Preparation method of novel DNA nano fluorescent probe based on APE1 enzymatic fluorescence signal amplification strategy and application of novel DNA nano fluorescent probe in liver disease typing

Abstract

The invention discloses a preparation method of a novel DNA nano fluorescent probe based on an APE1 enzymatic fluorescence signal amplification strategy and application of the novel DNA nano fluorescent probe in liver disease typing, and relates to the technical field of biomedical materials, the technical key points are as follows: step S1, preparing a metal organic framework carrier by adopting a hydrothermal method: ultrasonically dissolving TCPP-Fe (III), BPyDC, ZrOCl2. 8H2O and BA in a round-bottom flask containing DMF for reaction; step S2, preparing a DNA fluorescent probe: designing two highly related targets miR-122 and miR-222 capable of respectively identifying liver diseases; and step S3, preparing MOF (at) DNA: respectively attaching AH-122-FAM and AH-222-Cy5 to the surface of the MOF, so as to obtain the MOF (at) DNA nano probe. The novel DNA nano fluorescent probe can realize spatial control of signal amplification in a specific disease area, so that imaging signals in a normal area are reduced, the accuracy of miRNA imaging is improved, liver diseases are distinguished, and the novel DNA nano fluorescent probe has relatively low cytotoxicity and good biocompatibility.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, and particularly relates to a preparation method of a novel DNA nanofluorescent probe based on an APE1 enzyme-catalyzed fluorescence signal amplification strategy and its application in liver disease typing. Background Art

[0002] Liver diseases are a major global public health problem, having a wide impact on human health. The liver is one of the most important organs in the human body, performing multiple key biological functions including detoxification, metabolism, digestion, vitamin storage, etc. When the homeostasis and function of the liver are disrupted, it can lead to the occurrence of diseases such as alcoholic liver disease, drug-induced liver injury, hepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma. Currently, clinically, biomarkers such as transaminases, albumin, bilirubin, and coagulation factors are usually used to evaluate liver function, while the occurrence of hepatocellular carcinoma is usually evaluated by markers such as alpha-fetoprotein heterogeneity (AFP), alpha-L-fucosidase (AFU), and des-gamma-carboxyprothrombin (DCP). However, these biomarkers often face challenges such as a lag in the detection timing and difficulty in subtype differentiation in clinical applications, resulting in misdiagnosis and improper treatment. Therefore, improving diagnostic accuracy and advancing the diagnostic window are of crucial significance for the staging and grading of liver diseases, disease monitoring, and drug development.

[0003] In recent years, microRNAs (miRNAs), as a class of endogenous, non-coding small RNAs, regulate gene expression at the post-transcriptional level by inhibiting translation or degrading target mRNAs, and play a direct role in the onset and progression of hepatocellular carcinoma, making them ideal tumor markers for the early diagnosis of this malignant tumor. Studies have shown that miRNAs are closely related to different clinical stages of liver diseases. For example, miR-122, as a well-recognized liver-specific miRNA, plays an important role in the occurrence and progression of hepatitis, cirrhosis, and hepatocellular carcinoma. In addition, miR-29 is related to the deterioration of fibrosis and cirrhosis. miR-221 and miR-222 show significant differences among patients with hepatocellular carcinoma, patients with cirrhosis and hepatitis, and healthy individuals. Although many miRNAs show high effectiveness in differentiating liver disease patients from healthy individuals, there are still deficiencies in accurately differentiating hepatocellular carcinoma subtypes. Therefore, there is an urgent need to develop a strategy that can achieve powerful diagnostic capabilities through a small combination of miRNAs to reduce related costs.

[0004] Although various amplification methods, such as rolling circle amplification (RCA), strand displacement amplification (SDA), hybridization chain reaction (HCR), and catalytic hairpin assembly (CHA), have been developed in the past decade to address the low abundance of miRNAs and enhance their detection sensitivity, the current ability to perform chemical amplification and real-time miRNA imaging at the disease site in vivo remains extremely limited. For example, exogenous enzyme-assisted systems require cell fixation and permeabilization before detection, which limits their application in living cells and in vivo environments. Non-enzyme strategies, on the other hand, lack the ability to spatially control signal amplification. Since miRNAs upregulated in tumor or inflammatory cells are not unique to these conditions and also exist at low levels in normal cells, this limits the applicability of non-enzyme methods in the living cell environment. Therefore, inventing an enzymatic fluorescence amplification dual miRNA imaging strategy is expected to improve the accuracy of liver disease identification and achieve precision medicine. Summary of the Invention

[0005] The object of the present invention is to solve the above problems and provide a method for preparing a novel DNA nanofluorescent probe based on the APE1 enzymatic fluorescence signal amplification strategy and its application in liver disease typing.

[0006] To achieve the above object, the technical solution of the present invention is as follows:

[0007] The present invention provides a method for preparing a novel DNA nanofluorescent probe based on the APE1 enzymatic fluorescence signal amplification strategy, comprising the following steps:

[0008] Step S1: Prepare a metal-organic framework carrier (metal-organic frameworks, MOF) by hydrothermal method: Ultrasonically dissolve iron(III) tetrakis(4-carboxyphenyl)porphine chloride (TCPP-Fe(III)), 2,2'-bipyridine-5,5'-dicarboxylic acid (BPyDC), ZrOCl 2 ·8H 2 O and benzoic acid (BA) in a round-bottom flask containing N,N-dimethylformamide (DMF), stir the reaction solution at 90 °C for 3 h, cover the oil bath with aluminum foil to avoid light, after the reaction is completed, centrifuge to collect the nanoparticles, wash them 3 times with fresh DMF and absolute ethanol respectively, and finally, store the product in the dark for further use;

[0009] Step S2: Prepare a DNA fluorescent probe: Design 2 target miRNAs highly related to liver diseases, named miR-122 and miR-222, design molecular beacons MB, and modify FAM fluorophores and BHQ1 quenchers at both ends of the hairpin structure to form H-122-FAM, Cy5 fluorophores and BHQ2 quenchers to form H-222-Cy5,

[0010] Among them, H-122-FAM: FAM-ACA GTA CAA ACA CCA TTG TCA CAC TCC ATA CTG T-BHQ1,

[0011] H-222-Cy5: Cy5-ACA TCT ACC CAG TAG CCA GAT GTA GCT GAG ATG T-BHQ2,

[0012] Then, by modifying two apurinic / apyrimidinic sites (AP) into the hairpin loop of MB, DNA fluorescent probes AH-122-FAM: FAM-ACA GTA CAA ACA / idSp / CA TTG TC / idSp / CAC TCC ATA CTG T-BHQ1 and AH-222-Cy5: Cy5-ACA TCT ACC CAG / idSp / AG CCA G / idSp / T GTA GCT GGT ATG T-BHQ2 are formed;

[0013] Step S3: Prepare metal-organic framework @ DNA probe (MOF@DNA): Attach AH-122-FAM and AH-222-Cy5 to the surface of MOF respectively to obtain MOF@DNA nanoprobes; Mix AH-122-FAM, AH-222-Cy5, 500 μL of 2×HEPES buffer and 380 μL of water with 200 μL of MOF (500 μg / mL), react at room temperature for 1.5 h, centrifuge the reaction system, wash it 3 times with 1×HEPES buffer, and store the obtained MOF@DNA nanoprobes in 100 μL of ×HEPES buffer at 4 °C.

[0014] The present invention is further set as follows: In the step S1, DMF is 1 - 5 ml, TCPP-Fe(Ⅲ) is 0.2 - 1.0 mg, BPyDC is 10 - 50 mg, ZrOCl 2 ·8H 2 O is 10 - 50 mg, and BA is 100 - 300 ml.

[0015] The present invention is further set as follows: In the step S3, AH-122-FAM is 20 - 100 nM, AH-222-Cy5 is 20 - 100 nM, the pH value of 2×HEPES buffer is 7.3, 2×HEPES buffer includes 50 mM HEPES and 300 mM NaCl, the pH value of 1×HEPES buffer is 7.3, and 1×HEPES buffer includes 25 mM HEPES and 150 mM NaCl.

[0016] Application of a novel DNA nanofluorescent probe based on APE1 enzyme-catalyzed fluorescence signal amplification strategy in liver disease typing.

[0017] The present invention is further configured such that: the DNA nanofluorescent probe can achieve spatial control of signal amplification in a specific disease area, reduce the imaging signal in the normal area, improve the accuracy of miRNA imaging, and distinguish liver diseases.

[0018] Compared with the prior art, the beneficial effects of this solution are as follows: the novel DNA nanofluorescent probe can achieve spatial control of signal amplification in a specific disease area, thereby reducing the imaging signal in the normal area, improving the accuracy of miRNA imaging to distinguish liver diseases, and having low cytotoxicity and good biocompatibility. Brief Description of the Drawings

[0019] Figure 1 It is a diagram for verifying the feasibility of the probe and evaluating the detection performance in the embodiments of the present invention, where A is a schematic diagram of the DNA probe for detecting dual miRNAs; B is the fluorescence response of H-122-FAM (50 nM) and AH-122-FAM (50 nM) to miR-122 (20 nM) in the presence and absence of APE1 (5 U / mL); C is the fluorescence response of AH-222-Cy5 (50 nM) to miR-122 (20 nM) in the presence and absence of APE1 (5 U / mL); D is the time-dependent fluorescence intensity change diagram of AH-122-FAM (50 nM) with miR-122 (20 nM) and APE1 (1 U / mL); E is the FAM fluorescence spectrum of the AH-122-FAM (50 nM) probe in response to different concentrations of miR-122 (a - j curves: 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 15, 20 nM); F is the Cy5 fluorescence spectrum of the AH-222-Cy5 (50 nM) probe in response to different concentrations of miR-222 (a - j curves: 0, 0.05, 0.1, 0.5, 1, 2, 5, 10, 15, 20 nM) under optimized experimental conditions; H is the linear relationship between the FAM signal and the miR-122 concentration; I is the linear relationship between the Cy5 signal and the miR-222 concentration; J is the fluorescence reaction of AH-122-FAM and AH-222-Cy5 after incubation with miR-122, miR-222 (20 nM) and other different miRNA molecules (100 nM) respectively.

[0020] Figure 2It is the cytotoxicity diagram of MOF@DNA in the embodiments of the present invention. Among them, A is the cell survival rate of L02 cells after incubation with different concentrations of MOF@DNA for 24 h; B is the cell survival rate of Huh-7 cells after incubation with different concentrations of MOF@DNA for 24 h; C is the cell survival rate of HepG2 cells after incubation with different concentrations of MOF@DNA for 24 h;

[0021] Figure 3 It is the cell imaging diagram in the embodiments of the present invention. Among them, a is the confocal fluorescence imaging of miR-122 and miR-222 in L02 cells and the content of intracellular miR-122 and miR-222 modified by PMCH and transfection; b is the confocal fluorescence imaging of miR-122 and miR-222 in HepG2 cells and the content of intracellular miR-122 and miR-222 modified by PMCH and transfection; c is the confocal fluorescence imaging of miR-122 and miR-222 in Huh-7 cells and the content of intracellular miR-122 and miR-222 modified by MOF@DNA and transfection; d is L02+miR-222mimic; e is HepG2+anti-miR-222; f is Huh-7+anti-miR-122+anti-miR-222. The average fluorescence intensity of FAM and Cy5 in the cells corresponding to the histogram data (mean±SD, n = 3);

[0022] Figure 4 It is the in vivo tumor imaging diagram in the embodiments of the present invention. Among them, A is the fluorescence image of Huh-7 tumor-bearing mice after injection of MOF@DNA and the fluorescence enhancement in the tumor; B is the fluorescence image of HepG2 tumor-bearing mice after injection of MOF@DNA and the fluorescence enhancement in the tumor;

[0023] Figure 5 It is the fluorescence imaging diagram of liver injury in the embodiments of the present invention. A is the fluorescence imaging of the livers of CCl 4 -induced liver injury mice and normal mice; B is the fluorescence imaging of the tissue sections of CCl 4 -induced liver injury mice and normal mice. Detailed implementation manners

[0024] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0025] It should be noted that, without conflict, the embodiments in the present invention and the features in the embodiments can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0026] Embodiment:

[0027] Design two molecular beacons (MBs) that can respectively recognize two target miRNAs (miR-122 and miR-222) highly related to liver diseases, and modify FAM fluorophore and BHQ1 quencher, Cy5 fluorophore and BHQ2 quencher at both ends of the hairpin structure respectively (H-122-FAM, H-222-Cy5). Among them, H-122-FAM: FAM-ACA GTA CAA ACA CCA TTG TCA CAC TCCATA CTG T-BHQ1,

[0028] H-222-Cy5: Cy5-ACA TCT ACC CAG TAG CCA GAT GTA GCT GAG ATG T-BHQ2. Then, by modifying two apurinic / apyrimidinic sites (AP) into the hairpin loop of the MB, DNA fluorescent probes (AH-122-FAM, AH-222-Cy5) are formed.

[0029] Among them, AH-122-FAM: FAM-ACA GTA CAA ACA / idSp / CA TTG TC / idSp / CAC TCCATA CTGT-BHQ1,

[0030] AH-222-Cy5: Cy5-ACATCTACC CAG / idSp / AG CCA G / idSp / T GTA GCT GGT ATG T-BHQ2. After that, to improve the cellular uptake of the DNA fluorescent probe, the DNA fluorescent probe is adsorbed on the MOF carrier to form a novel DNA nanofluorescent probe (MOF@DNA).

[0031] Preparation of MOF carrier

[0032] Prepare MOF by hydrothermal method. Briefly, dissolve TCPP-Fe(Ⅲ) (0.53 mg), BPyDC (31.7 mg), ZrOCl 2 ·8H 2 O (20 mg) and BA (200 mg) in 3 ml of DMF by ultrasonic in a round-bottom flask. Stir the reaction solution at 90 °C for 3 h. Cover the oil bath with aluminum foil to avoid light. After the reaction is completed, centrifuge to collect the nanoparticles and wash them 3 times with fresh DMF and absolute ethanol respectively. Finally, store the product in the dark for further use.

[0033] Preparation of MOF@DNA

[0034] AH-122-FAM and AH-222-Cy5 were respectively attached to the surface of MOF to obtain the MOF@MB nanoprobe. Briefly, AH-122-FAM (50 nM), AH-222-Cy5 (50 nM), 2×HEPES buffer (50 mM HEPES, 300 mM NaCl, pH = 7.3, 500 μL) and 380 μL of water were mixed with 250 μg / mL of MOF and reacted at room temperature for 1.5 h. The reaction system was centrifuged and washed 3 times with 1×HEPES buffer. The obtained MOF@MB nanoprobe was stored in 100 μL of ×HEPES buffer (25 mM HEPES, 150 mM NaCl, pH = 7.3) at 4 °C.

[0035] Fluorescence detection of miR-122 and miR-222 in solution:

[0036] AH-122-FAM and AH-222-Cy5 were denatured at 95 °C for 5 min and then cooled to room temperature in HEPES buffer (50 mM HEPES, 100 mM NaCl, pH 7.4). Signal amplification was carried out in 1×NEBuffer 4. Fluorescence detection was performed using 50 nM of AH-122-FAM and 50 nM of AH-222-Cy5. 0 - 20 nM of miR-122 and miR-222 were respectively added and incubated with 5 U / mL of APE1 for 10 min. The fluorescence spectrum was measured with a fluorescence spectrophotometer. Under excitation at 488 nm, the FAM fluorescence data was accumulated from 505 - 600 nm. Under excitation at 620 nm, the fluorescence data of Cy5 was accumulated from 640 nm to 750 nm. As Figure 1 shown in E-I, as the target increased, the fluorescence intensity increased, indicating that the present invention can be used for the quantitative determination of miR-122 and miR-222. The specific detection method was the same as before, and the detection included miR-429, miR-630, miR-155 and miR-21 ( Figure 1 J).

[0037] Cell viability experiment:

[0038] The cytotoxicity of MOF@DNA was detected using a CCK-8 kit. Huh-7, HepG2 and L02 cells were seeded in a 96-well cell culture plate and incubated at 37 °C, 5% CO 2 for 24 h. After adding different concentrations (0, 10, 50, 100, 250, 500 μg / mL) of the medium and culturing for 24 h, MOF@MB was removed. Subsequently, after washing 3 times with PBS, 10 μL of CCK-8 was added and incubated for 0.5 h. Finally, the absorbance value at 450 nm was recorded with an enzyme-linked immunosorbent assay reader, as Figure 2The cell viability of all the cell lines shown exceeded 80%. The results indicate that the MOF@MB nanoprobe has low cytotoxicity and good biocompatibility.

[0039] Confocal fluorescence imaging of cellular miRNAs:

[0040] Huh-7, HepG2, and L02 cells were incubated in confocal dishes for 24 h, then MOF@DNA (500 μg / mL) was added and incubation continued for 5 h. The culture medium was removed, and the cells were washed 3 times with PBS, and confocal imaging was recorded. Meanwhile, to evaluate the feasibility of dynamically monitoring miR-122 and miR-222 in cells, we pre-transfected Huh-7, HepG2, and L02 cells with different DNA sequences via Lipo6000 TM The subsequent steps were the same as those described above.

[0041] L02 cells normally express miR-122 and have low expression of miR-222; HepG2 cells have low expression of miR-122 and high expression of miR-222; Huh-7 cells have high expression of both miR-122 and miR-222. These 3 cell lines were selected as imaging experimental models. As expected, in normal hepatocytes (L02), only a weak FAM fluorescence signal ( Figure 3 a) was observed, due to the low expression levels of miR-222 and APE1, and when miR-222 was upregulated in L02 cells, both FAM and Cy5 fluoresced ( Figure 3 b); in HepG2 cells, only a bright Cy5 fluorescence ( Figure 3 c) was observed, while the FAM fluorescence was almost negligible, and downregulation of miR-222 in HepG2 cells led to a significant decrease in Cy5 fluorescence ( Figure 3 d); Huh-7 cells showed strong FAM and Cy5 fluorescence ( Figure 3 e). However, after simultaneously downregulating intracellular miR-122 and miR-222, the fluorescence of Huh-7 cells was significantly weakened ( Figure 3 f). The experimental results show that the proposed nanoprobe can accurately detect dual miRNAs in living cells and distinguish different cell lines, providing a reliable basis for the specific diagnosis of liver diseases.

[0042] In vivo tumor fluorescence imaging:

[0043] Huh-7 and HepG2 cells were subcutaneously injected (8×10 6, 150 μL) to construct a tumor model for experiments. To perform miRNA imaging in live mice, we injected MOF@MB into the tumors of the mice. Real-time whole-body fluorescence imaging was performed on the Huh-7 group and HepG2 group of mice using an in vivo animal imaging system at 0, 2, 4, and 6 h after intratumoral injection. Since both miR-122 and miR-222 are highly expressed in Huh-7 cells, while miR-122 is lowly expressed and miR-222 is highly expressed in HepG2 cells. Therefore, at different time points after injection, the fluorescence of Cy3 and Cy5 at the tumor sites of mice bearing Huh-7 cells increased significantly ( Figure 4 A). In HepG2 tumor-bearing mice, only the fluorescence of Cy5 gradually increased at the tumor site ( Figure 4 B).

[0044] Liver injury fluorescence imaging:

[0045] Mice were intraperitoneally injected with an edible oil solution containing carbon tetrachloride (volume content 10%) (5 μL / g) and stimulated for 24 h to form a liver injury model. Then, the mice were sacrificed by cervical dislocation, and the liver tissues of the mice were removed and placed in pre-cooled PBS to wash away the blood for standby. The liver tissues were immersed in a solution containing the probe, and then an in vivo imaging system and a laser confocal imaging were used. At the same time, normal liver tissues were immersed in the probe solution as a control experiment. The detection ability of MOF@DNA was evaluated using a liver injury model induced by CCl 4 . The fluorescence intensities of FAM and Cy5 in the liver injury group treated with CCl 4 were significantly higher than those in the normal liver group ( Figure 5 A). Similarly, tissue section imaging showed consistent fluorescence signal results ( Figure 5 B).

[0046] In summary, the differential expression of miR-122 and miR-222 in liver injury and various liver cancers provides a means for the preliminary classification of liver diseases, facilitating personalized treatment plans. With this excellent performance, MOF@MB is expected to achieve true clinical monitoring applications.

[0047] The above specific embodiments are only explanations of the present invention, and they are not limitations of the present invention. Those skilled in the art can make modifications without creative contributions to the embodiments according to needs after reading this specification, but as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A novel method for preparing a DNA nanofluorescent probe based on APE1 enzymatic fluorescence signal amplification strategy, characterized in that: The following steps are involved: Step S1, preparing a metal organic framework carrier by a hydrothermal method: tetrakis(4-carboxyphenyl)porphine ferric chloride, 2,2'-bipyridine-5,5'-dicarboxylic acid, ZrOCl2·8H2O and benzoic acid are ultrasonically dissolved in a round-bottom flask containing N,N-dimethylformamide; the reaction solution is stirred at 90° C. for 3 h; the oil bath is covered with aluminum foil to avoid light exposure; after the reaction is completed, the nanoparticles are collected by centrifugation and washed three times with fresh DMF and anhydrous ethanol respectively; Finally, store the product in the dark for further use; Step S2, preparing DNA fluorescent probes: designing two target miRNAs that can respectively identify two highly related liver diseases and named them miR-122 and miR-222, designing molecular beacons MB, and modifying the two ends of the hairpin structure with FAM fluorophore and BHQ1 quencher to form H-122-FAM, and Cy5 fluorophore and BHQ2 quencher to form H-222-Cy5, respectively. Among them H-122-FAM:FAM-ACA GTA CAA ACA CCA TTG TCA CAC TCC ATA CTG T-BHQ1, H-222-Cy5:Cy5-ACA TCT ACC CAG TAG CCA GAT GTA GCT GAG ATG T-BHQ2, Then, two apurinic pyrimidine sites AP were modified into the hairpin loop of MB to form the DNA fluorescent probes AH-122-FAM: FAM-ACA GTA CAAACA / idSp / CA TTG TC / idSp / CAC TCC ATA CTG T-BHQ1 and AH-222-Cy5: Cy5-ACA TCT ACC CAG / idSp / AG CCAG / idSp / T GTA GCT GGTATG T-BHQ2; Step S3, preparation of metal organic framework @ DNA probe: AH-122-FAM and AH-222-Cy5 were attached to the surface of MOF respectively to obtain MOF@DNA nanoprobe; 50nM AH-122-FAM, 50nM AH-222-Cy5, 500μL 2×HEPES buffer and 380μL water were mixed with 200μL MOF (500μg / mL), reacted at room temperature for 1.5h, centrifuged the reaction system, washed 3 times with 1×HEPES buffer, and stored the obtained MOF@DNA nanoprobe in 100μL 1×HEPES buffer at 4°C.

2. The method for preparing a novel DNA nanofluorescent probe based on the APE1 enzymatic fluorescence signal amplification strategy as claimed in claim 1, characterized in that: In the step S1, the N,N-dimethylformamide is 1-5 ml, the tetrakis(4-carboxyphenyl)porphine ferric chloride is 0.2-1.0 mg, the 2,2'-bipyridine-5,5'-dicarboxylic acid is 10-50 mg, the ZrOCl2·8H2O is 10-50 mg, and the benzoic acid is 100-300 ml.

3. The method for preparing a novel DNA nanofluorescent probe based on the APE1 enzymatic fluorescence signal amplification strategy as claimed in claim 1, characterized in that: In step S3, AH-122-FAM is 20-100 nM, AH-222-Cy5 is 20-100 nM, the pH value of 2×HEPES buffer is 7.3, 2×HEPES buffer includes 50 mM HEPES and 300 mM NaCl, and the pH value of 1×HEPES buffer is 7.3, 1×HEPES buffer includes 25 mM HEPES and 150 mM NaCl.

4. Application of a new DNA nanofluorescent probe in liver disease typing based on APE1 enzymatic fluorescence signal amplification strategy.

5. The application of the novel DNA nano fluorescent probe preparation method based on APE1 enzymatic fluorescent signal amplification strategy in liver disease typing as claimed in claim 4, characterized in that: The DNA nanofluorescent probe can achieve spatial control of signal amplification in specific disease areas, reduce imaging signals in normal areas, improve the accuracy of miRNA imaging, and distinguish liver diseases.

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