Probe for simultaneous detection of gamma-glutamyl transpeptidase and peroxynitrite and method of preparation

Abstract

The application provides a probe for simultaneously detecting gamma-glutamyl transpeptidase and peroxynitrite and a preparation method thereof. The probe can realize the effect of simultaneously detecting gamma-glutamyl transpeptidase and peroxynitrite, has high sensitivity and specificity, and has the advantages of low cytotoxicity and fast reaction. Therefore, the probe has potential application value in early diagnosis and in-situ monitoring of AKI, and is expected to become a powerful tool for evaluating kidney injury and oxidative stress.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical detection, and particularly relates to a probe for simultaneously detecting gamma-glutamyl transpeptidase and peroxynitrite and a preparation method thereof. Background Art

[0002] Acute kidney injury (AKI) refers to the sudden loss of renal function, characterized by increased serum creatinine levels and decreased urine output within a week. If not treated early, it can lead to serious consequences or even death. Studies have shown that γ-glutamyl transpeptidase (γ-GGT) and peroxynitrite (ONOO - ) can be used as an important biomarker for the diagnosis and monitoring of AKI.

[0003] Currently, the research on γ-glutamyl transpeptidase (γ-GGT) and peroxynitrite (ONOO - ) probes can only detect γ-GGT or ONOO - For example, CN112409322B provides a GGT-activated chemiluminescent probe that can only detect γ-GGT, and the detection limit is only 16mU / L; CN112358440B provides an ONOO - Fluorescent probes that can only detect ONOO - , and the detection limit was only 85.7nM.

[0004] As far as the inventors know, people have not yet found how to develop a method for simultaneously detecting γ-GGT and ONOO. - A solution for a probe with high sensitivity. Summary of the Invention

[0005] In view of the defects of the prior art, the object of the present invention is to provide a probe with high sensitivity that can simultaneously detect γ-glutamyl transpeptidase and peroxynitrite and a preparation method thereof.

[0006] In order to achieve the above technical objectives, the present invention provides the following technical solutions:

[0007] A probe for simultaneously detecting γ-glutamyl transpeptidase and peroxynitrite, wherein the chemical structure of the probe is as follows:

[0008]

[0009] A method for preparing a probe for simultaneously detecting γ-glutamyl transpeptidase and peroxynitrite, wherein the probe for simultaneously detecting γ-glutamyl transpeptidase and peroxynitrite is as described in claim 1, and the preparation method comprises the following steps:

[0010] (1) Compound 1 and Compound 2 are reacted to prepare Compound 3;

[0011] (2) Compound 3 and Compound 4 are reacted to prepare Compound 5;

[0012] (3) preparing the probe for simultaneous detection of γ-glutamyl transpeptidase and peroxynitrite by reacting compound 5 and compound 6;

[0013] Wherein, the chemical structural formulas of compounds 1-6 are shown in Formula 1 to Formula 6 respectively;

[0014]

[0015]

[0016] Preferably, in step (1), compound 1, compound 2, NH4OAc and EtOH are mixed and reacted. More preferably, in step (1), the reaction temperature is 60°C and the reaction time is 12 hours. More preferably, in step (1), the added amounts of compound 1, compound 2, NH4OAc and EtOH are 150 mg, 251.53 mg, 27.95 mg and 5 mL, respectively.

[0017] Preferably, in step (2), compound 3, TEA, and DCM are mixed in an ice bath, and then compound 4 is added for reaction. More preferably, in step (2), the reaction temperature is room temperature, the reaction time is 12 hours, and the amounts of compound 3, TEA, DCM, and compound 4 added are 55.00 mg, 33.35 mg, 5 mL, and 14.92 mg, respectively.

[0018] Preferably, in step (3), compound 5, compound 6, methanol, and water are mixed and reacted after adding sodium hydroxide. More preferably, in step (3), the reaction temperature is room temperature and the reaction time is 2 hours; the added amounts of compound 5, compound 6, methanol, water, and sodium hydroxide are 50.00 mg, 60.16 mg, 1.50 mL, 1.50 mL, and 2 mg, respectively.

[0019] The present invention also provides the use of the probe for simultaneously detecting γ-glutamyl transpeptidase and peroxynitrite in preparing a probe product for simultaneously detecting γ-glutamyl transpeptidase and peroxynitrite in vivo.

[0020] Beneficial effects of the present invention:

[0021] The probe of the present invention can simultaneously detect γ-glutamyl transpeptidase and peroxynitrite with high sensitivity and specificity. Furthermore, the probe has the advantages of low cytotoxicity and rapid response. Therefore, the probe of the present invention has potential application value in the early diagnosis and in situ monitoring of AKI and is expected to become a powerful tool for assessing kidney damage and oxidative stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 For HX-GP 1 H NMR spectrum;

[0023] Figure 2 For HX-GP 13 C NMR spectrum;

[0024] Figure 3 is the MS spectrum of HX-GP;

[0025] Figure 4 The absorption spectra of HX-GP (10 μM) in the presence or absence of γ-GGT (1 U / mL) and the absorption spectra of HX-GP (10 μM) after adding γ-GGT (1 U / mL) and then adding ONOO - Absorption spectrum of (9.89μM);

[0026] Figure 5 The fluorescence emission spectrum experimental results of the present invention are as follows: Part A shows the fluorescence emission spectrum of HX-GP (10 μM) after the action of γ-GGT (0-1.0 U / mL) at 37°C for 30 minutes, λex / λem=465 / 613 nm; Part B shows the linear relationship between fluorescence intensity (λem=613) and γ-GGT concentration (0.001-0.05 U / mL); Part C shows the fluorescence emission spectrum of HX-GP (10 μM) pre-incubated with γ-GGT (1 U / mL) at 37°C for 30 minutes, followed by the addition of ONOO- (0-99.89 μM) and incubation for 5 minutes, λex / λem=365 / 518 nm; Part D shows the linear relationship between fluorescence intensity (λem=518 nm) and ONOO - Linear relationship with concentration (0.16-4.98 μM);

[0027] Figure 6The fluorescence emission spectrum experimental results of the present invention are shown in Figure 2. Part A shows the fluorescence intensity of HX-GP (10 μM, black curve) over time and the fluorescence intensity of HX-GP added with γ-GGT (1 U / mL, red curve) at 37°C over time; Part B shows the fluorescence intensity of HX-GP (10 μM, black curve) alone and with the addition of γ-GGT (1 U / mL, red curve) at different pH values; Part C shows the fluorescence response of HX-GP to different substances (K + Mg 2+ , Ca 2+ 、Fe 3+ 、Zn 2+ 、NO3 - 、SO4 2- , O 2- , ·OH, HClO, H2O2, GSH and Cys (100μM), NTR, ALP and LAP (1U / mL); Part D shows the fluorescence intensity of HX-GP and γ-GGT (1U / mL, blank curve) over time and the fluorescence intensity of HX-GP and γ-GGT (1U / mL) after adding ONOO - (9.89 μM, red curve) after the fluorescence intensity changes with time, λex / em=365 / 518 nm; Part E shows the fluorescence intensity of HX-GP and γ-GGT (1 U / mL, blank curve) at different pH and the fluorescence intensity of HX-GP and γ-GGT (1 U / mL) after adding ONOO - (9.89 μM, red curve) at different pH values; Part F shows the fluorescence response of HX-GP after adding γ-GGT (1 U / mL) and then adding different substances (K + Mg 2+ , Ca 2+ 、Fe 3+ 、Zn 2+ 、NO3 - 、SO4 2- , O 2- ,·OH, HClO, H2O2, GSH and Cys (100μM), ONOO - (9.89 μM);

[0028] Figure 7 is the ESI-HRMS spectrum after the reaction of HX-GP and γ-GGT;

[0029] Figure 8 The fluorescence intensity of 10 μM HX-GP in HK-2 cells at each stage (10, 20, 30, and 40 min);

[0030] Figure 9 Figure 2 shows the fluorescence imaging results of HX-GP in different cells. Part A shows the fluorescence imaging results of HX-GP (10 μM) in HK-2 cells and 3T3 cells. Part B shows the average fluorescence intensity of HX-GP in different cells. Red butcher knife: λex = 488 nm, λem = 580-620 nm. Scale bar: 25 μm.

[0031] Figure 10 HX-GP (10 μM), BODIPY 493 / 503 Colocalization assay results using LDs-Tracker, Lyso Tracker Green, and Mito-Tracker Green (1.0 μM); Green channel: λex = 488 nm, λem = 500-540 nm; Red channel: λex = 488 nm, λem = 580-620 nm; Scale bar: 25 μm.

[0032] Figure 11 Part A is the fluorescence imaging results of HX-GP (10 μM) in HK-2 cells, (a) HK-2 cells were treated with HX-GP (10 μM) for 30 minutes, (b) HK-2 cells were treated with HX-GP (10 μM) for 30 minutes and then incubated with 1 mM SIN-1 for 1 hour, (c) HK-2 cells were treated with HX-GP (10 μM) for 30 minutes and then incubated with 2 mM SIN-1 for 1 hour, (d) H K-2 cells were treated with 2 mM SIN-1 and 1 mM NAC for 1 hour and then incubated with HX-GP (10 μM) for 30 minutes. Part B shows the average red channel relative fluorescence intensity in the image in Part A (red channel: λex = 488 nm, λem = 580-620 nm). Part C shows the average green channel relative fluorescence intensity in the image in Part A (green channel: λex = 405 nm, λem = 500-540 nm). Scale bar: 25 μm.

[0033] Figure 12 γ-GGT and ONOO in HK-2 cell AKI model under different treatments - Fluorescence imaging results of activity, part A shows γ-GGT and ONOO in the HK-2 cell AKI model induced by cisplatin -Fluorescence imaging of activity, where (a) shows HK-2 cells treated with HX-GP (10 μM) only for 30 minutes, (b) shows HK-2 cells pretreated with 0.5 mM cisplatin for 12 hours, (c) shows HK-2 cells pretreated with 1 mM cisplatin for 12 hours and then incubated with 10 μM HX-GP for 30 minutes, and (d) shows HK-2 cells pretreated with 1 mM cisplatin and 1 mM LC for 12 hours and then incubated with 10 μM HX-GP for 30 minutes; Part B is the average red channel relative fluorescence intensity in the image of Part A, red channel: λex = 488 nm, λem = 580-620 nm; Part C is the average green channel relative fluorescence intensity in the image of Part A, green channel: λex = 405 nm, λem = 500-540 nm, scale bar: 25 μm. DETAILED DESCRIPTION

[0034] The present invention is described in detail below through examples. It is necessary to point out that the following examples are only used to further illustrate the present invention and are not to be construed as limiting the scope of protection of the present invention. Non-essential improvements and adjustments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0035] Example 1

[0036] 1. Experimental methods

[0037] 1.1 Preparation and characterization of the probe compound (abbreviated as HX-GP):

[0038] 1.1.1 Preparation: The preparation method is as follows:

[0039]

[0040] The specific plan is as follows:

[0041] (1) Preparation of compound 3:

[0042] Compound 1 (150 mg, 871.18 μmol), compound 2 (251.53 mg, 725.99 μmol), NH4OAc (27.95 mg, 362.99 μmol), and EtOH (5 mL) were added to a flame-dried flask at room temperature. The resulting mixture was stirred at 60°C for 12 hours, after which the reaction was complete. The mixture was then concentrated, and the residue phase was extracted with EA. The combined organic phases were filtered and concentrated under reduced pressure. Purification by MPLC afforded compound 3 (200 mg, 399.50 μmol, 55.03% yield) as a brown solid.

[0043] (2) Preparation of compound 5:

[0044] Compound 3 (55.00 mg, 109.86 μM), TEA (33.35 mg, 329.59 μmol, 45.97 μL), and DCM (5 mL) were added to a flame-dried flask in an ice bath, followed by stirring in an ice bath for 10 min. Compound 4 (14.92 mg, 164.79 μmol) was then added. The mixture was stirred at room temperature for 12 hours, then quenched with H₂O (1 mL), and extracted with EA (50 mL). The combined organic phases were washed with brine, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. Purification by MPLC gave Compound 5 (50 mg, 90.14 μmol, 82.05% yield) as a brown solid.

[0045] (3) Compound 5 (50.00 mg, 162.70 μmol), compound 6 (60.16 mg, 108 μmol), methanol (1.50 mL) and water (1.50 mL) were added to a flame-dried flask under ice bath. After stirring the suspension in ice bath for 10 min, sodium hydroxide (2 mg, 50 μmol) was added. The mixture was stirred at room temperature for 2 h, then quenched with water (1 mL) and extracted with ethyl acetate (EA, 50 mL). Under reduced pressure, the mixed organic phase was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The resultant was then purified by MPLC to obtain a brown solid product HX-GP (30.00 mg, 34.80 μmol, 32.09% yield).

[0046] 1.1.2 Characterization: The chemical formula of HX-GP is C 43 H 49 O 11 S2N4. HX-GP 1 H NMR spectrum Figure 1 As shown, HX-GP 13 C NMR spectrum Figure 2 As shown, the MS spectrum of HX-GP is as follows Figure 3 The H NMR spectrum information is as follows:

[0047] [M] + 861.3; found to be 861.1. 1H NMR (400 MHz, methanol-d4) δ 8.72-8.65 (m, 2H), 8.43 (d, J = 8.5 Hz, 1H), 8.31-8.21 (m, 2H), 8.20-8.10 (m, 2H), 8.08-7.96 (m, 2H), 7.95-7.78 (m, 2H), 7.77-7.68 (m, 2H), 7.41 (dd, J = 8.8, 2.2 Hz, 1H),4.67(dd,J=8.9,5.1Hz,1H),4.01-3.80(m,4H),3.35-3.30(m,1H),3.15(dd,J=14.0,5 .1Hz,1H),3.08-2.99(m,1H),2.95-2.84(m,1H),2.59(t,J=7.3Hz,2H),2.36-2.05(m,12H). 13 C NMR (100 MHz, methanol-d4) δ 182.97, 173.34, 171.76, 170.76, 162.06, 161.72, 153.50, 151.07, 139.10, 138.25, 136.32, 134.05, 133.52, 132.08, 131.45, 131.30, 130.94, 129.97, 128.71, 128.27, 127.33, 127.24, 124.91, 122.84, 122.48, 118.68, 118.08, 115.18, 112.43, 54.26, 52.96, 52.80, 49.71, 40.60, 34.37, 33.33, 31.17, 26.95, 26.67, 25.91, 25.09, 21.86. 1.2. Cell Culture Conditions and Fluorescence Imaging

[0048] HK-2 and 3T3 cells were cultured in DMEM at 37°C in a 5% CO2 atmosphere. Prior to imaging, the cells were seeded in 6-well plates and incubated for 24 hours. Subsequently, the cells were treated with 10 μM of the probe for 30 minutes, and excess probe was washed away before imaging. Imaging was performed using a Nikon confocal fluorescence microscope.

[0049] Cytotoxicity assay

[0050] The CCK-8 method was used to evaluate the cytotoxicity of the probe on cells. The cells were seeded in a 200 μL microplate and exposed to culture medium containing different concentrations of the probe (0 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM) for 24 hours. After incubation, the original culture medium was aspirated and the cells were washed with PBS. Subsequently, a PBS solution mixed with CCK-8 was added to each well and incubated for 1 hour. The absorbance (405 nm) of each well was then measured with a microplate reader to determine cell viability.

[0051] 1.4. Cell colocalization experiments

[0052] HK-2 cells were seeded in 6-well culture plates and cultured for 24 hours. The probe HX-GP (10 μM) was then added to the cell culture medium and incubated for 30 minutes. The cells were then washed with PBS to remove excess probe. Mito-Tracker Green / LysoTracker Green / BODIPY was then added. 493 / 503 (1.0μM) and incubated for another 30 minutes. Excess dye was removed with PBS solution, and then the cells were imaged with fluorescence. The excitation wavelength of HX-GP fluorescence imaging was 488nm, and the emission wavelength range was 580-620nm. 493 / 503 For fluorescence imaging, the excitation wavelength was 488 nm and the emission wavelength was 500-540 nm.

[0053] γ-GGT and ONOO in the cisplatin-induced AKI model of HK-2 cells - Fluorescence imaging activity

[0054] In the cisplatin-induced nephrotoxicity model established using HK-2 cells, the cell experiments were divided into four groups. The first group was to incubate HK-2 cells with HX-GP probe (10 μM) for 30 minutes. The second and third groups were pretreated with different concentrations of cisplatin (0.5 mM or 1 mM) for 12 hours in HK-2 cells and then incubated with 10 μM HX-GP for 30 minutes. In the fourth group, the cells were preincubated in the presence of L-carnitine (LC) and cisplatin (1 mM) for 12 hours and then incubated with 10 μM HX-GP for 30 minutes. Subsequently, all cells were washed with PBS to remove excess probes before fluorescence imaging.

[0055] 2. Experimental results

[0056] 2.1 Effect of HX-GP on γ-GGT and ONOO - Spectral response

[0057] The synthesis of HX-GP mainly involves three steps and was characterized by nuclear magnetic resonance and mass spectrometry ( Figure 1 - Figure 3 We first explored the effects of HX-GP on γ-glutamyl transpeptidase (γ-GGT) and peroxynitrite (ONOO - ) reaction. Under physiological conditions, we first tested the absorption spectra of HX-GP before and after the reaction with γ-GGT. Figure 4 As shown in the figure, after HX-GP was incubated with γ-GGT, its maximum absorption peak shifted from 458 nm to 468 nm. - When HX-GP was added to the solution, the original UV absorption peak disappeared and a new UV absorption peak appeared at 316 nm. - Fluorescence spectra before and after incubation. Due to the acylation of the hydroxyl groups on the probe, there is almost no fluorescence emission signal ( Figure 5 As the concentration of γ-GGT increases, the fluorescence intensity of the probe at 613 nm gradually increases and shows a good linear relationship with the concentration of γ-GGT in the range of 0.001-0.05 U / mL ( Figure 5 According to the formula, the detection limit LOD is 0.00042U / mL (3σ / k, where σ is the relative standard deviation of the blank sample, n=11, and k is the slope of the linear equation). Subsequently, we studied the effect of HX-GP on γ-GGT and ONOO - The fluorescence response of HX-GP in the presence of ONOO is shown in Table 1. The fluorophore HX-OH produced by the reaction of HX-GP with γ-GGT has almost no fluorescence at 518nm. However, the addition of ONOO - After that, under 365nm excitation, the generated fluorophore HNA showed a strong fluorescence signal at 518nm ( Figure 5 Part C). When ONOO - As the concentration of α-glucose increased from 0 to 9.89 μM, the fluorescence intensity at 518 nm also increased and showed a strong linear relationship in the range of 0.16-4.98 μM, with a detection limit of 76.2 nM ( Figure 5 (Section D).

[0058] Next, we further evaluated the probe's interaction with γ-GGT and ONOO - The reaction speed. Figure 6 As shown in part A, when 1 U / mL of γ-GGT was added to HX-GP and reacted at 37°C, the fluorescence intensity at 613 nm gradually increased over time and remained stable after about 20 minutes. In contrast, the fluorescence intensity of the probe without γ-GGT was almost unchanged. In addition, ONOO was continuously added to the solution. - After that, the fluorescence intensity of the probe at 518 nm increased rapidly and stabilized after 1 minute ( Figure 6 These findings indicate that the probe is sensitive to γ-GGT and ONOO - All showed rapid reactivity, which is beneficial for in vivo detection. We also studied the effect of pH on analytical detection. Figure 6 As shown in Part B of Figure 3, when γ-GGT was added to HX-GP in the pH range of 5.0-8.0, the fluorescence intensity at 613 nm reached a maximum at pH 7.4. - When added to the solution, the fluorescence intensity at 518 nm increases with the increase of pH value and reaches the maximum value at pH = 8 ( Figure 6 This indicates that HX-GP is suitable for the regulation of γ-GGT and ONOO in cells. - Detection.

[0059] In order to evaluate the effect of HX-GP on γ-GGT and ONOO - To determine the specificity of the cytotoxicity, we evaluated various reactive oxygen species and other biologically relevant competing substances. Figure 6 Part C and Figure 6 As shown in Part F of the figure, when the potential biological ion (K + Mg 2 + , Ca 2+ 、Fe 3+ 、Zn 2+ 、NO3 - 、SO4 2- , O 2- When other competing substances such as hydroxyl radicals (NH4O4, NH4O2, GSH, and Cys) and enzymes (NTR, ALP, and LAP) were added to HX-GP, the fluorescence signal change was negligible. However, only γ-GGT and ONOO - It can cause significant changes in the fluorescence spectrum of HX-GP, indicating that HX-GP has a strong effect on γ-GGT and ONOO under simulated physiological conditions. - Has higher specificity.

[0060] In subsequent studies, in order to further explore the reaction mechanism, we used high-resolution mass spectrometry (HRMS) technology for analysis. Figure 7 As shown in part A, when γ-GGTase acts on the probe HX-GP, we observe a characteristic peak at m / z 500.1886, corresponding to the fluorophore HX-OH ([M] + ,C 30 H 30 NO4S +, accurate molecular weight: 500.1890). This finding confirms that γ-GGTase catalyzes the cleavage of GSH in HX-GP, triggering the release of HX-OH, accompanied by the generation of red fluorescence. - After adding the solution containing HX-OH, we detected a peak at m / z 173.0597 by mass spectrometry analysis. ( Figure 7 Part B), which is consistent with the expected molecular weight of the fluorophore HNA ([M+H] + ,C 11 H9O2, accurate molecular weight: 173.0603). This result shows that ONOO - The reaction with HX-OH results in the oxidative cleavage of the olefin linker in the HX-OH molecule, which in turn causes the fluorescence emission wavelength to shift from red fluorescence to green fluorescence.

[0061] 2.2 γ-GGT and ONOO in living cells - Fluorescence imaging with HX-GP

[0062] We further investigated the relationship between γ-GGT and ONOO in living cells. - Fluorescence imaging activity of HX-GP. We first evaluated the cytotoxicity of HX-GP on HK-2 and 3T3 cells. The results showed that even at the highest concentration of HX-GP (20 μM) for 12 hours of incubation, the cell viability was over 80%, indicating that HX-GP has low cytotoxicity. Next, we evaluated the effectiveness of HX-GP in tracking the activity of endogenous γ-GGT in cells. As far as we know, GGT has a stable and high expression level in renal proximal tubular epithelial cells (HK-2). Therefore, during the experiment, we introduced the probe into the HK-2 cell culture system. From 0 to 40 minutes, we observed a gradual increase in the red fluorescence signal in the cells ( Figure 8 This observation confirms the strong cell permeability of this probe, making it suitable for fluorescence imaging analysis of endogenous γ-GGT. In addition, we used HX-GP to compare the expression levels of γ-GGT in different cell lines. Figure 9As shown, the fluorescence intensity of the HK-2 cell line is significantly higher than that of the 3T3 cell line, which indicates that the expression level of γ-GGT in HK-2 cells is higher than that in 3T3 cells. This finding is consistent with existing research data in this field. According to the above experimental results, HX-GP can effectively monitor the expression of γ-GGT in cells. In order to further clarify the specific location of HX-GP in cells, we used HX-GP and three well-known commercial organelle staining dyes BODIPY493 / 503 (LDsTracker), Lyso-Tracker Green and Mito-Tracker Green to conduct co-localization experiments on HK-2 cells. Figure 10 As shown, cells loaded with Lyso-Tracker Green and HX-GP overlapped well, with the highest Pearson colocalization coefficient of 0.89. These results indicate that HX-GP can be used to visually monitor γ-GGT in lysosomes.

[0063] Subsequently, we evaluated the effects of HX-GP on ONOO in living cells. - Potential for detection and imaging. Figure 11 As shown in part A, HK-2 cells treated with HX-GP showed no SIN1 (ONOO - donor) showed obvious red fluorescence and weak green fluorescence. Afterwards, as the concentration of SIN1 increased, the fluorescence of the green channel was greatly enhanced, while the fluorescence of the red channel dropped sharply ( Figure 11 Therefore, for ONOO - A clear ratio fluorescence response (green / red) was established. In addition, when non-acetylcysteine ​​(NAC, a reactive oxygen species scavenger) was added to the cells, the fluorescence intensity of the red fluorescence channel in the cells was relatively enhanced, while the fluorescence intensity of the green channel was relatively weakened ( Figure 11 ). This may be because the addition of NAC leads to ONOO - This further reduces the further oxidation of HX-OH, resulting in a decrease in the formation of the green fluorophore HNA, and thus a relative decrease in the fluorescence intensity of the green channel. These experimental results indicate that the probe HX-GP can produce a sensitive fluorescence response to changes in the levels of intracellular γ-GGT and ONOO.

[0064] 2.3HX-GP monitoring of γ-GGT and ONOO in cisplatin-induced AKI cell model

[0065] Based on the above experimental results, we further explored the feasibility of HX-GP for detecting γ-GGT and ONOO in a cisplatin-induced AKI cell model. Existing literature has confirmed that excessive cisplatin can lead to upregulation of intracellular γ-GGT expression and may trigger oxidative stress response. HK-2 cells incubated with HX-GP showed only weak fluorescence signals in the red fluorescence channel, and almost no fluorescence signals were detected in the green fluorescence channel ( Figure 12 ). This phenomenon indicates that the intracellular γ-GGT and ONOO - The levels of γ-GGT and ONOO in HK-2 cells were relatively low under conditions not affected by cisplatin. However, after exposing HK-2 cells to 0.5 mM and 1 mM cisplatin, we found that the fluorescence intensity of both the red and green channels increased, indicating that cisplatin treatment significantly increased the intracellular γ-GGT and ONOO - concentration( Figure 12 In addition, when cells were treated with cisplatin and L-carnitine (LC, an antioxidant) was added, the fluorescence of the green channel was significantly reduced and the fluorescence signal intensity of the red channel was slightly reduced compared with the cells treated with cisplatin alone, indicating that LC has an effect on ONOO - These experimental results indicate that HX-GP can simultaneously monitor γ-GGT and ONOO through different fluorescence channels in the cisplatin-induced AKI cell model. - This characteristic of the probe indicates that it has potential application value in the early diagnosis of AKI and is expected to become a powerful tool for evaluating renal injury and oxidative stress status.

Claims

1. A probe for simultaneous detection of γ-glutamyl transpeptidase and peroxynitrite, characterized in that: The chemical structural formula of the probe is as follows:

2. A method for preparing a probe for simultaneous detection of γ-glutamyl transpeptidase and peroxynitrite, characterized in that: The probe for simultaneously detecting γ-glutamyl transpeptidase and peroxynitrite is as described in claim 1, and the preparation method comprises the following steps: (1) Compound 1 and Compound 2 are reacted to prepare Compound 3; (2) Compound 3 and Compound 4 are reacted to prepare Compound 5; (3) preparing the probe for simultaneous detection of γ-glutamyl transpeptidase and peroxynitrite by reacting compound 5 and compound 6; Wherein, the chemical structural formulas of compounds 1-6 are shown in Formula 1 to Formula 6 respectively; 3. The preparation method according to claim 2, characterized in that In the step (1), compound 1, compound 2, NH4OAc and EtOH are mixed and reacted.

4. The preparation method according to claim 3, characterized in that In the step (1), the reaction temperature is 60° C. and the reaction time is 12 hours.

5. The preparation method according to claim 4, characterized in that In the step (1), the added amounts of compound 1, compound 2, NH4OAc and EtOH were 150 mg, 251.53 mg, 27.95 mg and 5 mL, respectively.

6. The preparation method according to claim 2, characterized in that In the step (2), compound 3, TEA and DCM are mixed in an ice bath, and then compound 4 is added to react.

7. The preparation method according to claim 6, characterized in that In the step (2), the reaction temperature is room temperature and the reaction time is 12 hours; the added amounts of compound 3, TEA, DCM and compound 4 are 55.00 mg, 33.35 mg, 5 mL and 14.92 mg, respectively.

8. The preparation method according to claim 2, characterized in that In the step (3), compound 5, compound 6, methanol and water are mixed, and sodium hydroxide is added to carry out the reaction.

9. The preparation method according to claim 8, characterized in that In the step (3), the reaction temperature is room temperature and the reaction time is 2 hours; the added amounts of compound 5, compound 6, methanol, water and sodium hydroxide are 50.00 mg, 60.16 mg, 1.50 mL, 1.50 mL and 2 mg, respectively.

10. Use of the probe for simultaneous detection of γ-glutamyl transpeptidase and peroxynitrite according to claim 1 in the preparation of a probe product for simultaneous detection of γ-glutamyl transpeptidase and peroxynitrite in vivo.

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