Fluorescent sensor for detecting cardiac troponin I and preparation method thereof

Abstract

The invention discloses a fluorescence sensor for detecting cardiac troponin I and a preparation method of the fluorescence sensor. The preparation method comprises the following steps: S1, adding a capture antibody of the cardiac troponin I into an elisa plate; s2, adding the prepared bovine serum albumin, and incubating; s3, adding prepared antigen cardiac troponin I with different concentrations, and incubating; s4, adding a prepared horse radish peroxidase modified detection antibody for detecting cardiac troponin I, and incubating; s5, preparing an EuMOF nanosheet solution; and S6, adding H2O2, o-phenylenediamine and an EuMOF nanosheet solution, reacting in a Tris buffer solution at room temperature, detecting the fluorescence intensity in the presence of cardiac troponin I with different concentrations by using a luminoscope, and constructing a fluorescence intensity-concentration working curve, thereby constructing the ratio fluorescence sensor. According to the invention, high-sensitivity quantitative analysis detection of cardiac troponin I is realized.

Description

Technical Field

[0001] This invention belongs to the field of lanthanide metal-organic framework materials for the analysis and detection of disease biomarkers, specifically relating to a fluorescent sensor for detecting cardiac troponin I and its preparation method. Background Technology

[0002] Cardiovascular disease (CVD) encompasses any disease that damages the cardiovascular system, such as coronary artery disease, arrhythmias, stroke (CVA), vascular lesions, and peripheral vascular disease. It is a leading cause of death and disability worldwide, significantly impacting people's lives. Despite advances in CVD diagnosis, the prevalence of CVD continues to increase globally due to an aging population. Acute myocardial infarction (AMI) is a very common and serious cardiovascular disease characterized by high morbidity, high mortality, and high disability rates. Traditional electrocardiography (ECG) cannot specifically detect AMI. cTnI has been selected as an ideal biomarker for detecting AMI.

[0003] cTn comprises three subunits: T, I, and C, each playing a different role in the regulation of muscle contraction. cTnI exhibits a unique myocardial-specific isoform. Its amino acid sequence differs significantly from the troponin I isoform in other muscle types (fast-twitch and slow-twitch skeletal muscle). In skeletal muscle, the isoform identical to myocardial cTnI is not expressed, neither in basal nor during regeneration or injury. This makes it a nearly absolute marker of myocardial injury. Following cardiomyocyte injury, cTnI undergoes proteolytic modification and oxidation in the blood, forming different fragments. Modern detection antibodies typically target epitopes of stable regions of the cTnI molecule (especially the central region). These epitopes are relatively stable in circulation and are usually designed to have high affinity for myocardial-specific peptide sequences (such as N-terminal or C-terminal extensions), further enhancing the myocardial specificity of the detection. While cTnT also has a myocardial-specific isoform, its specificity is slightly lower than that of cTnI. During embryonic development, skeletal muscle regeneration (e.g., muscular dystrophy, polymyositis, myopathy caused by chronic renal failure), or severe injury, skeletal muscle may re-express small amounts of fetal or regenerative subtypes that are very similar to cardiac cTnT. This can lead to the detection of elevated cTnT in non-myocardial injury (especially severe skeletal muscle injury combined with renal insufficiency), resulting in "false positives." The amino acid sequence of cTnC is highly homologous (very similar) to troponin C of skeletal muscle (fast and slow muscle). They are encoded by the same gene or have very similar encoding genes. Therefore, cTnC also significantly increases in skeletal muscle injury, lacking myocardial specificity. This is the fundamental reason why cTnC has never been developed as a clinical biomarker for myocardial injury. Detecting cTnC cannot distinguish between myocardial and skeletal muscle injury. Therefore, cTnI is the ideal biomarker for detecting acute myocardial infarction (AMI), making early diagnosis of cTnI extremely important.

[0004] Metal-organic frameworks (MOFs) are a class of porous crystalline materials formed by the self-assembly of metal ions or metal clusters with organic ligands. They possess high specific surface area, tunable pore size, and excellent chemical and thermal stability, making them widely applicable in gas adsorption, catalysis, sensing, and drug delivery. Lanthanide metal-organic frameworks (Ln-MOFs) offer significant advantages in the detection of disease biomarkers due to their unique optical properties and structural characteristics. However, traditional single-wavelength fluorescence emission is often susceptible to multiple external variables, affecting the accuracy of detection results. Ratio fluorescence sensors overcome these drawbacks by measuring the ratio of fluorescence signals at two different wavelengths and calibrating them relative to each other, thereby improving the accuracy of measurements. The self-calibration function improves the signal-to-noise ratio by correcting background interference, thus contributing to more accurate detection results. However, ratio fluorescence sensing still has certain limitations. Typically, it involves a change in one signal while the other remains constant, which limits its resolution and field of view. Therefore, developing a simple multi-response method for detecting disease biomarkers (cTnIs) is particularly important. Summary of the Invention

[0005] In view of this, the purpose of this invention is to address the shortcomings of existing technologies by providing a fluorescent sensor for detecting cardiac troponin I and its preparation method. This colorimetric sensor utilizes the red fluorescence properties of the synthesized two-dimensional metal-organic framework material EuMOF and constructs an analytical detection method for cardiac troponin I based on a horseradish peroxidase-induced enzyme-linked immunosorbent assay (ELISA), achieving highly sensitive quantitative analysis and detection of cardiac troponin I.

[0006] The technical solution adopted is as follows: The present invention discloses a method for preparing a fluorescent sensor for detecting cardiac troponin I, comprising the following steps: S1. Add the capture antibody for cardiac troponin I to the ELISA plate; S2. Add the prepared bovine serum albumin and incubate; S3. Add the prepared antigen cardiac troponin I at different concentrations and incubate; S4. Add the prepared horseradish peroxidase-modified antibody for detecting cardiac troponin I and incubate. S5. Fluorescent two-dimensional europium metal-organic framework material EuMOF nanosheets were ultrasonically dispersed in ultrapure water and oscillated to prepare EuMOF nanosheet solution; S6. Add H2O2, o-phenylenediamine and EuMOF nanosheet solution, react in Tris buffer solution at room temperature, and use a fluorescence spectrometer to detect the fluorescence intensity in the presence of different concentrations of cardiac troponin I, construct a fluorescence intensity-concentration working curve, thereby constructing a ratiometric fluorescence sensor.

[0007] Furthermore, in S5, nanoflowers formed by stacking fluorescent two-dimensional europium metal-organic framework material EuMOF nanosheets were dispersed in ultrapure water and sonicated for 30 min, followed by oscillation, to prepare a EuMOF nanosheet solution with a concentration of 400 μg / mL.

[0008] Furthermore, in S6, the preparation method of EuMOF nanosheets includes the following steps: (1) Dissolve 10.0-300.0 mg europium(III) nitrate hexahydrate in 1-10 mL N,N-dimethylformamide solvent, and sonicate and vortex. (2) Dissolve 10.0-300.0 mg of 4,4'-([2,2'-bipyridine]-5,5'-diyl)benzoic acid in 1-10 mL of N,N-dimethylformamide solvent, and sonicate and vortex. (3) Add 1-500 μL of concentrated nitric acid to the solution in step (2), and sonicate and vortex. (4) Mix the solutions from steps (1) and (3) and transfer them to a stainless steel reactor lined with polytetrafluoroethylene, and react at 50.0-200.0 °C for 6-48 hours; (5) A white product was obtained. N,N-dimethylformamide and concentrated nitric acid were washed away by filtration. The product was then dried overnight in a vacuum oven at 60-100 °C to obtain EuMOF nanosheets.

[0009] Further, in S1, 100 μL of 5 μg / mL cardiac troponin I capture antibody was added to a 96-well microplate and incubated overnight at 4°C. The plate was then washed three times with TBST to remove any cardiac troponin I capture antibody that was not immobilized on the microplate.

[0010] Further, in S2, 100 µL of prepared 1 vol% bovine serum albumin was added and incubated at 37°C for 1 h. The mixture was then washed three times with TBST to remove excess bovine serum albumin.

[0011] Further, in S3, 100 µL of prepared antigen cardiac troponin I solutions of different concentrations were added, and the mixture was incubated at 37°C for 1 h. The mixture was then washed three times with TBST to remove unbound antigen cardiac troponin I.

[0012] Further, in S4, 100 μL of the prepared 20 μg / mL horseradish peroxidase-modified antibody Ab2-HRP for detecting cardiac troponin I was added, and the mixture was incubated at 37 °C for 1 h. The mixture was then washed three times with 50 mM Tris buffer solution at pH 5.0 to remove unbound Ab2-HRP.

[0013] Further, in S6, 10 μL of 50 mM H2O2, 10 μL of 40 mM o-phenylenediamine, and a 400 μg / mL EuMOF nanosheet solution were added, and the mixture was reacted at room temperature for 15 min in 50 mM Tris buffer solution at pH 5.0.

[0014] The present invention provides a fluorescent sensor for detecting cardiac troponin I, which is prepared by the preparation method described above.

[0015] The beneficial technical effects of this invention are as follows: This invention utilizes an enzyme-linked immunosorbent assay (ELISA) induced by horseradish peroxidase (HPP) to detect the fluorescence intensity of cardiac troponin I antigen at different concentrations, constructing a fluorescence intensity-concentration working curve to create a fluorescence sensor. By detecting the fluorescence intensity of cardiac troponin I in the sample using a fluorometer and then substituting it into the working curve, quantitative analysis of cardiac troponin I in the sample can be achieved. This invention overcomes the shortcomings of existing cardiac troponin I detection techniques, such as single-detection methods. It leverages the fluorescence properties of the synthesized two-dimensional metal-organic framework material EuMOF, constructs an HPP-induced ELISA based on cardiac troponin I, and builds a biosensor for the quantitative analysis and detection of cardiac troponin I. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of a fluorescent sensor based on EuMOF nanosheets for detecting cardiac troponin I. Figure 2 The first scanning electron microscope (SEM) image of EuMOF nanosheets; Figure 3 This is the second scanning electron microscope (SEM) characterization image of the EuMOF nanosheets; Figure 4 Figure (bar chart) shows the feasibility study of detecting different concentrations of cardiac troponin I using a fluorescence sensor constructed based on EuMOF nanosheets. Figure 5 Figure (curve graph) shows the feasibility study of detecting different concentrations of cardiac troponin I using a fluorescence sensor constructed based on EuMOF nanosheets. Figure 6 Fluorescence spectra of different concentrations of cardiac troponin I detected by a fluorescence sensor constructed based on EuMOF nanosheets; Figure 7 The working curve of fluorescence detection of cardiac troponin I by a fluorescence sensor based on EuMOF nanosheets (I) 390 / I 556 ); Figure 8The working curve of fluorescence detection of cardiac troponin I by a fluorescence sensor based on EuMOF nanosheets (I) 617 / I 556 ). Detailed Implementation

[0017] The present invention will be described in detail below through specific embodiments. However, the uses and purposes of these exemplary embodiments are only for illustrating the present invention and do not constitute any limitation on the actual protection scope of the present invention, nor are they intended to limit the protection scope of the present invention to this.

[0018] DAP stands for 2,3-diaminophenazine. OPD stands for o-phenylenediamine. TBST is a solution containing Tris-HCl buffer and Tween-20, which is a commonly used buffer in biological experiments.

[0019] The working principle of the fluorescence sensor of this invention is as follows: In this invention, a horseradish peroxidase-induced enzyme-linked immunosorbent assay (ELISA) is constructed based on cardiac troponin I. In the presence of cardiac troponin I, horseradish peroxidase generates hydroxyl radicals from hydrogen peroxide, oxidizing OPD to 2,3-diaminophenazine (DAP). The solution changes from colorless to yellow. Simultaneously, the UV spectrum of DAP overlaps with the fluorescence emission spectrum of EuMOF nanoflowers. Due to fluorescence energy resonance transfer (FRET), the fluorescence of EuMOF nanosheets increases at 556 nm and decreases at 390 nm and 617 nm, forming ratio fluorescence. The detection principle is shown in Figure 1.

[0020] The analysis and detection of cardiac troponin I are achieved by varying the fluorescence intensity based on its presence and concentration in the system. The feasibility of this detection is as follows: Figure 2 - Figure 3 As shown. Example 1

[0021] The preparation method of a fluorescent sensor for detecting cardiac troponin I enzyme according to this embodiment includes the following specific steps: 1. First, synthesize EuMOF nanosheet materials, following these steps: (1) Dissolve 10.0-300.0 mg europium(III) nitrate hexahydrate in 1-10 mL N,N-dimethylformamide solvent, and sonicate and vortex. (2) Dissolve 10.0-300.0 mg of 4,4'-([2,2'-bipyridine]-5,5'-diyl)benzoic acid in 1-10 mL of N,N-dimethylformamide solvent, and sonicate and vortex. (3) Add 1-500 μL of concentrated nitric acid to the solution in step (2), and sonicate and vortex. (4) Mix the solutions from steps (1) and (3) and transfer them to a stainless steel reactor lined with polytetrafluoroethylene, and react at 50.0-200.0 °C for 6-48 hours; (5) A white product was obtained. N,N-dimethylformamide and concentrated nitric acid were washed away by filtration. The product was then dried overnight in a vacuum oven at 60-100 °C to obtain EuMOF nanosheets. 2. Add the cardiac troponin I capture antibody (5 μg / mL, 100 μL) to a 96-well microplate, incubate overnight at 4°C, and wash three times with TBST to remove cardiac troponin I capture antibody that is not immobilized on the microplate. 3. Add the prepared bovine serum albumin (1%, 100 µL), incubate at 37°C for 1 h, and wash three times with TBST to remove excess bovine serum albumin; 4. Add different concentrations of the prepared antigen cardiac troponin I (100 µL), incubate at 37°C for 1 h, and wash three times with TBST to remove unbound cardiac troponin I; 5. Add the prepared horseradish peroxidase-modified detection antibody for cardiac troponin I (Ab2-HRP, 20 μg / mL, 100 μL), incubate at 37 °C for 1 h, and wash three times with Tris buffer (5 mM, pH=5.0) to remove unbound Ab2-HRP. 6. Disperse the fluorescent two-dimensional metal-organic framework material EuMOF nanosheets into ultrapure water and sonicate for 30 min, then oscillate. The concentration of EuMOF material in the solution is 400 μg / mL. 7. Add H2O2 (10 μL, 50 mM), o-phenylenediamine (OPD, 40 mM, 10 µL) and 400 μg / mL EuMOF nanosheet solution, and react at room temperature for 15 min in Tris buffer (50 mM, pH=5.0). Detect the fluorescence intensity in the presence of different concentrations of cardiac troponin I using a fluorescence spectrometer to construct a fluorescence intensity-concentration working curve.

[0022] The results showed that when the concentration of cardiac troponin I was between 5-800 ng / mL -1 Within a certain concentration range, the fluorescence intensity of the system exhibits a linear relationship with the concentration: I 390 / I 556 (FL=-7.316 lgC cTnI +21.7723, R 2= 0.990), with a detection limit of 0.349 ng·mL. -1 ;I 617 / I 556 (FL=-2.081 lgCcTnI +6.542, R) 2 = 0.932), with a detection limit of 0.777 ng·mL. -1 ,like Figure 4-8 As shown.

[0023] Among them, "I" 390 / I 556 ” and “I 617 / I 556 "These are two ratio fluorescence working curves constructed when the fluorescence sensor detects cardiac troponin I (cTnI)."

[0024] I 390 / I 556 The fluorescence intensity (I) of the system at a wavelength of 390 nm represents the fluorescence intensity of the system. 390 ) and fluorescence intensity at 556 nm wavelength (I 556 The ratio of ).

[0025] I 617 / I 556 The fluorescence intensity (I) of the system at a wavelength of 617 nm represents the fluorescence intensity of the system. 617 ) and fluorescence intensity at 556 nm wavelength (I 556 The ratio of ).

[0026] I 390 These are fluorescence peaks of the ligands in EuMOF nanosheets; see [link / reference]. Figure 6 As shown, EuMOF nanosheets exhibit a characteristic peak at 390 nm.

[0027] I 556 The fluorescence emission mainly comes from the enzyme reaction product 2,3-diaminophenazine (DAP), see [link to relevant documentation]. Figure 6 As shown, DAP has a characteristic peak near 556 nm.

[0028] I 617 This is the characteristic emission peak of Eu³⁺ ions, see [link / reference]. Figure 6 As shown, the characteristic peak of Eu³⁺ ions is at 617 nm.

[0029] The purpose of designing two linear relationships is twofold: First, it can improve detection reliability. A single fluorescence signal is easily interfered with by environmental factors (such as temperature, pH, and instrument fluctuations). Ratio fluorescence method calculates the signal ratio of two wavelengths (e.g., IL). 390 / I 556 This cancels out common background interference, achieving self-calibration. Secondly, it can expand the scope of detection applications.390 / I 556 Based on the FRET effect of DAP and EuMOF, it has high sensitivity (0.349 ng / mL) and is suitable for the detection of low concentrations of cTnI. 617 / I 556 Directly utilizing the Eu³⁺ characteristic peak minimizes interference from the sample matrix (e.g., impurities in blood absorb 390 nm light), making it suitable for complex biological samples. Thirdly, cross-validation improves accuracy; two curves respond to cTnI through different mechanisms, allowing for mutual verification and avoiding false positives caused by non-specific DAP binding.

[0030] The detailed descriptions listed above are merely specific illustrations of feasible embodiments of the present invention and are not intended to limit the scope of protection of the present invention. All equivalent embodiments or modifications made without departing from the spirit of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a fluorescent sensor for detecting cardiac troponin I, characterized in that, Includes the following steps: S1. Add the capture antibody for cardiac troponin I to the ELISA plate; S2. Add the prepared bovine serum albumin and incubate; S3. Add the prepared antigen cardiac troponin I at different concentrations and incubate; S4. Add the prepared horseradish peroxidase-modified antibody for detecting cardiac troponin I and incubate. S5. Fluorescent two-dimensional europium metal-organic framework material EuMOF nanosheets were ultrasonically dispersed in ultrapure water and oscillated to prepare EuMOF nanosheet solution; S6. Add H2O2, o-phenylenediamine and EuMOF nanosheet solution, react in Tris buffer solution at room temperature, and use a fluorescence spectrometer to detect the fluorescence intensity in the presence of different concentrations of cardiac troponin I, construct a fluorescence intensity-concentration working curve, thereby constructing a ratiometric fluorescence sensor.

2. The method for preparing a fluorescent sensor for detecting cardiac troponin I according to claim 1, characterized in that, In S5, nanoflowers formed by stacking fluorescent two-dimensional europium metal-organic framework material EuMOF nanosheets were dispersed in ultrapure water and sonicated for 30 min, followed by oscillation, to prepare a EuMOF nanosheet solution with a concentration of 400 μg / mL.

3. The method for preparing a fluorescent sensor for detecting cardiac troponin I according to claim 1, characterized in that, The preparation method of EuMOF nanosheets in S6 includes the following steps: (1) Dissolve 10.0-300.0 mg europium(III) nitrate hexahydrate in 1-10 mL N,N-dimethylformamide solvent, and sonicate and vortex. (2) Dissolve 10.0-300.0 mg of 4,4'-([2,2'-bipyridine]-5,5'-diyl)benzoic acid in 1-10 mL of N,N-dimethylformamide solvent, and sonicate and vortex. (3) Add 1-500 μL of concentrated nitric acid to the solution in step (2), and sonicate and vortex. (4) Mix the solutions from steps (1) and (3) and transfer them to a stainless steel reactor lined with polytetrafluoroethylene, and react at 50.0-200.0 °C for 6-48 hours; (5) A white product was obtained. N,N-dimethylformamide and concentrated nitric acid were washed away by filtration. The product was then dried overnight in a vacuum oven at 60-100℃ to obtain EuMOF nanosheets.

4. The method for preparing a fluorescent sensor for detecting cardiac troponin I according to claim 1, characterized in that, In S1, 100 μL of 5 μg / mL cardiac troponin I capture antibody was added to a 96-well microplate and incubated overnight at 4°C. The plate was then washed three times with TBST to remove any cardiac troponin I capture antibody that was not immobilized on the microplate.

5. The method for preparing a fluorescent sensor for detecting cardiac troponin I according to claim 4, characterized in that, In S2, add 100 µL of the prepared 1 vol% bovine serum albumin and incubate at 37°C for 1 h. Wash three times with TBST to remove excess bovine serum albumin.

6. The method for preparing a fluorescent sensor for detecting cardiac troponin I according to claim 5, characterized in that, In S3, 100 µL of prepared antigen cardiac troponin I solutions of different concentrations were added and incubated at 37°C for 1 h. The mixture was then washed three times with TBST to remove unbound antigen cardiac troponin I.

7. The method for preparing a fluorescent sensor for detecting cardiac troponin I according to claim 6, characterized in that, In step S4, add 100 μL of the prepared 20 μg / mL horseradish peroxidase-modified antibody Ab2-HRP for detecting cardiac troponin I, incubate at 37 °C for 1 h, and wash three times with 50 mM Tris buffer solution at pH 5.0 to remove unbound Ab2-HRP.

8. The method for preparing a fluorescent sensor for detecting cardiac troponin I according to claim 7, characterized in that, In S6, 10 μL of 50 mM H2O2, 10 μL of 40 mM o-phenylenediamine, and a 400 μg / mL EuMOF nanosheet solution were added, and the mixture was reacted at room temperature for 15 min in 50 mM Tris buffer solution at pH 5.

0.

9. A fluorescent sensor for detecting cardiac troponin I, which is prepared by the preparation method according to any one of claims 1-8.

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