Multi-channel nano-probe, preparation method thereof and application of multi-channel nano-probe in detection of liver ischemia reperfusion injury biomarkers

Abstract

The invention discloses a multi-channel nanoprobe, a preparation method thereof and application of the multi-channel nanoprobe in detection of liver ischemia reperfusion injury biomarkers, and belongs to the technical field of in-situ detection and nanoprobe preparation. The nanoprobe is a functionalized metal organic framework nanomaterial which is loaded with gold nanoparticles and a fluorescent dye in sequence and is coated with a hyaluronic acid film; the metal organic framework is prepared by taking 2-methylimidazole and zinc nitrate as raw materials; raman active molecule cytochrome C is modified by the gold nanoparticles through an amidation reaction. The preparation process is simple, the cost is low, the operation is easy, the prepared multi-channel nanoprobe has high sensitivity and excellent biocompatibility, fluorescence and SERS dual-channel simultaneous detection of ATP and O2 <.-> biomarkers in the liver cell injury process can be achieved, and the application prospect is wide. The method can be used for clinically, accurately, efficiently and quickly diagnosing the liver ischemia reperfusion injury and monitoring the disease course in real time.

Description

Technical Field

[0001] The present invention relates to the technical field of in-situ detection and nanoprobe preparation, and in particular to a multi-channel nanoprobe and a preparation method thereof, and application in detecting biomarkers of liver ischemia-reperfusion injury. Background Art

[0002] Hepatic ischemia-reperfusion injury (HIRI) is a common complication in clinical procedures such as liver resection or transplantation, as well as in hemorrhagic shock. It consists of two interrelated phases: local ischemia and reperfusion injury. Temporarily interrupting the liver's blood supply (the ischemic phase) can effectively reduce intraoperative bleeding. During the ischemic phase, insufficient arterial blood supply disrupts the metabolic supply-demand balance, leading to hepatic hypoxia. However, the restoration of oxygen supply during reperfusion can cause oxidative stress and inflammation. HIRI and the resulting systemic inflammation can not only lead to transplant organ failure but also cause acute and chronic rejection in liver transplant recipients, leading to liver surgery failure and even death. Up to 13% of liver resection patients experience liver failure, and 11-18% of liver transplant recipients experience early graft failure. HIRI is a major factor affecting surgical success and patient survival. Traditional methods for monitoring HIRI rely on biological methods, such as blood tests, liver biopsy, and imaging. However, these methods often face numerous challenges in practical application. Due to the lag and insensitivity of blood liver function indicators, blood tests are often unable to definitively identify the onset of early liver injury. Invasive biopsy, recognized as the gold standard for clinical diagnosis of liver injury, can only provide a static picture of the pathological state, and the sampling procedure may produce secondary damage. Commonly used non-invasive imaging techniques in clinical practice, including computed tomography, magnetic resonance imaging, and positron emission tomography, all have limitations due to low resolution and are only suitable for detecting late, severe tissue damage. Early diagnosis and dynamic tracking of HIRI processes are currently unavailable. Therefore, accurate, real-time monitoring of the dynamic development of early HIRI provides an important opportunity for timely intervention and treatment.

[0003] Because during the HIRI process, abnormalities at the molecular level occur before abnormalities at the histological level. If HIRI can be clearly identified in the early stages, it will be convenient to use effective prevention and treatment methods to minimize HIRI. Therefore, monitoring the molecular level changes of chemical substances during the HIRI process is a more sensitive and effective method to detect HIRI. HIRI is a time-dependent multi-stage process involving a series of biochemical events triggered by abnormal changes in multiple biomarkers, with the characteristics of short life span, dynamic changes and multiple coordinated regulations. At present, researchers have conducted extensive research on the diagnostic targets of HIRI, including mitochondrial dysfunction, excessive production of reactive oxygen species (ROS), etc. Among them, adenosine triphosphate (ATP) and superoxide anion radicals (O2 .- ) has been shown to be a potential biomarker for detecting HIRI levels at the molecular level.

[0004] However, the current O2 .- Most methods for measuring HIRI and ATP rely on single-signal identification and focus on a single biomarker, which cannot effectively reveal the full picture. Furthermore, due to the low absolute amount and low variability of HIRI markers, as well as the complexity of tissue structure and composition, single-target control systems may lack specificity, produce off-target effects, and generate false-positive signals. Therefore, the development of new detection strategies that can simultaneously identify multiple signals and analyze multiple components is urgently needed. Summary of the Invention

[0005] The purpose of the present invention is to provide a multi-channel nanoprobe and its preparation method and application in detecting biomarkers of liver ischemia-reperfusion injury. The preparation process is simple, low-cost and easy to operate. The prepared multi-channel nanoprobe has high sensitivity and excellent biocompatibility, and can realize the ATP and O2 .- Simultaneous fluorescence and SERS dual-channel detection of two biomarkers.

[0006] To achieve the above object, the present invention provides a method for preparing a multi-channel nanoprobe, comprising the following steps:

[0007] S1. Preparation of metal organic framework: Using 2-methylimidazole as ligand, zinc ion as metal center, and methanol as solvent, the reaction was carried out at room temperature, followed by centrifugal washing and drying to obtain metal organic framework ZIF-8;

[0008] S2. Preparation of a multi-channel nanoprobe: The ZIF-8 prepared in step S1 is mixed with gold nanoparticles modified with cytochrome c and the fluorescent dye fluorescein isothiocyanate, and the gold nanoparticles modified with cytochrome c and the fluorescent dye fluorescein isothiocyanate are loaded into the pores of the ZIF-8 by pore adsorption. After reacting at room temperature, the nanoprobe is washed by centrifugation to obtain a nanoprobe having both SERS and fluorescence response characteristics.

[0009] S3. Encapsulating probes: The nanoprobes obtained in step S2 and hyaluronic acid are prepared into solutions, mixed thoroughly, reacted at room temperature, and washed by centrifugation to obtain multi-channel nanoprobes.

[0010] Preferably, in step S1, zinc nitrate hexahydrate provides zinc ions, the concentrations of 2-methylimidazole and zinc nitrate hexahydrate are 0.61 mol / L and 0.07 mol / L, respectively. The reaction is carried out for 5 minutes, centrifuged at 13,000 rpm for 10 minutes, washed with methanol three times, and placed in a 60°C oven for drying overnight to obtain the metal organic framework ZIF-8.

[0011] Furthermore, the specific steps of step S2 are as follows: the gold nanoparticles modified with cytochrome c are fully mixed with ZIF-8, ultrapure water is used as a solvent, the reaction is carried out for 4 hours, the mixture is centrifuged at a speed of 13000 rpm for 10 minutes, and washed with water three times to obtain a nanoprobe with SERS response performance; then the fluorescent dye fluorescein isothiocyanate and the nanoprobe with SERS response performance are fully mixed, ultrapure water is used as a solvent, the reaction is carried out at room temperature for 24 hours, the mixture is centrifuged at a speed of 13000 rpm for 10 minutes, and washed with water three times to obtain a nanoprobe with both SERS and fluorescence response characteristics.

[0012] Furthermore, the specific steps of step S3 are: dissolving hyaluronic acid in ultrapure water to obtain a hyaluronic acid solution with a concentration of 5 mg / mL; dissolving the nanoprobe prepared in step S2 in ultrapure water, and ultrasonically obtaining a nanoprobe solution with a concentration of 1 mg / mL; adding the nanoprobe solution dropwise to the hyaluronic acid solution at a volume ratio of 1:1, placing it at room temperature with stirring for 3 hours, centrifuging it at a speed of 13000 rpm for 10 minutes, and washing it with water 3 times to obtain a multi-channel nanoprobe.

[0013] Furthermore, in step S2, the preparation method of the gold nanoparticles modified with cytochrome c is as follows: first, gold nanoparticles are added to a mercaptopropionic acid solution, and mercaptopropionic acid is modified onto the gold nanoparticles using the bonding principle of the Au-S bond; then, cytochrome c is added thereto, and cytochrome c is connected to the gold nanoparticles through an amidation reaction to obtain gold nanoparticles modified with cytochrome c.

[0014] Furthermore, in step S2, the preparation method of the gold nanoparticles modified with cytochrome c is as follows: ultrapure water is used as a solvent, 10 -6 mol / L mercaptopropionic acid solution, drop gold nanoparticles into the mercaptopropionic acid solution, stir and react at room temperature for 10 minutes, centrifuge at 10000 rpm for 8 minutes, and wash with water 3 times to obtain amino-modified gold nanoparticles; use ultrapure water as solvent and prepare 10 -6 mol / L cytochrome c solution, the aminated gold nanoparticles were added dropwise into the cytochrome c solution, the volume ratio between the aminated gold nanoparticles and the cytochrome c solution was 1:5, the reaction was stirred at room temperature for 30 minutes, and finally centrifuged at a speed of 10000 rpm for 8 minutes, and washed with water 3 times to obtain cytochrome c modified gold nanoparticles.

[0015] Furthermore, in step S2, the preparation method of the gold nanoparticles is: mixing chloroauric acid with ultrapure water, heating and boiling at 330° C., adding sodium citrate solution, continuing heating and stirring for 3-5 minutes, and centrifuging at a speed of 10,000 rpm to obtain gold nanoparticles.

[0016] To achieve the above object, the present invention provides a multi-channel nanoprobe prepared by the above preparation method.

[0017] To achieve the above objectives, the present invention also provides the use of the multi-channel nanoprobe in detecting biomarkers of liver ischemia-reperfusion injury, wherein the biomarkers are adenosine triphosphate and superoxide anion free radicals.

[0018] Furthermore, the specific application process is as follows: the multi-channel nanoprobe is placed in normal liver cells THLE and liver cancer cells HepG-2, and the peak position and fluorescence intensity of fluorescein isothiocyanate released after adenosine triphosphate decomposes ZIF-8 are determined by fluorescence spectroscopy, and the change in fluorescence intensity is recorded. When the signal intensity at 520nm in the fluorescence spectrum increases, the change in ATP content can be detected; then the gold nanoparticles modified with cytochrome c released after adenosine triphosphate decomposes ZIF-8 are determined by SERS spectroscopy, and the peak position of cytochrome c and the signal change before and after the reaction with superoxide anion radicals are recorded. When the signal intensity at 1314cm in the SERS spectrum increases, the ATP content can be detected. -1 The change in the content of superoxide anion radicals can be detected by increasing the intensity of the characteristic peak at

[0019] The technical principle of the present invention is that the functionalized MOFs nanoprobe provided by the present invention decomposes the MOFs carrier under the stimulation of adenosine triphosphate (ATP), releases fluorescent (FL) dye and generates fluorescent signals, and at the same time, the released gold nanoparticles react with superoxide anion free radicals (O2 .-) will specifically generate surface enhanced Raman scattering (SERS) signals under the stimulation of fluorescence. By monitoring the changes of fluorescence and SERS dual signals, the detection of ATP and O2 .- FL-SERS dual-mode sensing of two biomarkers.

[0020] Compared with the prior art, the present invention has the following advantages:

[0021] (1) The present invention utilizes the advantages of high porosity, good adsorption and good stability of ZIF-8, and constructs functionalized MOFs nanoprobes by loading gold nanoparticles and fluorescent dyes respectively through a step-by-step modification method, and coating them with hyaluronic acid films. The preparation process is simple, low-cost, easy to operate, and easy to prepare in large quantities.

[0022] (2) The functionalized MOFs nanoprobe prepared by the present invention modifies the hyaluronic acid film, further improving the stability of the probe and avoiding interference from the complex intracellular environment and false positive signals.

[0023] (3) The functionalized MOFs nanoprobes prepared by the present invention have good biocompatibility. When the concentration of the nanomaterial is as high as 80 μg / mL, the cell survival rate is still greater than 90%, effectively avoiding the toxic side effects on normal cells.

[0024] (4) The functionalized MOFs nanoprobe prepared by the present invention has high sensitivity to ATP, O2 .- The detection limits were as low as 10 -8 mol / L, 10 -14 mol / L, and achieved intracellular ATP, O2 .- Fluorescence and SERS imaging.

[0025] (5) The functionalized MOFs nanoprobe prepared by the present invention can monitor ATP, O2 in real time during normal liver cell damage. .- dynamic changes.

[0026] In summary, the preparation process of the present invention is simple, low-cost, and easy to operate. The prepared multi-channel nanoprobe has high sensitivity and excellent biocompatibility, which can realize the ATP and O2 .- The development and application of this probe, which uses fluorescence and SERS dual channels to simultaneously detect two biomarkers, is expected to provide a more convenient and efficient monitoring method for HIRI and even ischemia-reperfusion injury in organs such as the myocardium and kidneys, and to offer new ideas for the detection of biomarkers for other diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1Schematic diagram of the preparation process and principle of the functionalized MOFs nanoprobe ZAFH of the present invention.

[0028] Figure 2 Scanning electron microscope characterization images of Au-MPA-Cyt-c (a), ZIF-8 (b) and functionalized MOFs nanoprobe ZAFH (c) in the examples.

[0029] Figure 3 This is a fluorescence curve diagram of FITC released by functionalized MOFs nanoprobe ZAFH at different concentrations in ATP solution in the example.

[0030] Figure 4 The fluorescence curve diagram (A) and linear correlation diagram (B) of the functionalized MOFs nanoprobe ZAFH detecting ATP in the embodiment.

[0031] Figure 5 Functional MOFs nanoprobe ZAFH detects O2 in the embodiment .- SERS spectrum curve (A) and linear correlation diagram (B).

[0032] Figure 6 The figure is a bar graph showing the cytotoxicity of the functionalized MOFs nanoprobe ZAFH to liver cancer cells HepG-2 (A) and human normal liver cells THLE (B).

[0033] Figure 7 These are fluorescence confocal images of the functionalized MOFs nanoprobe ZAFH incubated with HepG-2 cells for different times in the examples.

[0034] Figure 8 This is a fluorescence confocal imaging image of the functionalized MOFs nanoprobe ZAFH in the example to detect changes in ATP content in HIRI cells.

[0035] Figure 9 The functionalized MOFs nanoprobe ZAFH was used to detect O2 in HIRI cells. .- SERS confocal imaging of content changes. DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0037] Unless otherwise specified, the raw materials and reagents used in the following examples are all commercially available.

[0038] Example

[0039] like Figure 1 As shown, a method for preparing a multi-channel nanoprobe comprises the following steps:

[0040] S1. Preparation of metal organic framework: 2-methylimidazole was used as the matrix, zinc nitrate hexahydrate provided zinc ions as the metal center, methanol was used as the dispersion solvent, the concentrations of 2-methylimidazole and zinc nitrate hexahydrate were 0.61 mol / L and 0.07 mol / L, respectively. The mixture was stirred at room temperature for 5 min, centrifuged at 13000 rpm for 10 min, washed with methanol three times, and dried in a 60°C oven overnight to obtain white particles ZIF-8. The results were characterized by scanning electron microscopy. Figure 2 As shown in b, the particle size of ZIF-8 is about 50 nm;

[0041] S2. Preparation of multi-channel nanoprobes: The ZIF-8 prepared in step S1 is mixed with gold nanoparticles modified with cytochrome c (Au-MPA-Cyt-c) and the fluorescent dye fluorescein isothiocyanate (FITC). The Au-MPA-Cyt-c and FITC are loaded into the pores of the ZIF-8 by pore adsorption. After reaction at room temperature, the nanoprobes are washed by centrifugation to obtain nanoprobes with both SERS and fluorescence response characteristics.

[0042] The specific steps are as follows: using ultrapure water as solvent, 5 mg ZIF-8 was added to 5 mL 10 -6 mol / LAu-MPA-Cyt-c solution, stirred thoroughly, reacted at room temperature for 4 h, centrifuged at 13000 rpm for 10 min, washed with water 3 times, and obtained ZIF-8 nanomaterials ZA modified with Au-MPA-Cyt-c with SERS response performance; ultrapure water was used as solvent, and 5 mL of 1 mg / mL ZA was mixed with 5 mL of 10 -6 The mixture was thoroughly mixed with 10 mol / L FITC, stirred and reacted at room temperature for 24 h, centrifuged at 13,000 rpm for 10 min, and washed with water three times to obtain the ZIF-8 nanomaterial ZAF modified with Au-MPA-Cyt-c and FITC. ZAF has both SERS and fluorescence response characteristics.

[0043] The preparation method of Au-MPA-Cyt-c is as follows: 1 mL of 1% chloroauric acid is mixed with 100 mL of ultrapure water, heated to boil at 330°C, 2.5 mL of 0.1% sodium citrate solution is added dropwise, heating and stirring are continued for 3-5 minutes, and centrifuged at a speed of 10000 rpm for 8 minutes to obtain gold nanoparticles; ultrapure water is used as a solvent, and 10 -6 mol / L mercaptopropionic acid solution, drop gold nanoparticles into the mercaptopropionic acid solution, stir and react at room temperature for 10 minutes, centrifuge at 10000 rpm for 8 minutes, wash with water 3 times to obtain amino gold nanoparticles; finally, use ultrapure water as solvent to prepare 10 -6mol / L cytochrome c solution, the aminated gold nanoparticles were added dropwise to the cytochrome c solution, the volume ratio between the aminated gold nanoparticles and the cytochrome c solution was 1:5, the reaction was stirred at room temperature for 30 minutes, centrifuged at 10000 rpm for 8 minutes, and washed with water three times to obtain Au-MPA-Cyt-c. The results were characterized by scanning electron microscopy. Figure 2 As shown in a, Au-MPA-Cyt-c is a spherical particle with uniform size, and the particle size is 15 nm;

[0044] S3, encapsulating the probe: preparing the nanoprobe ZAF obtained in step S2 and hyaluronic acid into solutions, mixing them thoroughly, reacting them at room temperature, and washing them by centrifugation to obtain the multi-channel nanoprobe ZAFH;

[0045] The specific steps are as follows: using ultrapure water as solvent, respectively preparing a 1 mg / mL ZAF nanoprobe solution and a 5 mg / mL hyaluronic acid solution; adding 5 mL of 1 mg / mL ZAF nanomaterial dropwise to 5 mL of 5 mg / mL hyaluronic acid solution, stirring and reacting at room temperature for 3 hours, centrifuging at 13000 rpm for 10 minutes, and washing with water three times to obtain hyaluronic acid-coated ZAFH nanoprobes, i.e., multi-channel nanoprobes, which were characterized by scanning electron microscopy. The results are as follows: Figure 2 As shown in c, the particle size of the ZAFH nanoprobe is 50 nm.

[0046] Application Example 1

[0047] The ZAFH nanoprobes prepared in the example were prepared into different concentrations (5 μg / mL, 50 μg / mL, 100 μg / mL), and then the ZAFH nanoprobe solutions of different concentrations were mixed with 10 -3 mol / L ATP solution for different reaction times (0-24h), and the changes in fluorescence intensity at 520nm were recorded to obtain the release curve of the ZAFH nanoprobe, as shown in Figure 3 As shown, the nanoprobe - 3 In an environment of 10 mol / L ATP, the loaded FITC and Au-MPA-Cyt-c can be released completely within 4 h.

[0048] The feasibility of the multi-channel nanoprobe ZAFH prepared in the embodiment to detect ATP was investigated. -3The reaction was carried out in a 10 mol / L ATP solution. After 40 minutes, the mixture was centrifuged at 13000 rpm for 10 minutes. The supernatant was aspirated to remove the unreacted probe. 200 μL of the supernatant was placed in a fluorescence spectrophotometer with the excitation wavelength set at 490 nm. The fluorescence spectrum was recorded. The results are shown in the attached figure. Figure 4 .Depend on Figure 4 It can be seen that after the probe ZAFH reacted with different concentrations of ATP, the characteristic peak signal intensity of FITC at 520nm increased significantly, and at 10 -8 mol / L-10 -3 There is a good linear correlation between the logarithm of ATP concentration and the logarithm of fluorescence intensity at 520 nm in the mol / L range, and the detection limit of ATP is 10 -8 mol / L, indicating that the probe ZAFH can realize fluorescent sensing of ATP with high sensitivity.

[0049] The multi-channel nanoprobe ZAFH prepared in the embodiment was used to detect O2 .- To investigate the feasibility of the multi-channel nanoprobe ZAFH, -3 The reaction was carried out in a 10 mol / L ATP solution. After 40 min of reaction, the mixture was centrifuged at 13000 rpm for 10 min. The supernatant was aspirated to remove the unreacted probe. The solution was then mixed with different concentrations of O2 .- After 20 minutes of reaction, 2 μL of the reaction system was aspirated and placed on a silicon wafer, which was then placed on the sample stage of a Raman spectrometer. The excitation wavelength was set to 532 nm, the laser intensity was 10%, and the integration time was 15 s. The SERS spectrum was recorded as follows: Figure 5 As shown. Figure 5 It can be seen that the multi-channel nanoprobe ZAFH is sensitive to O2 .- The detection limits were 10 -14 mol / L, and at 10 -14 mol / L-10 -12 mol / L and 10 -12 mol / L-10 -4 In the range of mol / L, O2 .- The logarithm of the concentration and 1314 cm -1 There is a good linear relationship between the logarithms of the SERS signal intensity, which proves that the probe ZAFH can achieve O2 .- Highly sensitive sensing.

[0050] Application Example 2

[0051] The biocompatibility of the multi-channel nanoprobe ZAFH prepared in the embodiment was investigated. Normal human liver cells (THLE) and liver cancer cells (HepG-2) were seeded into 96 wells, 100 μL of Dulbecco's modified Eagle medium (DMEM) was added to the culture medium, heat-inactivated bovine serum (10%) and double antibodies (penicillin and streptomycin) were added, and the cells were cultured in a cell culture incubator at 37°C and 5% carbon dioxide for 24 hours. The blank group was only added with DMEM culture medium, and the experimental group was added with different concentrations of nanoprobe ZAFH (0-200 μg / mL). Three replicate wells were set for each sample and cultured for 24 hours. 10 μL of MTT indicator was added, and the cells were incubated in the incubator for another 24 hours. 150 μL of DMSO solution was added, and after slight shaking to mix evenly, the absorbance of each well at 490 nm was measured with an enzyme reader, and the cell activity of the experimental group was calculated. The results are shown in the figure. Figure 6 As shown in the figure, when the probe concentration was below 80 μg / mL, the cell activity exceeded 80%, indicating that the probe ZAFH had good biocompatibility.

[0052] The multi-channel nanoprobe ZAFH prepared in the example was subjected to a cell endocytosis experiment. HepG-2 cells were seeded in a confocal culture dish and cultured in a cell culture incubator at 37°C and 5% carbon dioxide for 24 hours. After the cells attached to the wall and grew, 80 μg / mL of the probe ZAFH was added. After incubation for different time periods (0-8 hours), the cells were imaged using a laser confocal fluorescence microscope. Figure 7 As shown in the figure, with the increase of incubation time, the green fluorescence gradually increased. When the incubation time was greater than or equal to 4 h, the fluorescence intensity remained almost unchanged, indicating that the optimal cell uptake time of the probe ZAFH was 4 h.

[0053] The multi-channel nanoprobe ZAFH prepared in the embodiment was used to detect ATP, O2 .- HepG-2 cells were seeded in a confocal culture dish and cultured in a 37°C, 5% carbon dioxide cell culture incubator for 24 hours. 80 μg / mL of the probe ZAFH was added. After 4 hours of cellular endocytosis, different concentrations of 2-methylestradiol (2-ME) were added to the cells to establish a HIRI injury model. The cells were imaged using a laser confocal fluorescence microscope and a SERS imaging microscope, respectively. The results are shown in Figure 2. Figure 8 and Figure 9 As shown in Figure 2, as the concentration of 2-ME molecules increases, the fluorescence intensity of FITC decreases, indicating that HIRI damage will cause a decrease in the intracellular ATP content. On the contrary, as the degree of cell HIRI damage increases, the fluorescence intensity of 1314 cm -1 The SERS signal intensity at the site gradually increases, proving that HIRI damage can cause intracellular O2 .-The results show that the probe ZAFH can achieve the expression of ATP and O2, the markers of liver ischemia-reperfusion injury in cells. .- Fluorescence-SERS dual-channel real-time monitoring of detection.

Claims

1. A method for preparing a multi-channel nanoprobe, characterized in that: The following steps are involved: S1. Preparation of metal organic framework: Using 2-methylimidazole as ligand, zinc ion as metal center, and methanol as solvent, the reaction was carried out at room temperature, followed by centrifugal washing and drying to obtain metal organic framework ZIF-8; S2. Preparation of a multi-channel nanoprobe: The ZIF-8 prepared in step S1 is mixed with gold nanoparticles modified with cytochrome c and the fluorescent dye fluorescein isothiocyanate, and the gold nanoparticles modified with cytochrome c and the fluorescent dye fluorescein isothiocyanate are loaded into the pores of the ZIF-8 by pore adsorption. After reacting at room temperature, the nanoprobe is washed by centrifugation to obtain a nanoprobe having both SERS and fluorescence response characteristics. S3. Encapsulating probes: The nanoprobes obtained in step S2 and hyaluronic acid are prepared into solutions, mixed thoroughly, reacted at room temperature, and washed by centrifugation to obtain multi-channel nanoprobes.

2. The method for preparing a multi-channel nanoprobe according to claim 1, characterized in that: In step S1, zinc nitrate hexahydrate provides zinc ions, the concentrations of 2-methylimidazole and zinc nitrate hexahydrate are 0.61 mol / L and 0.07 mol / L, respectively. The reaction is carried out for 5 minutes, and the mixture is centrifuged at 13,000 rpm for 10 minutes. The mixture is washed with methanol three times and dried in an oven at 60°C overnight to obtain the metal-organic framework ZIF-8.

3. The method for preparing a multi-channel nanoprobe according to claim 1 or 2, characterized in that: The specific steps of step S2 are as follows: the gold nanoparticles modified with cytochrome c are fully mixed with ZIF-8, the mixture is reacted for 4 hours using ultrapure water as a solvent, the mixture is centrifuged at a speed of 13,000 rpm for 10 minutes, and the mixture is washed with water three times to obtain a nanoprobe with SERS response performance; then the fluorescent dye fluorescein isothiocyanate and the nanoprobe with SERS response performance are fully mixed, the mixture is reacted at room temperature for 24 hours using ultrapure water as a solvent, the mixture is centrifuged at a speed of 13,000 rpm for 10 minutes, and the mixture is washed with water three times to obtain a nanoprobe with both SERS and fluorescence response characteristics.

4. The method for preparing a multi-channel nanoprobe according to claim 1 or 2, characterized in that: The specific steps of step S3 are as follows: dissolving hyaluronic acid in ultrapure water to obtain a hyaluronic acid solution with a concentration of 5 mg / mL; dissolving the nanoprobe prepared in step S2 in ultrapure water, and ultrasonically obtaining a nanoprobe solution with a concentration of 1 mg / mL; adding the nanoprobe solution dropwise to the hyaluronic acid solution at a volume ratio of 1:1, stirring and reacting at room temperature for 3 hours, centrifuging at a speed of 13000 rpm for 10 minutes, and washing with water 3 times to obtain a multichannel nanoprobe.

5. The method for preparing a multi-channel nanoprobe according to claim 1 or 2, characterized in that: In step S2, the preparation method of the gold nanoparticles modified with cytochrome c is as follows: first, gold nanoparticles are added to a mercaptopropionic acid solution, and mercaptopropionic acid is modified onto the gold nanoparticles using the bonding principle of the Au-S bond; then, cytochrome c is added thereto, and cytochrome c is connected to the gold nanoparticles through an amidation reaction to obtain gold nanoparticles modified with cytochrome c.

6. The method for preparing a multi-channel nanoprobe according to claim 5, characterized in that: In step S2, the preparation method of the gold nanoparticles modified with cytochrome c is as follows: using ultrapure water as solvent, preparing 10 -6 mol / L mercaptopropionic acid solution, drop gold nanoparticles into the mercaptopropionic acid solution, stir and react at room temperature for 10 minutes, centrifuge at 10000 rpm for 8 minutes, and wash with water 3 times to obtain amino-modified gold nanoparticles; use ultrapure water as solvent and prepare 10 -6 mol / L cytochrome c solution, the aminated gold nanoparticles were added dropwise into the cytochrome c solution, the volume ratio between the aminated gold nanoparticles and the cytochrome c solution was 1:5, the reaction was stirred at room temperature for 30 minutes, and finally centrifuged at a speed of 10000 rpm for 8 minutes, and washed with water 3 times to obtain cytochrome c modified gold nanoparticles.

7. The method for preparing a multi-channel nanoprobe according to claim 1 or 2, characterized in that: In step S2, the gold nanoparticles are prepared by mixing chloroauric acid with ultrapure water, heating and boiling at 330° C., adding sodium citrate solution, continuing heating and stirring for 3-5 minutes, and centrifuging at a speed of 10,000 rpm to obtain gold nanoparticles.

8. A multi-channel nanoprobe prepared by the preparation method according to any one of claims 1 to 7. 9 . Use of the multi-channel nanoprobe according to claim 8 in detecting biomarkers of liver ischemia-reperfusion injury, wherein the biomarkers are adenosine triphosphate and superoxide anion free radicals.

10. The use according to claim 9, characterized in that The specific application process is as follows: the multi-channel nanoprobe described in claim 8 is placed in normal liver cells THLE and liver cancer cells HepG-2, and the peak position and fluorescence intensity of fluorescein isothiocyanate released after adenosine triphosphate decomposes ZIF-8 are determined by fluorescence spectroscopy, and the change in fluorescence intensity is recorded. When the signal intensity at 520nm in the fluorescence spectrum is enhanced, the change in ATP content can be detected; and the gold nanoparticles modified with cytochrome c released after adenosine triphosphate decomposes ZIF-8 are determined by SERS spectroscopy, and the peak position of cytochrome c and the signal change before and after the reaction with superoxide anion free radicals are recorded. When the signal intensity at 1314cm in the SERS spectrum is enhanced, the change in ATP content can be detected. -1 The change in the content of superoxide anion radicals can be detected by increasing the intensity of the characteristic peak at

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