by intravenous systemic injection, but also by local injection, and the detection of pathological ROS in local areas can be achieved through urine detection.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A method for preparing a zwitterionic chemiluminescent reactive oxygen species probe that is cleared by the kidney, characterized in that: The steps include: S1. Using NaBH3CN as a reducing agent, MSA-AuNPs protected by thiol ligand MSA were prepared by chemical reduction method; The chemical reduction method specifically includes the following steps: Add 100mM HAuCl4 solution dropwise to 60-80mM MSA aqueous solution, and stir at room temperature until the solution color changes completely from golden yellow to colorless and transparent; continue to add 120mM NaBH3CN aqueous solution, and stir at room temperature for 2-4 hours; after the reaction is completed, centrifuge at 15000rpm to obtain MSA-AuNPs; The volume ratio of MSA aqueous solution: HAuCl4 solution: NaBH3CN aqueous solution is 3:0.01:1; S2, preparing nanoprobe ABEI-MSA-AuNPs by chemically coupling ABEI to the surface of MSA-AuNPs prepared in step S1 through covalent bonds; The specific steps of the coupling reaction include: Mix MSA-AuNPs, carbodiimide hydrochloride EDCI, 1-hydroxybenzotriazole HOBT, and N, N-diisopropylethylamine DIPEA, and stir to react for 1 hour; continue to add ABEI and stir to react for 3-5 hours; after the reaction, purify the solution with a 1500Da dialysis bag, and the purified solution is centrifuged at 15000rpm to obtain ABEI-MSA-AuNPs; MSA-AuNPs: EDCI: HOBT: DIPEA: ABEI= (1-2) eq: (1-2) eq: (1-2) eq: (1-2) eq: (3-4) eq; MSA is mercaptosuccinic acid; ABEI is N-(4-aminobutyl)-N-ethylisoluminol.

2. A nanoprobe ABEI-MSA-AuNPs prepared by the method for preparing a zwitterionic chemiluminescent reactive oxygen species probe cleared by the kidney as claimed in claim 1.

3. Use of the nanoprobe ABEI-MSA-AuNPs as claimed in claim 2 in detecting pathological ROS.

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