Fluorescent compound for detecting noradrenaline and epinephrine and application thereof

Abstract

The invention discloses a fluorescent probe as shown in a formula I or II for detecting noradrenaline and epinephrine and application of the fluorescent probe in cell imaging and biosensing. The fluorescent probe for detecting noradrenaline can be specifically combined with noradrenaline, two-channel ratio-type fluorescence change is presented, a ratio-type signal has a self-calibration function, and higher accuracy is provided for quantitative analysis. The fluorescent probe for detecting the epinephrine can be specifically combined with the epinephrine and shows low-background opening type fluorescence change. The fluorescent probe has high sensitivity and selectivity, can realize quantitative detection of noradrenaline and epinephrine in serum and fluorescence imaging in cells, has the potential to be used as an effective tool for evaluating the sympathetic nerve activity degree in diseases such as hypertension, has a wide application prospect, and has a wide application prospect. And the detection means of catecholamine substances are enriched.

Description

Technical Field

[0001] This invention belongs to the field of fluorescence imaging and biosensing technology. Specifically, this invention relates to a novel fluorescent probe for detecting norepinephrine and epinephrine and its application in cell imaging and biosensing. Background Technology

[0002] Catecholamines, primarily including dopamine (DA), norepinephrine (NE), and epinephrine (EP), act as neurotransmitters when released into the synaptic cleft and as hormones when released into the bloodstream, playing multiple roles in the nervous and endocrine systems. Norepinephrine, as an important neurotransmitter of the sympathetic nervous system, is closely related to neurodegenerative diseases such as depression, Parkinson's disease, hypertension, and neuroendocrine tumors, as well as cardiovascular diseases. Epinephrine reflects the body's stress state and is a core effector molecule of the sympathetic-adrenal medullary axis, primarily functioning in the peripheral system. Long-term chronic stress leads to persistently abnormally elevated epinephrine levels, increasing cardiac load and potentially inducing cardiovascular disease. Therefore, accurate monitoring of norepinephrine and epinephrine levels helps reveal the level of sympathetic nerve activity and assess disease states related to the sympathetic nervous system, such as hypertension.

[0003] However, the levels of norepinephrine and epinephrine in organisms are extremely low, there are many interfering substances, and the two differ by only one methyl group. Accurately distinguishing and quantifying the two poses a significant challenge to detection methods.

[0004] Therefore, developing a highly sensitive, selective, and low-cost method for analyzing norepinephrine and epinephrine is crucial for assessing sympathetic nerve activity and monitoring and diagnosing cardiovascular diseases related to the sympathetic nervous system. Summary of the Invention

[0005] The purpose of this invention is to provide a novel fluorescent compound for detecting norepinephrine and epinephrine.

[0006] Another object of the present invention is to provide a detection reagent or kit for detecting norepinephrine and epinephrine comprising the above-mentioned compounds.

[0007] Another objective of this invention is to provide a method for detecting norepinephrine and epinephrine in a sample.

[0008] In a first aspect, the present invention provides a method for detecting catecholamines in a sample, the method comprising the step of contacting the sample to be tested with a compound to detect the presence and / or content of catecholamines in the sample.

[0009] The compound is the compound shown in Formula I or II:

[0010]

[0011] In Formula I, L1 is selected from: substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S, substituted or unsubstituted C3-C7 cycloalkylene, substituted or unsubstituted C3-C7 heterocyclic group containing one, two or three heteroatoms independently selected from N, O or S;

[0012] L2 is selected from: substituted or unsubstituted C1-C 10 Alkylene, substituted or unsubstituted C1-C containing one, two or three heteroatoms independently selected from N, O or S. 10 Alkylene;

[0013] In Formula II, X is selected from: -O-, -S-, -NR1-;

[0014] R1 is selected from: H, substituted or unsubstituted C1-C6 alkylene groups.

[0015] In a preferred embodiment, in Formula I, L1 is selected from: substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C1-C4 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S;

[0016] L2 is selected from: substituted or unsubstituted C1-C8 alkylene groups, substituted or unsubstituted C1-C8 alkylene groups containing 1, 2 or 3 heteroatoms independently selected from N, O or S;

[0017] In Formula II, X is selected from: -O-, -S-, -NH-, with -O- being preferred.

[0018] In a preferred embodiment, in formula I, L1 is selected from the following group:

[0019] Preferred

[0020] L2 is independently selected from the following group:

[0021] Preferred

[0022]

[0023] In a preferred embodiment, the compound of formula I is selected from:

[0024]

[0025] Preferably, the compound of formula I is: The compound of formula II is selected from:

[0026]

[0027] Preferably, the compound of formula II is:

[0028] In specific embodiments, the catecholamines include, but are not limited to, dopamine (DA), norepinephrine (NE), epinephrine (EP), and combinations thereof;

[0029] Preferably, the catecholamines are selected from norepinephrine (NE), epinephrine (EP), and combinations thereof.

[0030] In a specific embodiment, the method is a method for fluorescence imaging of intracellular norepinephrine or epinephrine, and the method includes the following steps:

[0031] 1) Incubate the cells with the compound shown in Formula I; and

[0032] 2) Using 405nm as the excitation wavelength, the fluorescence intensity of the 526nm channel was observed to gradually increase through confocal microscopy, thereby achieving imaging analysis of changes in intracellular norepinephrine levels;

[0033] or,

[0034] 1') Incubate the cells with the compound shown in Formula II; and

[0035] 2') Using 488nm as the excitation wavelength, the fluorescence intensity at 530nm was observed, thereby enabling imaging analysis of changes in intracellular adrenaline levels.

[0036] In a preferred embodiment, the fluorescence intensity at 530 nm is observed using a confocal microscope.

[0037] In a specific implementation, the method for quantitatively detecting non-intracellular norepinephrine or epinephrine in a sample includes the following steps:

[0038] 1) A standard curve for detecting norepinephrine was prepared using the compound shown in Formula I and a standard of norepinephrine;

[0039] 2) Add the compound shown in Formula I to the sample; and

[0040] 3) Quantitatively detect the content of norepinephrine in the sample;

[0041] or,

[0042] 1') A standard curve for detecting norepinephrine was prepared using the compound shown in Formula II and a standard of adrenaline;

[0043] 2') Add the compound shown in Formula II to the sample; and

[0044] 3') Quantitatively detect the content of adrenaline in the sample.

[0045] In a preferred embodiment, in step 1), a solution of the compound of formula I with a determined concentration is mixed with norepinephrine solutions of different concentrations, and the fluorescence intensity at 400 nm and 526 nm is recorded under excitation at a wavelength of 350 nm. A standard curve is established between the ratio of fluorescence intensity at 526 nm to 400 nm and the concentration of norepinephrine.

[0046] In a preferred embodiment, in step 1'), a solution of the compound of formula II with a determined concentration is mixed with epinephrine solutions of different concentrations, the fluorescence intensity at 530 nm is recorded under excitation at a wavelength of 430 nm, and a standard curve of fluorescence intensity at 530 nm versus epinephrine concentration is established.

[0047] In a preferred embodiment, in step 3), the fluorescence intensity at 400 nm and 526 nm under excitation at a wavelength of 350 nm is recorded, and the content of norepinephrine in the sample is quantitatively detected using the standard curve prepared in step 1).

[0048] In a preferred embodiment, in step 3'), the fluorescence intensity at 530 nm under excitation at a wavelength of 430 nm is recorded, and the content of adrenaline in the sample is quantitatively detected using the standard curve prepared in step 1').

[0049] In a preferred embodiment, a microplate reader is used to detect the fluorescence signal.

[0050] In a preferred embodiment, the non-intracellular component is in a free state.

[0051] In a preferred embodiment, the sample is serum.

[0052] In a preferred embodiment, the method is for non-diagnostic purposes and is performed in vitro.

[0053] In a preferred embodiment, the method further includes a step of treating the sample to remove proteins therein before step 1) or step 1').

[0054] In a preferred embodiment, the sample treatment to remove proteins therefrom includes precipitating proteins from the sample using an organic solvent.

[0055] In a preferred embodiment, the organic solvent includes, but is not limited to, acetonitrile.

[0056] In a second aspect, the present invention provides compounds represented by Formula I or Formula II:

[0057]

[0058] In Formula I, L1 is selected from: substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S, substituted or unsubstituted C3-C7 cycloalkylene, substituted or unsubstituted C3-C7 heterocyclic group containing one, two or three heteroatoms independently selected from N, O or S;

[0059] L2 is selected from: substituted or unsubstituted C1-C 10 Alkylene, substituted or unsubstituted C1-C containing one, two or three heteroatoms independently selected from N, O or S. 10 Alkylene;

[0060] In Formula II, X is selected from: -O-, -S-, -NR1-;

[0061] R1 is selected from: H, substituted or unsubstituted C1-C6 alkylene groups.

[0062] In a specific embodiment, in Formula I, L1 is selected from: substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C1-C4 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S;

[0063] L2 is selected from: substituted or unsubstituted C1-C8 alkylene groups, substituted or unsubstituted C1-C8 alkylene groups containing 1, 2 or 3 heteroatoms independently selected from N, O or S;

[0064] In Formula II, X is selected from: -O-, -S-, -NH-, with -O- being preferred.

[0065] In a specific implementation, in formula I, L1 is selected from the following group:

[0066] Preferred

[0067] L2 is independently selected from the following group:

[0068] Preferred

[0069] In a specific embodiment, the compound of formula I is selected from:

[0070]

[0071] The compound of formula II is selected from:

[0072] In a specific embodiment, the compound of formula I is:

[0073] The compound of formula II is:

[0074] In a third aspect, the present invention provides the use of the compounds of Formula I or Formula II in the preparation of detection reagents or kits for detecting catecholamines in samples:

[0075]

[0076] In Formula I, L1 is selected from: substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S, substituted or unsubstituted C3-C7 cycloalkylene, substituted or unsubstituted C3-C7 heterocyclic group containing one, two or three heteroatoms independently selected from N, O or S;

[0077] L2 is selected from: substituted or unsubstituted C1-C 10 Alkylene, substituted or unsubstituted C1-C containing one, two or three heteroatoms independently selected from N, O or S. 10 Alkylene;

[0078] In Formula II, X is selected from: -O-, -S-, -NR1-;

[0079] R1 is selected from: H, substituted or unsubstituted C1-C6 alkylene groups.

[0080] In a preferred embodiment, in Formula I, L1 is selected from: substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C1-C4 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S;

[0081] L2 is selected from: substituted or unsubstituted C1-C8 alkylene groups, substituted or unsubstituted C1-C8 alkylene groups containing 1, 2 or 3 heteroatoms independently selected from N, O or S;

[0082] In Formula II, X is selected from: -O-, -S-, -NH-, with -O- being preferred.

[0083] In a preferred embodiment, in formula I, L1 is selected from the following group:

[0084] Preferred

[0085] L2 is independently selected from the following group:

[0086] Preferred

[0087] In a preferred embodiment, the compound of formula I is selected from:

[0088]

[0089] The compound of formula II is selected from:

[0090]

[0091] In a preferred embodiment, the compound of formula I is:

[0092] The compound of formula II is:

[0093] In a preferred embodiment, the catecholamines include, but are not limited to, dopamine (DA), norepinephrine (NE), epinephrine (EP), and combinations thereof;

[0094] Preferably, the catecholamines are selected from norepinephrine (NE), epinephrine (EP), and combinations thereof.

[0095] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0096] Figure 1 The emission spectra of the fluorescent probe PBN-5 reacting with different concentrations (2μM, 4μM, 8μM, 15μM, 20μM, 30μM, 40μM, 50μM) of norepinephrine in PBS buffer (10mM, pH=7.4);

[0097] Figure 2 The standard curve for detecting norepinephrine using the fluorescent probe PBN-5 in PBS buffer (10 mM, pH = 7.4) is shown.

[0098] Figure 3 To investigate the selectivity of the fluorescent probe PBN-5 for detecting norepinephrine, the selectivity of the probe for catecholamine homologs, catecholamine metabolites, neurotransmitters, and various amino acids was examined.

[0099] Figure 4 The emission spectra of the fluorescent probe PCA-Fu reacting with different concentrations (5 μM, 10 μM, 20 μM, 50 μM, 75 μM, 0.1 mM, 0.13 mM, 0.16 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, 0.8 mM) of adrenaline in PBS buffer (10 mM, pH = 7.4).

[0100] Figure 5 The standard curve for detecting adrenaline using the fluorescent probe PCA-Fu in PBS buffer (10 mM, pH = 7.4);

[0101] Figure 6 , 7 To investigate the selectivity of the fluorescent probe PCA-Fu for detecting adrenaline, the selectivity of the probe for catecholamine homologs, catecholamine metabolites, neurotransmitters, and various amino acids was examined.

[0102] Figure 8 This is a graph showing the cytotoxicity assay of the norepinephrine fluorescent probe PBN-5.

[0103] Figure 9 Fluorescence imaging for detecting endogenous norepinephrine in cells using the fluorescent probe PBN-5;

[0104] Figure 10 Fluorescence imaging for detecting exogenous norepinephrine in cells using the fluorescent probe PBN-5;

[0105] Figure 11 This is a graph showing the cytotoxicity assay of the epinephrine fluorescent probe PCA-Fu.

[0106] Figure 12 Fluorescence imaging for detecting exogenous adrenaline in cells using the fluorescent probe PCA-Fu;

[0107] Figure 13 Fluorescence imaging for detecting endogenous adrenaline in cells using the fluorescent probe PCA-Fu;

[0108] Figure 14 A standard curve for detecting norepinephrine using the fluorescent probe PBN-5 in PBS buffer (10 mM, pH = 8.5, 50% DMSO);

[0109] Figure 15, 16 An experiment to detect serum norepinephrine levels in normal and hypertensive rats using the fluorescent probe PBN-5;

[0110] Figure 17 A standard curve for detecting adrenaline using the fluorescent probe PCA-Fu in PBS buffer (10 mM, pH = 10, 30% DMSO);

[0111] Figure 18 , 19 An experiment using the fluorescent probe PCA-Fu to detect adrenaline in the serum of normal and hypertensive rats. Detailed Implementation

[0112] Through extensive and in-depth research, the inventors unexpectedly discovered a series of novel fluorescent compounds capable of detecting norepinephrine and epinephrine. These compounds can be applied to fluorescence imaging and biosensing, and possess advantages such as high sensitivity and high selectivity. Based on this, the present invention was completed.

[0113] The fluorescent compounds or fluorescent probes of the present invention and their applications

[0114] The terms "probe of the present invention," "compound of the present invention," or "fluorescent compound of the present invention" used in this article have the same meaning. They all refer to compounds that can produce fluorescence of a certain wavelength when excited by light of a certain wavelength. At the same time, such compounds can specifically bind to norepinephrine and epinephrine, thereby enabling the detection of norepinephrine and epinephrine with high sensitivity, high selectivity, and low cost.

[0115] In specific embodiments, the fluorescent compound of the present invention is a compound of formula I or formula II:

[0116]

[0117] In the formula, L1, L2 and X are as described above.

[0118] In the compounds of this invention, the compound represented by Formula I is a fluorescent probe that specifically binds to norepinephrine. This norepinephrine fluorescent probe is a dual-site ratiometric fluorescent probe, combining a cyclization recognition strategy and a ratiometric signal to achieve highly selective quantitative detection of norepinephrine. As the norepinephrine concentration increases, the fluorescence emission peak of the probe decreases at 400 nm and increases at 526 nm. This ratiometric signal has a self-calibrating function, providing higher accuracy for quantitative analysis. Compared with other probes, the norepinephrine fluorescent probe of this invention has excellent anti-interference ability, good biocompatibility, fast response speed, and high stability, offering significant advantages in fluorescence imaging and biosensing.

[0119] In a specific embodiment, the compound of formula I is selected from the following compounds:

[0120]

[0121] Preferred

[0122] In the compounds of this invention, the compound represented by Formula II is a fluorescent probe that specifically binds to adrenaline. The adrenaline fluorescent probe is a low-background-on fluorescent probe, which specifically recognizes adrenaline through a cascade nucleophilic-cyclization reaction. As the adrenaline concentration increases, the fluorescence emission peak at 530 nm of the probe increases. Compared with other probes, the adrenaline fluorescent probe of this invention exhibits excellent sensitivity and anti-interference ability, good biocompatibility, and high stability, providing significant advantages in fluorescence imaging and biosensing.

[0123] In a specific embodiment, the compound of formula II is selected from the following compounds:

[0124]

[0125] Preferred

[0126] Method of using the compound of the present invention

[0127] Based on the fact that the compounds of this invention can specifically bind to catecholamines such as norepinephrine or epinephrine, this invention provides a method for detecting catecholamines in a sample, comprising contacting the sample to be tested with the compounds of this invention to detect the presence and / or content of catecholamines in the sample.

[0128] The method of the present invention can perform fluorescence imaging of intracellular norepinephrine or epinephrine. The method includes co-incubating cells with a compound of Formula I; observing the gradual increase in fluorescence intensity of the 526nm channel using a confocal microscope at an excitation wavelength of 405nm, thereby achieving imaging analysis of changes in intracellular norepinephrine levels; or...

[0129] Cells were co-incubated with the compound shown in Formula II; fluorescence intensity at 530 nm was observed using 488 nm as the excitation wavelength, thereby enabling imaging analysis of changes in intracellular adrenaline levels.

[0130] In a specific implementation, the method includes the following steps: co-incubating cultured cells with the norepinephrine fluorescent probe, observing them using a confocal microscope, and exciting them with a 405nm excitation wavelength. As the norepinephrine level increases, the fluorescence intensity of the 526nm channel gradually increases, thereby achieving imaging analysis of changes in intracellular norepinephrine; co-incubating cultured cells with the adrenaline fluorescent probe, observing them using a confocal microscope, and exciting them with a 488nm excitation wavelength. As the adrenaline level increases, the fluorescence intensity of the 530nm channel gradually increases, thereby achieving imaging analysis of changes in intracellular adrenaline.

[0131] Those skilled in the art can appropriately adjust the incubation time between the cells and the norepinephrine probe. The incubation time is typically 0.5-2 hours; preferably 0.5 hours. Those skilled in the art can also appropriately adjust the incubation time between the cells and the epinephrine probe. The incubation time is typically 0.5-2 hours; preferably 1 hour. The incubation temperature between the cells and the norepinephrine probe or the epinephrine probe is typically 37°C.

[0132] The method of the present invention can also be used to quantitatively detect non-intracellular norepinephrine or epinephrine in a sample, including preparing a standard curve for detecting norepinephrine using the compound shown in Formula I and a standard of norepinephrine; adding the compound shown in Formula I to the sample; and quantitatively detecting the content of norepinephrine in the sample.

[0133] or,

[0134] A standard curve for detecting norepinephrine was prepared using the compound shown in Formula II and a standard of adrenaline; the compound shown in Formula II was added to the sample; and the content of adrenaline in the sample was quantitatively detected.

[0135] Those skilled in the art know how to prepare standard curves. For example, a solution of a compound of formula I at a determined concentration is mixed with solutions of norepinephrine at different concentrations, and the fluorescence intensity at 400 nm and 526 nm is recorded under excitation at a wavelength of 350 nm, and a standard curve is established between the ratio of fluorescence intensity at 526 nm to 400 nm and the concentration of norepinephrine; or, a solution of a compound of formula II at a determined concentration is mixed with solutions of epinephrine at different concentrations, and the fluorescence intensity at 530 nm is recorded under excitation at a wavelength of 430 nm, and a standard curve is established between the fluorescence intensity at 530 nm and the concentration of epinephrine.

[0136] Accordingly, in actual testing, the fluorescence intensity at 400 nm and 526 nm under excitation at a wavelength of 350 nm is recorded, and the content of norepinephrine in the sample is quantitatively detected using the prepared standard curve; or, the fluorescence intensity at 530 nm under excitation at a wavelength of 430 nm is recorded, and the content of norepinephrine in the sample is quantitatively detected using the prepared standard curve.

[0137] To avoid the influence of proteins in the sample, such as serum, on the probe signal, it is preferable to include a step of treating the sample to remove the proteins therein. For example, proteins in the sample are precipitated using an organic solvent. The organic solvent includes, but is not limited to, acetonitrile.

[0138] In a specific implementation, the method includes the following steps: First, an equal volume of acetonitrile is added to the serum to precipitate the protein, and the mixture is vortexed using a vortex mixer. Then, the mixture is placed in a centrifuge at 4°C and centrifuged at 14,000 rpm for 30 minutes. The supernatant is then collected and lyophilized to avoid the proteins in the serum affecting the probe signal and to reduce the serum autofluorescence.

[0139] Secondly, for the detection of norepinephrine, the lyophilized serum was dissolved in the detection system, the norepinephrine probe was added, and the mixture was stirred to allow it to react fully.

[0140] Finally, the reaction solution was added to a 96-well plate, and the fluorescence signals at 400 nm and 526 nm were detected using a microplate reader with an excitation wavelength of 350 nm.

[0141] For the detection of adrenaline, the serum was dissolved and lyophilized using the detection system, the adrenaline probe was added, the mixture was stirred to allow it to react fully, the reaction solution was added to a 96-well plate, and the fluorescence signal at 530 nm was detected using an ELISA reader with an excitation wavelength of 430 nm.

[0142] For the detection of norepinephrine, the detection system is a phosphate buffer containing 50% DMSO; preferably, the concentration of the phosphate buffer is 10 mM and the pH is 8.5. For the detection of adrenaline, the detection system is a phosphate buffer containing 30% DMSO; preferably, the concentration of the phosphate buffer is 10 mM and the pH is 10.

[0143] The reaction temperature is 25-37℃; preferably, it is 37℃.

[0144] For the detection of norepinephrine, the reaction time is 10-25 min; preferably, it is 15 min. For the detection of adrenaline, the reaction time is 10-25 min; preferably, it is 20 min.

[0145] The linear range for detecting norepinephrine is 0-40 nM, and the limit of detection is 1.8 nM; the linear range for detecting epinephrine is 0-36 nM, and the limit of detection is 1.5 nM.

[0146] Advantages or beneficial technical effects of the present invention:

[0147] This invention synthesizes two small-molecule fluorescent probes with high selectivity for norepinephrine and epinephrine, respectively. The norepinephrine fluorescent probe specifically binds to norepinephrine, achieving a dual-channel ratiometric fluorescence change. This ratiometric signal has a self-calibrating function, providing higher accuracy for quantitative analysis. The epinephrine fluorescent probe specifically binds to epinephrine, achieving a low-background-on fluorescence change. The fluorescent probes of this invention can achieve quantitative detection of norepinephrine and epinephrine in serum, as well as fluorescence imaging of norepinephrine and epinephrine in cells. They have broad application prospects in cell imaging and biosensing, enriching the detection methods for catecholamines.

[0148] The technical solution of the present invention is further described below with reference to specific implementation examples. However, the following implementation examples do not constitute a limitation on the present invention. All application methods based on the principles and technical means of the present invention are within the scope of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are by weight.

[0149] Example 1. Synthesis and characterization of the norepinephrine fluorescent probe PBN-5

[0150] The structural formula of compound PBN-5 is as follows:

[0151]

[0152] The synthetic route for PBN-5 is as follows:

[0153]

[0154] Synthesis and characterization of compound A: (S)-1,1'-bis-2-naphthol (2.00 g, 7.00 mmol) and diisopropylethylamine (2.43 mL, 14.70 mmol) were dissolved in 20 mL of tetrahydrofuran. A solution of bromomethyl methyl ether (0.96 g, 7.70 mmol) in tetrahydrofuran was slowly added dropwise to the mixture at 0 °C. After the reaction was complete, the solvent was removed by vacuum distillation, and the product was purified by column chromatography (PE / EA = 4:1) to give compound A as a white solid, 1.80 g, in yield of 72%.

[0155] 1H NMR (400MHz, DMSO-d6) δ9.39(s,1H),8.02(d,J=9.2Hz,1H),7.94(d,J=8.0Hz,1H),7.87(t,J=8.2Hz,2H),7.60(d,J=9.2Hz,1H),7.37-7 .33(m,2H),7.24(t,J=7.2Hz,2H),7.17(t,J=7.2Hz,1H),7.01(d,J=8.8Hz,1H),6.89(d,J=8.4Hz,1H),5.15-5.01(m,2H),3.10(s,3H).

[0156] Synthesis and characterization of compound B: Compound A (1.00 g, 3.03 mmol) was dissolved in 20 mL of anhydrous tetrahydrofuran and cooled to -78 °C under nitrogen protection. Then, n-butyllithium (2.54 mL, 6.06 mmol, 2.5 M n-hexane solution) was slowly added dropwise using a syringe over 30 min. After the addition was complete, the reaction was stirred at room temperature for 1 h, then cooled to -78 °C and anhydrous DMF was slowly added dropwise. After the addition was complete, the reaction mixture was stirred at room temperature for another 12 h. After the reaction was complete, the reaction was quenched with saturated ammonium chloride solution and extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The product was purified by column chromatography (PE / EA = 20:1) to give compound B as a white solid, 0.71 g, in 65% yield.

[0157] 1 H NMR(600MHz,DMSO-d6)δ10.47(s,1H),9.74(s,1H),8.58(s,1H),8.23(d,J=8.4Hz,1H), 7.96(d,J=9.0Hz,1H),7.89(d,J=7.8Hz,1H),7.53(t,J=7.2Hz,1H),7.45(t,J=7.2Hz,1 H),7.39(d,J=8.4Hz,1H),7.28(t,J=7.2Hz,1H),7.23(t,J=7.2Hz,1H),7.13(d,J=9.0H z,1H),6.92(d,J=8.4Hz,1H),4.82(d,J=5.4Hz,1H),4.65(d,J=6.0Hz,1H),2.97(s,3H).

[0158] Synthesis and characterization of compound Nap-Br: 1,8-Naphthalenedicarboxylic anhydride (5.00 g, 25.23 mmol) was dissolved in 20 mL of concentrated sulfuric acid. Then, N-bromosuccinimide (5.39 g, 30.28 mmol) was added in portions with stirring at room temperature. The mixture was stirred at 60 °C for 4 h. After the reaction was complete, the reaction solution was slowly quenched dropwise in a large amount of ice water, and the pH was adjusted to neutral. The white solid was collected by filtration. The crude product was purified by column chromatography (DCM / MeOH = 200:1) to give 3.78 g of compound Nap-Br as a white solid, with a yield of 54%.

[0159] 1 H NMR (400MHz, Chloroform-d) δ8.69 (s, 1H), 8.64 (d, J = 7.4Hz, 1H), 8.48 (s, 1H), 8.25 (d, J = 8.4Hz, 1H), 7.86 (t, J = 8.0Hz, 1H).

[0160] Synthesis and characterization of compound 5a: 3-bromo-1,8-naphthalenedicarboxylic anhydride (2.00 g, 7.22 mmol) was dissolved in 20 mL of ethanol, followed by the addition of 3-[2-(2-aminoethoxy)ethoxy]propionic acid (1.28 g, 10.83 mmol), and the mixture was heated under reflux for 4 h. After the reaction was complete, the solvent was removed by vacuum distillation and the product was purified by column chromatography (DCM / MeOH = 40:1) to give compound 5a as a pale yellow oil, 2.74 g, in yield of 87%.

[0161] 1 H NMR (400MHz, DMSO-d6) δ12.13(s,1H),8.68(d,J=2.0Hz,1H),8.44(dd,J=7.2,1.2Hz,1H),8.39–8.31(m,2H),7.87(t,J =8.4Hz,1H),4.20(t,J=6.4Hz,2H),3.65(t,J=6.4Hz,2H),3.59–3.52(m,4H),3.47–3.45(m,2H),2.35(t,J=6.4Hz,2H).

[0162] Synthesis and characterization of compound 5b: Compound 5a (2.00 g, 4.58 mmol), 2,6-bis(bromomethyl)pyridine (1.22 g, 4.58 mmol), and potassium carbonate (0.63 g, 4.58 mmol) were dissolved in 25 mL of DMF and stirred at room temperature for 4 h. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The product was then purified by column chromatography (PE / EA = 5:1) to give 2.24 g of compound 5b as a pale yellow oil, with a yield of 79%.

[0163] 1 H NMR(400MHz,Chloroform-d)δ8.63(d,J=1.6Hz,1H),8.57(d,J=7.2Hz,1H),8.36(d,J= 1.6Hz,1H),8.12(d,J=8.0Hz,1H),7.77(t,J=7.6Hz,2H),7.41(d,J=7.6Hz,1H),7.32( d,J=8.0Hz,1H),5.26(s,2H),4.59(s,2H),4.41(t,J=6.0Hz,2H),3.81(t,J=6.0Hz,2H ),3.74(t,J=6.4Hz,2H),3.68–3.66(m,2H),3.60–3.58(m,2H),2.62(t,J=6.4Hz,2H).

[0164] Synthesis and characterization of compound 5c: Compound 5b (1 g, 1.61 mmol), compound B (0.58 g, 1.61 mmol), and potassium carbonate (0.22 g, 1.61 mmol) were dissolved in 15 mL of DMF and stirred at 50 °C for 2 h. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The product was then purified by column chromatography (PE / EA = 5:1) to give 1.03 g of compound 5c as a pale yellow solid, with a yield of 71%.

[0165] 1H NMR (400MHz, Chloroform-d) δ10.58(s,1H),8.61(s,1H),8.59(s,1H),8.55(d,J=7.2Hz,1H),8.33(s,1H),8.07(t,J=8.8Hz,2H),8.00(d, J=8.8Hz,1H),7.89(d,J=8.0Hz,1H),7.74(d,J=9.2Hz,1H),7.48–7.45(m,2H),7.37–7.35(m,2H),7.32(d,J=9.2Hz,2H),7.22(t,J=9.2Hz, 2H),7.12(d,J=7.6Hz,1H),6.66(d,J=7.6Hz,1H),5.28–5.19(m,2H),5.14(s,2H),4.73(d,J=5.2Hz,1H),4.65(d,J=5.2Hz,1H),4.40(t,J= 6.4Hz,2H),3.80(t,J=5.2Hz,2H),3.72(t,J=5.6Hz,2H),3.66(t,J=3.6Hz,2H),3.58(t,J=4.0Hz,2H),2.95(s,1H),2.60(t,J=5.6Hz,2H).

[0166] Synthesis and characterization of compound 5d: Compound 5c (1.00 g, 1.11 mmol), pinacol diboronate (0.56 g, 2.21 mmol), potassium acetate (0.46 g, 3.32 mmol), and PdCl(dppf) (0.07 g, 0.09 mmol) were dissolved in 20 mL of anhydrous 1,4-dioxane and stirred at 85 °C for 6 h under nitrogen protection. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The product was then purified by column chromatography (PE / EA = 2:1) to give compound 5d as a yellow solid, 0.60 g, in a yield of 63%.

[0167] 1H NMR(400MHz,DMSO-d6)δ10.42(s,1H),8.77(s,1H),8.63–8.61(m,2H),8.52( dd,J=16.4,8.4Hz,2H),8.24(d,J=8.4Hz,1H),8.11(d,J=9.1Hz,1H),7.96(d, J=8.4Hz,1H),7.84(t,J=7.6Hz,1H),7.64(d,J=9.2Hz,1H),7.52(q,J=6.4Hz, 2H),7.44(t,J=8.0Hz,1H),7.37(t,J=7.6Hz,1H),7.30(t,J=8.0Hz,1H),7.15 (d,J=7.6Hz,1H),7.10(d,J=8.4Hz,1H),7.01(d,J=8.5Hz,1H),6.74(d,J=7.6 Hz,1H),5.23(s,2H),5.00(s,2H),4.77(d,J=6.0Hz,1H),4.66(d,J=6.0Hz,1H ),4.22(t,J=6.4Hz,2H),3.64(t,J=6.4Hz,2H),3.57(t,J=6.0Hz,2H),3.53–3 .51(m,2H),3.46–3.43(m,2H),2.80(s,3H),2.50–2.48(m,2H),1.35(s,12H).

[0168] Synthesis and characterization of probe PBN-5: Compound 5d (0.20 g, 0.21 mmol) and sodium periodate (0.90 g, 0.42 mmol) were dissolved in a mixture of 10 mL tetrahydrofuran and 2 mL ultrapure water. Then, 2 mL of 1 N hydrochloric acid was slowly added dropwise, and the mixture was stirred at room temperature for 2 h. After the reaction was complete, the mixture was extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The product was then purified by column chromatography (DCM / MeOH = 100:1) to obtain 0.12 g of compound PBN-5 as a yellow solid, with a yield of 69%.

[0169] 1H NMR (400MHz, DMSO-d6) δ10.33(s,1H),10.22(s,1H),8.91(s,1H),8.83(s,1H),8.64(s,1H),8.58(s,2H),8.47(d,J=6.8Hz,1H),8.44(d,J=8.0Hz, 1H),8.14–8.11(m,1H),8.05(d,J=9.2Hz,1H),7.95(d,J=8.4Hz,1H),7.8 1(t,J=7.6Hz,1H),7.58(d,J=9.2Hz,1H),7.52(t,J=8.0Hz,1H),7.43–7.3 8(m,2H),7.36(t,J=7.6Hz,1H),7.28(t,J=7.2Hz,1H),7.16(d,J=7.6Hz, 1H),7.03(d,J=8.4Hz,1H),6.99–6.96(m,1H),6.75(d,J=8.0Hz,1H),5.21 (s,2H),5.02(s,2H),4.24(t,J=6.0Hz,2H),3.65(t,J=6.4Hz,2H),3.58( t,J=6.0Hz,2H),3.54–3.52(m,2H),3.46–3.44(m,2H),2.50–2.48(m,2H).

[0170] Example 2. Synthesis and characterization of the epinephrine fluorescent probe PCA-Fu

[0171] The structural formula of the compound is as follows:

[0172]

[0173] The synthesis route is as follows:

[0174]

[0175] Synthesis of compound Br-ID: 0.68 g (3.00 mmol) of 3-bromophthalic anhydride was weighed and added to a 50 mL two-necked flask. 0.73 mL of acetic anhydride and 0.88 mL of triethylamine were added. Under nitrogen protection, 0.73 mL (4.41 mmol) of ethyl acetoacetate was added dropwise using a syringe. The reaction was carried out at room temperature for 12 h, followed by reflux with 20 mL of 5N hydrochloric acid for 1 h. After the reaction was complete, the mixture was cooled to room temperature, extracted with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation to obtain compound Br-ID.

[0176] Synthesis of compound Fu-ID: Compound Br-ID (0.50 g, 2.22 mmol), furan-2-boronic acid (0.34 g, 2.67 mmol), and tetraphenylphosphine palladium (0.03 g, 0.02 mmol) were weighed and added to a 250 mL double-necked flask. 50 mL of tetrahydrofuran and 30 mL of 2 M sodium carbonate solution were added. Under nitrogen protection, the mixture was reacted at 80 °C for 3 h. After the reaction was complete, tetrahydrofuran was removed by rotary evaporation. The mixture was extracted with dichloromethane, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by rotary evaporation to obtain compound Fu-ID.

[0177] Synthesis and characterization of probe PCA-Fu: Compound Fu-ID (2.0 g, 9.43 mmol) was dissolved in 20 mL of anhydrous DMSO. Triethylamine (2.61 mL, 18.85 mmol) was added under nitrogen protection, followed by dropwise addition of carbon disulfide (0.56 mL, 9.40 mmol). The mixture was stirred at room temperature for 2 h. Subsequently, iodomethane (1.17 mL, 18.82 mmol) was added dropwise at 0 °C, and the reaction was continued at room temperature for 4 h. After the reaction was completed, the mixture was quenched with ice water. The reaction solution was extracted with ethyl acetate. The organic phase was washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, and the solvent was removed by vacuum distillation. The product was purified by column chromatography (PE / EA = 20:1) to obtain compound PCA-Fu as a yellow solid, 0.27 g, with a yield of 9%.

[0178] 1 H NMR(400MHz,Chloroform-d)δ8.05(d,J=1.6Hz,1H),7.94(dd,J=7.9,1.6Hz,1H),7.82(d,J=7.9 Hz,1H),7.53(d,J=1.8Hz,1H),6.86(d,J=3.5Hz,1H),6.52(dd,J=3.5,1.8Hz,1H),2.63(s,6H).

[0179] Example 3. Performance test of fluorescent probe PBN-5 in detecting norepinephrine

[0180] (1) Fluorescence emission spectroscopy determination of the reaction between probe PBN-5 and different concentrations of norepinephrine

[0181] Take an appropriate amount of the probe prepared in Example 1 of this invention and dissolve it in DMSO to prepare a stock solution with a concentration of 10 mM. Take 3 μL of the probe stock solution and add it to 3 mL of PBS buffer (10 mM, pH = 7.4) to make the final probe concentration 10 μM. Take different volumes of norepinephrine stock solution and add them to the above buffer to make the final norepinephrine concentrations 2 μM, 4 μM, 8 μM, 15 μM, 20 μM, 30 μM, 40 μM, and 50 μM, respectively. Place the mixture at 37 °C and stir to react. Record the fluorescence spectra of the probe response to different concentrations of norepinephrine under 350 nm excitation. Each concentration gradient is repeated three times, and the mean and standard deviation are calculated.

[0182] As the concentration of added norepinephrine increased, the fluorescence intensity at 526 nm increased, accompanied by a decrease in fluorescence intensity at 400 nm. Figure 1 A standard curve was established by extracting the fluorescence intensity ratio at 526 nm and 400 nm and norepinephrine concentration, and linear fitting was performed. The curve showed a good linear relationship in the range of 0-50 μM, and the linear regression equation was F1. 526 / F 400 =0.0957×[NE](μM)+0.0383, R 2 =0.998, detection limit as low as 8.5 nM ( Figure 2 ).

[0183] (2) Selectivity test of probe PBN-5

[0184] Take an appropriate amount of the probe prepared in Example 1 of this invention and dissolve it in DMSO to prepare a stock solution with a concentration of 10 mM. Take 3 μL of the probe stock solution and add it to 3 mL of PBS buffer (10 mM, pH = 7.4) to make the final probe concentration 10 μM. Add the analytes norepinephrine, catecholamine homologs (DA, EP), catecholamine metabolites (HVA, MHPG, VMA), neurotransmitters (GABA, 5-HT), and solutions of various amino acids (Lys, Cys, Ser, Thr, Glu, Asp, Pro, Ala) to the buffer solution to make the final concentration of each analyte 50 μM. Place the mixture in a 37°C environment and stir to react. Record the fluorescence intensity of the probe at 400 nm and 526 nm under 350 nm excitation.

[0185] like Figure 3 As shown, the ratio of the fluorescence intensities of the two channels (F) 526 / F 400 It exhibits high selectivity for norepinephrine and is virtually unaffected by other analytes.

[0186] The above experimental results fully demonstrate that the fluorescent probe PBN-5 prepared in this invention has high sensitivity and selectivity, and can be used for the effective detection of norepinephrine.

[0187] Example 4. Performance test of the fluorescent probe PCA-Fu for detecting adrenaline

[0188] (1) Fluorescence emission spectroscopy determination of the reaction between probe PCA-Fu and different concentrations of adrenaline

[0189] Take an appropriate amount of the probe prepared in Example 2 of this invention and dissolve it in DMSO to prepare a stock solution with a concentration of 10 mM. Take 3 μL of the probe stock solution and add it to 3 mL of PBS buffer (10 mM, pH = 7.4) to make the final probe concentration 10 μM. Take different volumes of adrenaline stock solution and add them to the above PBS buffer to make the final adrenaline concentrations 5 μM, 10 μM, 20 μM, 50 μM, 75 μM, 0.1 mM, 0.13 mM, 0.16 mM, 0.2 mM, 0.3 mM, 0.4 mM, 0.5 mM, 0.6 mM, and 0.8 mM, respectively. Place the mixture at 37 °C and stir to react. Record the fluorescence spectra of the probe response to different concentrations of adrenaline at an excitation wavelength of 430 nm. Extract the fluorescence intensity at 530 nm and establish a standard curve with adrenaline concentration and perform linear fitting. Each concentration gradient is repeated three times, and the mean and standard deviation are calculated.

[0190] As the concentration of added epinephrine standard increased, the probe exhibited a new fluorescence emission peak at 530 nm, and the signal intensity gradually increased with increasing epinephrine concentration. When the epinephrine concentration reached 0.8 mM, the fluorescence intensity increased by approximately 273 times, indicating that the probe possesses excellent low-background fluorescence-on detection performance. Figure 4 Meanwhile, the fluorescence intensity of the probe at 530 nm showed a positive correlation with adrenaline concentration, and exhibited a good linear relationship within the adrenaline concentration range of 0-160 μM. The linear regression equation was F = 1.50391 × 10 × [EP] (mM) + 186767.1, R0 2 =0.980, detection limit as low as 3.2 nM ( Figure 5 ).

[0191] (2) Selectivity experiment of probe PCA-Fu

[0192] Take an appropriate amount of the probe prepared in Example 2 of this invention and dissolve it in DMSO to prepare a stock solution with a concentration of 10 mM. Take 3 μL of the probe stock solution and add it to 3 mL of PBS buffer (10 mM, pH = 7.4) to make the final probe concentration 10 μM. Add the stock solutions of the analytes adrenaline, catecholamine homologs (DA, NE), catecholamine metabolites (HVA, MHPG, VMA), neurotransmitters (GABA, 5-HT), and various amino acids (Ala, Asn, Cys, Gln, Leu, Pro, Thr, Tyr, Ser, Lys, Met, Phe, Glu, Trp) to the buffer solution to make the final concentration of each analyte 500 μM. Place the mixture at 37 °C and stir to react. Record the fluorescence spectrum of the probe under 430 nm excitation.

[0193] like Figure 6 , Figure 7 As shown, the probe exhibits a distinct fluorescence emission peak at 530 nm only after the addition of adrenaline. The fluorescence signal at this point is almost unaffected by the other analytes, indicating that the probe has high specificity for the recognition of adrenaline.

[0194] The above experimental results fully demonstrate that the fluorescent probe PCA-Fu prepared in this invention can be used for the effective detection of adrenaline.

[0195] Example 5. Detection of norepinephrine in cells using fluorescent probe PBN-5

[0196] (1) Cytotoxicity test of probe PBN-5

[0197] To evaluate the biotoxicity of the fluorescent probe PBN-5 prepared in Example 1 of this invention, CCK8 assays were performed in HepG2 liver cancer cells, 4T1 breast cancer cells, and PC12 pheochromocytoma cells. The three cell types were incubated with different concentrations (2-20 μM) of the probe for 24 h, and cell viability was evaluated after incubation with CCK8 reagent for 2 h.

[0198] like Figure 8 As shown, the survival rate of all three cell types remained above 85%, indicating that the probe PBN-5 has low cytotoxicity and is suitable for the detection of norepinephrine in biological samples.

[0199] (2) Detection of endogenous norepinephrine in cells using probe PBN-5

[0200] To evaluate the ability of the fluorescent probe PBN-5 prepared in Example 1 of this invention to detect endogenous norepinephrine in cells, HepG2, 4T1 and PC12 cells were incubated with the probe (10 μM) for 30 min and then washed off, followed by confocal fluorescence imaging.

[0201] like Figure 9 As shown, HepG2 and 4T1 cells showed no obvious fluorescence signal due to the absence of endogenous norepinephrine. PC12 cells, however, exhibited a significant green fluorescence signal due to the presence of abundant endogenous norepinephrine. These results demonstrate that the fluorescent probe PBN-5 prepared in this invention can be used for fluorescence imaging of endogenous norepinephrine in PC12 cells.

[0202] (3) Detection of exogenous norepinephrine in cells using probe PBN-5

[0203] To evaluate the ability of the fluorescent probe PBN-5 prepared in Example 1 of this invention to detect exogenous norepinephrine in cells, PC12 cells were incubated with different concentrations of norepinephrine (0, 100 μM, 500 μM, 1 mM) for 30 min, washed off, and then incubated with probe PBN-5 for another 30 min, followed by confocal fluorescence imaging.

[0204] like Figure 10 As shown, the intensity of green fluorescence in cells is positively correlated with the concentration of exogenous norepinephrine during incubation, indicating that the probe PBN-5 can effectively detect the level of exogenous norepinephrine in cells.

[0205] The above experimental results fully demonstrate that the fluorescent probe PBN-5 prepared in this invention can be used for the effective detection of endogenous and exogenous norepinephrine in cells.

[0206] Example 6. Detection of adrenaline in cells using the fluorescent probe PCA-Fu

[0207] (1) Cytotoxicity test of probe PCA-Fu

[0208] To evaluate the biotoxicity of the fluorescent probe PCA-Fu prepared in Example 2 of this invention, CCK8 assays were performed in cervical cancer cells (HeLa), breast cancer cells (4T1), and pheochromocytoma cells (PC12). The three cell types were incubated with different concentrations (2-20 μM) of the probe for 12 h, and then incubated with CCK8 reagent for 2 h before cell viability was evaluated.

[0209] like Figure 11 As shown, the survival rate of all three cell types remained above 80%, indicating that the probe has low cytotoxicity and is suitable for the detection of adrenaline in biological samples.

[0210] (1) Detection of exogenous adrenaline in cells using probe PCA-Fu

[0211] To evaluate the ability of the fluorescent probe PCA-Fu prepared in Example 2 of this invention to detect exogenous adrenaline in cells, PC12 cells were incubated with different concentrations of adrenaline (0, 5 μM, 10 μM, 25 μM, 50 μM, 100 μM) for 30 min, washed off, and then incubated with probe PCA-Fu for another 30 min, followed by confocal fluorescence imaging.

[0212] like Figure 12 As shown, the intensity of green fluorescence in cells is positively correlated with the concentration of exogenous adrenaline during incubation, indicating that the probe PCA-Fu can effectively detect the level of exogenous adrenaline in cells.

[0213] (2) Detection of endogenous adrenaline in cells using probe PCA-Fu

[0214] To evaluate the ability of the fluorescent probe PCA-Fu prepared in Example 2 of this invention to detect endogenous adrenaline in cells, PC12 cells were treated with dexamethasone (1 μM) for 2 days to induce endogenous adrenaline secretion. Subsequently, the cells were incubated with the probe (10 μM) for 30 min, washed off, and subjected to confocal fluorescence imaging. HepG2 cells, 4T1 cells, and untreated PC12 cells were used as control groups.

[0215] like Figure 13 As shown, only dexamethasone-induced PC12 cells showed obvious green fluorescence signals, while HepG2 cells, 4T1 cells and PC12 cells without dexamethasone treatment showed almost no green fluorescence. This indicates that the probe PCA-Fu can reflect changes in endogenous adrenaline levels in cells.

[0216] The above experimental results fully demonstrate that the fluorescent probe PCA-Fu prepared in this invention can be used for the effective detection of endogenous and exogenous adrenaline in cells.

[0217] Example 7. Detection of norepinephrine in rat serum using fluorescent probe PBN-5

[0218] (1) Determination of the standard curve for the detection of norepinephrine using probe PBN-5 in a serum detection system

[0219] Add 1 μL of the probe stock solution prepared in Example 1 of this invention to 100 μL of PBS buffer (10 mM, pH = 8.5, 50% DMSO) to make the final probe concentration 10 μM. Add different volumes of norepinephrine stock solution to the above buffer to make the final norepinephrine concentrations 2 nM, 4 nM, 8 nM, 12 nM, 18 nM, 24 nM, 30 nM, and 40 nM, respectively. After stirring the mixture at 37°C for 15 min, transfer it to a 96-well plate. Use a microplate reader to record the fluorescence intensity of the probe at 400 nm and 526 nm under 350 nm excitation and establish a standard curve with the norepinephrine concentration. Each concentration gradient is repeated ten times, and the mean and standard deviation are calculated.

[0220] like Figure 14 As shown, the ratio of the fluorescence intensity of the probe at 526 nm to 400 nm showed a good linear correlation with the norepinephrine concentration (0-40 nM), and the linear equation was F. 526 / F 400 =0.04732×[NE](nM)+0.4142,R 2 =0.981, with a detection limit as low as 1.8 nM, indicating that the probe PBN-5 is highly sensitive to the extremely low actual concentrations of norepinephrine in serum.

[0221] (2) Determination of serum norepinephrine concentration in normal rats and hypertensive rats

[0222] Blood was collected from 10 normal rats and 10 hypertensive rats via the abdominal aorta. Serum was obtained by centrifugation (3000 rpm, 15 min, 4℃). An equal volume of acetonitrile was added to the serum, vortexed for 30 s, and incubated at -20℃ for 30 min to precipitate proteins. The supernatant was then collected by centrifugation (14000 rpm, 30 min, 4℃). The supernatant was lyophilized and rehydrated with PBS buffer (10 mM, pH 8.5, 50% DMSO). After adding the probe, the reaction mixture was stirred at 37℃ for 15 min. The reaction mixture was then transferred to a 96-well plate, and fluorescence signals at 400 nm and 526 nm were recorded using a microplate reader.

[0223] The test results were quantitatively analyzed using the probe standard curve established above. For example... Figure 15 , Figure 16As shown, the serum norepinephrine concentrations in 10 normal rats were 7.47 nM, 7.80 nM, 6.24 nM, 14.17 nM, 15.36 nM, 4.50 nM, 4.24 nM, 6.22 nM, 8.09 nM, and 12.43 nM, respectively. The serum norepinephrine concentrations in 10 hypertensive rats were 19.90 nM, 31.51 nM, 16.49 nM, 9.80 nM, 17.88 nM, 16.89 nM, 12.42 nM, 15.70 nM, 15.05 nM, and 6.21 nM, respectively. Statistical analysis showed that the serum norepinephrine concentration in hypertensive rats was significantly higher than that in normal rats.

[0224] This study demonstrates that the level of norepinephrine in rat serum changes under hypertensive conditions, and the PBN-5 probe described in this invention can effectively detect this change.

[0225] Example 8. Detection of adrenaline in serum of hypertensive rats using the fluorescent probe PCA-Fu

[0226] (1) Determination of the standard curve for the detection of adrenaline by probe PCA-Fu in a serum detection system

[0227] Take 1 μL of the probe stock solution prepared in Example 2 of this invention and add it to 100 μL of PBS buffer (10 mM, pH = 10, 30% DMSO) to make the final probe concentration 10 μM. Take different volumes of epinephrine stock solution and add them to the above buffer to make the final epinephrine concentrations 3 nM, 6 nM, 9 nM, 12 nM, 18 nM, 24 nM, and 36 nM, respectively. Place the mixture in a 37°C environment and stir for 20 min. Then transfer it to a 96-well plate. Use a microplate reader to record the fluorescence intensity of the probe at 530 nm under 430 nm excitation and establish a standard curve with the epinephrine concentration. Each concentration gradient is repeated ten times, and the mean and standard deviation are calculated.

[0228] like Figure 17 As shown, the fluorescence intensity of the probe at 530 nm showed a good linear correlation with the adrenaline concentration (0-36 nM), with the linear equation being F = 41.60 × [EP](nM) + 1073.38, R 2 =0.98, with a detection limit as low as 1.5 nM, indicating that the probe is highly sensitive to the extremely low actual concentrations of adrenaline in serum.

[0229] (2) Determination of serum adrenaline concentration in normal rats and hypertensive rats

[0230] Blood was collected from 10 normal rats and 10 hypertensive rats via abdominal aortic sampling. Serum was obtained by centrifugation (3000 rpm, 15 min, 4℃). An equal volume of acetonitrile was added to the serum, vortexed, and incubated at -20℃ for 30 min to precipitate proteins. The supernatant was then collected by centrifugation (14000 rpm, 30 min, 4℃). The supernatant was lyophilized and reconstituted with PBS buffer (10 mM, pH = 10, 30% DMSO). After adding the probe, the mixture was stirred at 37℃ for 20 min. The reaction mixture was then transferred to a 96-well plate, and the fluorescence signal at 530 nm was recorded using a microplate reader.

[0231] The test results were quantitatively analyzed using the probe standard curve established above. For example... Figure 18 , Figure 19 As shown, the serum adrenaline concentrations in 10 normal rats were 4.80 nM, 3.81 nM, 11.46 nM, 9.15 nM, 6.57 nM, 7.03 nM, 2.86 nM, 7.79 nM, 5.63 nM, and 5.47 nM, respectively. The serum adrenaline concentrations in 10 hypertensive rats were 25.35 nM, 11.85 nM, 23.01 nM, 20.43 nM, 18.12 nM, 13.02 nM, 8.78 nM, 14.72 nM, 10.84 nM, and 20.18 nM, respectively. Statistical analysis showed that the serum adrenaline concentration in hypertensive rats was significantly higher than that in normal rats.

[0232] This study demonstrates that the level of adrenaline in rat serum changes under hypertensive conditions, and the PCA-Fu probe described in this invention can effectively detect this change.

[0233] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A method for detecting catecholamines in a sample, the method comprising the steps of contacting the sample to be tested with the following compounds to detect the presence and / or content of catecholamines in the sample. The compound is the compound shown in Formula I or II: In Formula I, L1 is selected from: substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S, substituted or unsubstituted C3-C7 cycloalkylene, substituted or unsubstituted C3-C7 heterocyclic group containing one, two or three heteroatoms independently selected from N, O or S; L2 is selected from: substituted or unsubstituted C1-C 10 Alkylene, substituted or unsubstituted C1-C containing one, two or three heteroatoms independently selected from N, O or S. 10 Alkylene; In Formula II, X is selected from: -O-, -S-, -NR1-; R1 is selected from: H, substituted or unsubstituted C1-C6 alkylene groups.

2. The method as described in claim 1, characterized in that, The catecholamines include, but are not limited to, dopamine (DA), norepinephrine (NE), epinephrine (EP), and combinations thereof; Preferably, the catecholamines are selected from norepinephrine (NE), epinephrine (EP), and combinations thereof.

3. The method as described in claim 2, characterized in that, The method is a fluorescence imaging technique for intracellular norepinephrine or epinephrine, and the method includes the following steps: 1) Incubate the cells with the compound shown in Formula I; and 2) Using 405nm as the excitation wavelength, the fluorescence intensity of the 526nm channel was observed to gradually increase through confocal microscopy, thereby achieving imaging analysis of changes in intracellular norepinephrine levels; or, 1') Incubate the cells with the compound shown in Formula II; and 2') Using 488nm as the excitation wavelength, the fluorescence intensity at 530nm was observed, thereby enabling imaging analysis of changes in intracellular adrenaline levels.

4. The method as described in claim 2, characterized in that, The method is a method for quantitatively detecting non-intracellular norepinephrine or epinephrine in a sample, comprising the following steps: 1) A standard curve for detecting norepinephrine was prepared using the compound shown in Formula I and a standard of norepinephrine; 2) Add the compound shown in Formula I to the sample; and 3) Quantitatively detect the content of norepinephrine in the sample; or, 1') A standard curve for detecting norepinephrine was prepared using the compound shown in Formula II and a standard of adrenaline; 2') Add the compound shown in Formula II to the sample; and 3') Quantitatively detect the content of adrenaline in the sample.

5. Compounds represented by Formula I or Formula II: In Formula I, L1 is selected from: substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S, substituted or unsubstituted C3-C7 cycloalkylene, substituted or unsubstituted C3-C7 heterocyclic group containing one, two or three heteroatoms independently selected from N, O or S; L2 is selected from: substituted or unsubstituted C1-C 10 Alkylene, substituted or unsubstituted C1-C containing one, two or three heteroatoms independently selected from N, O or S. 10 Alkylene; In Formula II, X is selected from: -O-, -S-, -NR1-; R1 is selected from: H, substituted or unsubstituted C1-C6 alkylene groups.

6. The compound according to claim 5, characterized in that, In Formula I, L1 is selected from: substituted or unsubstituted C1-C4 alkylene, substituted or unsubstituted C1-C4 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S; L2 is selected from: substituted or unsubstituted C1-C8 alkylene groups, substituted or unsubstituted C1-C8 alkylene groups containing 1, 2 or 3 heteroatoms independently selected from N, O or S; In Formula II, X is selected from: -O-, -S-, -NH-, with -O- being preferred.

7. The compound according to claim 6, characterized in that, In Equation I, L1 is selected from the following group: Preferred L2 is independently selected from the following group: Preferred 8. The compound according to claim 7, characterized in that, The compound of formula I is selected from: The compound of formula II is selected from:

9. The compound according to claim 8, characterized in that, The compound of formula I is: The compound of formula II is:

10. Use of the compound shown in Formula I or Formula II in the preparation of reagents or kits for detecting catecholamines in samples: In Formula I, L1 is selected from: substituted or unsubstituted C1-C6 alkylene, substituted or unsubstituted C1-C6 alkylene containing one or two heteroatoms independently selected from N, O or S, substituted or unsubstituted C5-C8 arylene, substituted or unsubstituted C5-C8 heteroarylene containing one, two or three heteroatoms independently selected from N, O or S, substituted or unsubstituted C3-C7 cycloalkylene, substituted or unsubstituted C3-C7 heterocyclic group containing one, two or three heteroatoms independently selected from N, O or S; L2 is selected from: substituted or unsubstituted C1-C 10 Alkylene, substituted or unsubstituted C1-C containing one, two or three heteroatoms independently selected from N, O or S. 10 Alkylene; In Formula II, X is selected from: -O-, -S-, -NR1-; R1 is selected from: H, substituted or unsubstituted C1-C6 alkylene groups.

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