A fluorescent probe for detecting cysteine and a preparation method and use method thereof

Abstract

The application discloses a fluorescent probe for detecting cysteine and a preparation method and use method thereof. The probe is (1-(5-(acryloyloxy)-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium (NACYS), and a structure is shown as formula (I). The probe is based on a Michael addition / intramolecular lactonization tandem reaction induced by cysteine, releases a naphthalimide fluorophore, and realizes an 'on' type fluorescent response (λ em = 591 nm). The probe has good water solubility, high selectivity and mitochondrial targeting ability, can be used for quantitative detection of mitochondrial cysteine in an aqueous solution and cells, and has application prospects in the field of biomedical research.

Description

Technical Field

[0001] This invention belongs to the field of organic small molecule fluorescent probes, specifically relating to (1-(5-(acryloyloxy)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium as a cysteine ​​fluorescent probe, and its preparation and usage methods. Background Technology

[0002] Endogenous biothiols such as cysteine ​​(Cys), homocysteine ​​(Hcy), and glutathione (GSH) are widely present in organisms and play important roles in maintaining intracellular redox homeostasis. Among them, GSH (1–10 mM) is the most abundant biothiols in cells and is an important component of the body's antioxidant defense system. Cys (30–200 μM) has a significantly higher concentration in vivo than Hcy (5–12 μM) and is more easily oxidized than GSH, playing an independent and important role in regulating cellular redox homeostasis. Studies have shown that in the development of neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, and amyotrophic lateral sclerosis (ALS), the expression of cystathionine-γ-lyase (CSE), a key enzyme involved in Cys production, is inhibited, leading to abnormal Cys metabolism and consequently causing intracellular redox imbalance and increased oxidative stress levels. Therefore, Cys can serve as an important biomarker reflecting the body's oxidative stress state, and its accurate detection has significant biomedical implications.

[0003] Molecular fluorescent probe detection methods have advantages such as high selectivity, high stability, low toxicity, low cost, and no damage to samples, making them the most powerful tool for detecting cell and tissue levels.

[0004] Currently developed small-molecule fluorescent probes for detecting cysteine ​​are mainly based on cysteine's nucleophilic addition reactions, substitution reactions, and cysteine ​​thiol-induced metal substitution reactions. In the presence of cysteine, the detection group in the probe molecule specifically interacts with cysteine, altering the molecule's original PET or ICT effects, leading to changes in the probe molecule's fluorescence properties and achieving specific recognition of cysteine.

[0005] Currently, based on the unique redox properties and strong nucleophilicity of the thiol group in cysteine, a variety of luminescent probes specifically for cysteine ​​detection have been developed (see review Run Zhang, Jiaxi Yong, Jingli Yuan, Zhi PingXu. Recent advances in the development of responsive probes for selective detection of cysteine. Coordination Chemistry Reviews, 2020, 408, 213182.DOI:10.1016 / j.ccr.2020.213182). 1) In the presence of cysteine, its thiol group first nucleophilically attacks the imine carbon atom of the Schiff base, forming a hemithioacetal amine intermediate; subsequently, this intermediate undergoes a proton transfer reaction, the C=N double bond breaks, generating an adduct of cysteine ​​and an aldehyde, as well as a free amine compound; 2) In the presence of cysteine, its thiol group undergoes aromatic nucleophilic substitution (S) of activated halogens (such as chlorine, bromine, and fluorine) on the aromatic ring. N Ar), generating a thioether-linked intermediate and releasing a halide anion; subsequently, this intermediate may undergo intramolecular rearrangement (such as the Smiles rearrangement), in which the amino group of cysteine ​​or another nucleophilic group attacks the appropriate position on the aromatic ring, ultimately forming a stable product with structural rearrangement; 3) In the presence of cysteine, its amino group first undergoes a nucleophilic addition-elimination reaction with the aldehyde group to generate an imine (Schiff base) intermediate; then, the thiol group of cysteine ​​rapidly attacks the carbon atom of the imine, undergoing intramolecular cyclization to form a stable five-membered thiazolyl ring, thereby irreversibly capturing and consuming the aldehyde group; 4) In the presence of cysteine, its thiol group acts as a nucleophile to attack the sulfonate. The sulfur atom in the amide or sulfonate forms a four-coordinate sulfur intermediate; subsequently, the intermediate undergoes an elimination reaction, releasing an amine (or ammonia) or an alcohol, respectively, to generate a cysteine-S-sulfonic acid adduct (R-SO2-S-Cys), thereby breaking the original sulfonamide or sulfonate bond; 5) In the presence of cysteine, its thiol and amino groups act as strong bidentate ligands, forming complexes with metal ions that are more stable than the original ligands; because cysteine ​​has a higher binding constant with these metal ions, it can competitively displace the small molecule ligands or protein side chains originally coordinated to the metal ions, leading to the breaking of the metal-ligand bond, and at the same time generating a cysteine-metal complex.

[0006] These reported probes still suffer from selectivity or poor solubility, primarily due to the nucleophilic addition of biothiols such as Cys and GSH to the responding group. In contrast, two consecutive intermolecular and intramolecular nucleophilic addition reactions can significantly improve probe selectivity for cysteine. However, many reported probes require mixed systems containing organic solvents (such as DMSO and acetonitrile) to function stably, limiting their application in pure water environments or under physiological conditions. Some probes have poor water solubility, easily aggregate or precipitate, thus affecting detection sensitivity and repeatability. Furthermore, some probes have slow response rates or insignificant fluorescence signal changes, making it difficult to meet the needs of rapid detection, avoid interference from other biothiols, and achieve specific recognition of cysteine. Summary of the Invention

[0007] The present invention aims to provide an "on" type cysteine ​​fluorescent probe based on the mechanisms of intramolecular charge transfer (ICT) and photoinduced electron transfer (PET), which has high selectivity, good water solubility and mitochondrial targeting ability.

[0008] In a first aspect, the present invention provides a fluorescent probe for detecting cysteine, which is (1-(5-(acryloyloxy)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium (abbreviated as NACYS), with the structure shown in formula (I):

[0009]

[0010] The working principle of the probe of this invention is as follows: In the probe molecule, the naphthimide fluorophore is linked to a triphenylphosphine cation (mitochondrial targeting group) via a PEG chain, and its hydroxyl group is protected by an acrylate. The acrylate group acts as the recognition unit for cysteine ​​and simultaneously quenches fluorescence through the PET effect. When cysteine ​​is present, its thiol group first undergoes Michael addition to the double bond of the acrylate, followed by intramolecular amino group attacking the ester carbonyl group, resulting in intramolecular esterification, forming a five-membered ring thiazolidinone and releasing the free hydroxyl group of the naphthimide fluorophore, thereby restoring the ICT effect and emitting bright orange fluorescence at 591 nm. Since other biothiols (such as glutathione and homocysteine) cannot complete the two-step continuous intramolecular cyclization reaction, this probe has excellent selectivity for cysteine.

[0011] In a second aspect, the present invention provides a method for preparing the fluorescent probe, the synthetic route of which is as follows:

[0012]

[0013] The specific steps are as described in claim 3.

[0014] A third aspect of the present invention provides a method for using the fluorescent probe to detect the cysteine ​​content in a sample for non-diagnostic purposes, wherein quantitative detection is achieved by establishing a standard curve of fluorescence intensity versus cysteine ​​concentration.

[0015] The usage method of the above-mentioned cysteine ​​fluorescent probe is as follows:

[0016] Step 1: Add the same concentration of the compound shown in formula (I) to phosphate buffer solutions (10 mM, pH = 7.4) containing different concentrations of cysteine ​​to prepare at least 5 standard solutions with different cysteine ​​concentrations.

[0017] The concentration of the compound represented by formula (I) in the standard solution shown is 1 nM to 10 μM.

[0018] The cysteine ​​content in the standard solution shown is 0.1 nM to 1 mM;

[0019] Step 2: Measure the fluorescence emission spectra of the standard solutions, with an excitation wavelength of 382 nm. Plot the cysteine ​​concentration on the x-axis and Ig on the y-axis. 591 Establish a standard curve with the vertical axis as the ordinate;

[0020] I 591 This indicates the fluorescence emission intensity of the standard solution at a wavelength of 591 nm;

[0021] Step 3: Add the compound shown in formula (I) to the sample to be tested, so that its concentration is equal to that of the compound shown in formula (I) in the standard solution. Measure the fluorescence emission spectrum of the sample to be tested at an excitation wavelength of 382 nm. Calculate the cysteine ​​content in the sample to be tested based on the standard curve.

[0022] This invention has the following characteristics:

[0023] 1) The fluorescent probe provided by this invention is a colorless oily substance with a stable structure.

[0024] 2) The fluorescent probe provided by this invention is sensitive to the concentration of cysteine ​​in its solution. As the concentration of cysteine ​​increases, its fluorescence in the test solution changes from weak blue light to bright orange fluorescence under a 365 nm ultraviolet lamp.

[0025] 3) The fluorescent probe provided by this invention emits light at a wavelength of 591 nm after reacting with cysteine. It is a fluorescence-on response, and the fluorescence intensity changes significantly before and after the reaction. This can greatly eliminate the influence of differences in detection conditions on the results and improve the sensitivity of the detection.

[0026] 4) The fluorescent probe provided by this invention has a linear relationship with cysteine ​​concentration and can be used for accurate measurement of cysteine ​​concentration.

[0027] The activating cysteine ​​probe based on (1-(5-(acryloyloxy)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium provided by this invention exhibits a good response to cysteine ​​solutions, enabling sensitive quantitative detection of cysteine ​​in samples. It can also co-localize to mitochondria, efficiently detecting changes in cysteine ​​levels in mitochondria. This probe offers advantages such as ease of operation, low cost, sensitive response, and ease of promotion and application. Attached Figure Description

[0028] Figure 1 : 1H NMR spectrum of the fluorescent probe NACYS (400 MHz, CDCl3).

[0029] Figure 2 Fluorescence response of fluorescent probe NACYS (10 μM) before and after the addition of 200 μM cysteine ​​in phosphate buffer (pH 7.4) (under 365 nm UV lamp).

[0030] Figure 3 Photographs showing the color change of the fluorescent probe NACYS (10 μM) before and after the addition of 200 μM cysteine ​​under the same conditions (in sunlight).

[0031] Figure 4 UV-Vis absorption spectra of fluorescent probe NACYS (10 μM) reacted with different concentrations of cysteine ​​(0–300 μM) in phosphate buffer (pH 7.4).

[0032] Figure 5 Fluorescence emission spectra (λ) of fluorescent probe NACYS (10 μM) reacting with different concentrations of cysteine ​​(0–300 μM) under the same conditions. ex = 382 nm), the inset shows the linear relationship between fluorescence intensity and cysteine ​​concentration.

[0033] Figure 6 : A bar chart of the fluorescence response of the fluorescent probe NACYS (10 μM) to common biothiols and metal ions (200 μM each) under the same conditions (λ) ex = 382 nm, λ em = 591 nm). Detailed Implementation

[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0036] The compound numbers in the examples correspond to the numbers in the compounds described above.

[0037] Example 1: Synthesis of the fluorescent probe NACYS

[0038] (1) Synthesis of intermediate (1-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium (3)

[0039] Under nitrogen protection, 200.0 mg (0.43 mmol) 3-(2-(2-(2-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethoxy)ethoxy)ethoxy)propionic acid (1), 183.7 mg (0.96 mmol) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 64.7 mg (0.48 mmol) 1-hydroxybenzotriazole (HOBT), 242.4 mg (2.40 mmol) triethylamine and 222.1 mg (0.52 mmol) (2-aminoethyl)triphenylphosphine bromide (2) were dissolved in 5 mL of anhydrous N,N-dimethylformamide (DMF). The reaction was stirred at room temperature for 3 hours. After the reaction was monitored by thin-layer chromatography, the solvent was removed by vacuum distillation, and the product was purified by column chromatography (dichloromethane / methanol = 10:1) to obtain 304.2 mg of pale yellow oily intermediate (3), with a yield of 90%.

[0040] (2) Synthesis of fluorescent probe NACYS(5)

[0041] Under nitrogen protection, 100.0 mg (0.12 mmol) of intermediate (3) and 28.7 mg (0.28 mmol) of triethylamine were dissolved in 5 mL of anhydrous dichloromethane and cooled to 0°C in an ice bath. 15.4 mg (0.17 mmol) of acryloyl chloride (4) was slowly added dropwise, and the reaction was maintained at 0°C for 0.5 hours. After the reaction was monitored by thin-layer chromatography to ensure complete reaction, the solvent was removed under reduced pressure, and the product was purified by column chromatography (dichloromethane / methanol = 15:1) to give 104.8 mg of pale yellow oily product NACYS (5), with a yield of 98%.

[0042] like Figure 1As shown, the 1H NMR spectrum (400 MHz, Chloroform-d) is as follows: 8.64 (t, J = 5.9 Hz, 1H), 8.56 (dd, J = 7.3, 1.1 Hz, 1H), 8.34 (d, J = 2.3 Hz, 1H), 8.19 (dd, J = 8.4, 1.1 Hz, 1H), 8.02 (d, J = 2.3 Hz, 1H), 7.85 – 7.74 (m, 10H), 7.70 (ddd, J = 8.6, 6.9, 3.4 Hz, 6H), 6.70 (dd, J = 17.3, 1.1 Hz, 1H), 6.41 (dd, J = 17.3, 10.5 Hz, 1H), 6.13 (dd, J = 10.5, 1.1 Hz, 1H), 4.40 (t, J = 6.1 Hz, 2H), 2.42 (t, J = 6.4 Hz, 2H).

[0043] Example 2: Synthesis of the fluorescent probe NACYS

[0044] (1) Synthesis of intermediate (1-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium (3)

[0045] Under nitrogen protection, 150.0 mg (0.32 mmol) 3-(2-(2-(2-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethoxy)ethoxy)ethoxy)propionic acid (1), 122.7 mg (0.64 mmol) EDCI, 64.9 mg (0.48 mmol) HOBT, 161.9 mg (1.60 mmol) triethylamine, and 164.0 mg (0.38 mmol) (2-aminoethyl)triphenylphosphine bromide (2) were dissolved in 4 mL of anhydrous N,N-dimethylformamide. The reaction was stirred at room temperature for 4 hours. After the reaction was monitored by thin-layer chromatography to ensure complete reaction, the solvent was removed by vacuum distillation, and the product was purified by silica gel column chromatography (dichloromethane / methanol = 12:1) to give 218.5 mg of a pale yellow oily intermediate (3), with a yield of 86%.

[0046] (2) Synthesis of fluorescent probe NACYS(5)

[0047] Under nitrogen protection, 80.0 mg (0.10 mmol) of intermediate (3) and 20.2 mg (0.20 mmol) of triethylamine were dissolved in 8 mL of anhydrous dichloromethane and cooled to 0–5 °C in an ice bath. 10.9 mg (0.12 mmol) of acryloyl chloride (4) was slowly added dropwise, and the reaction was maintained at 0–5 °C for 45 minutes. After the reaction was monitored by thin-layer chromatography to ensure complete reaction, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography (dichloromethane / methanol = 15:1) to give 77.4 mg of pale yellow oily product NACYS (5), with a yield of 90%.

[0048] Example 3: Synthesis of the fluorescent probe NACYS

[0049] (1) Synthesis of intermediate (1-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium (3)

[0050] Under nitrogen protection, 300.0 mg (0.65 mmol) 3-(2-(2-(2-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethoxy)ethoxy)ethoxy)propionic acid (1), 311.5 mg (1.63 mmol) EDCI, 131.6 mg (0.97 mmol) HOBT, 328.8 mg (3.25 mmol) triethylamine, and 333.6 mg (0.78 mmol) (2-aminoethyl)triphenylphosphine bromide (2) were dissolved in 7 mL of anhydrous N,N-dimethylformamide. The reaction was stirred at room temperature for 6 hours. After the reaction was completed, the solvent was removed under reduced pressure, and the product was purified by silica gel column chromatography (dichloromethane / methanol = 10:1) to give 446.8 mg of a pale yellow oily intermediate (3), with a yield of 87%.

[0051] (2) Synthesis of fluorescent probe NACYS(5)

[0052] Under nitrogen protection, 120.0 mg (0.15 mmol) of intermediate (3) and 30.4 mg (0.30 mmol) of triethylamine were dissolved in 10 mL of anhydrous dichloromethane and cooled to 0 °C in an ice bath. 16.3 mg (0.18 mmol) of acryloyl chloride (4) was slowly added dropwise, and the reaction was maintained at 0 °C for 1 hour. After the reaction was completed, the solvent was removed by vacuum distillation, and the product was purified by silica gel column chromatography (dichloromethane / methanol = 20:1) to give 125.6 mg of pale yellow oily product NACYS (5), with a yield of 96%.

[0053] Example 4: Fluorescence response of compound NACYS to cysteine

[0054] Prepare a 1 mM N,N-dimethylformamide (DMF) stock solution of NACYS. Add 50 μL of this stock solution to 5 mL of phosphate buffered saline (PBS, 10 mM, pH 7.4) to bring the final probe concentration to 10 μM. Add cysteine ​​to bring the final concentration to 200 μM. Figure 2 As shown, under a 365 nm UV lamp, the solution changed from weak blue fluorescence to bright orange fluorescence; as Figure 3 As shown, the solution color changes from colorless to pale yellow under sunlight. This indicates that the probe has a direct "on" response to cysteine.

[0055] Example 5: UV titration detection of NACYS by different concentrations of cysteine

[0056] A series of samples were prepared using PBS solutions (10 mM, pH 7.4) with a probe concentration of 10 μM. Cysteine ​​was added to each solution to final concentrations of 0, 20, 40, 60, 80, 100, 120, 160, 200, 250, and 300 μM. The UV-Vis absorption spectra of each system were measured. Figure 4 As shown, the absorbance at 339 nm gradually decreases and the absorbance at 382 nm gradually increases with increasing cysteine ​​concentration, indicating that the probe responds in a concentration-dependent manner to cysteine.

[0057] Example 6: Fluorescent titration detection of NACYS by different concentrations of cysteine

[0058] Using the same series of solutions as in Example 3, fluorescence emission spectra were measured at an excitation wavelength of 382 nm. Figure 5 As shown, the fluorescence intensity at 591 nm gradually increases with increasing cysteine ​​concentration, reaching a plateau at 200 μM cysteine.

[0059] Example 7: Selectivity of compound NACYS against common interfering substances

[0060] Prepare PBS solutions (10 mM, pH 7.4) with a probe concentration of 10 μM, and add various common interfering agents (final concentration 200 μM). Measure the fluorescence intensity of each sample at 591 nm at an excitation wavelength of 382 nm. Figure 6 As shown, only cysteine ​​caused a significant fluorescence enhancement, while other interfering substances had almost no response, indicating that the probe has excellent selectivity for cysteine.

Claims

1. A fluorescent probe for detecting cysteine, characterized in that, The fluorescent probe is (1-(5-(acryloyloxy)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium, with the molecular formula C 44 H 44 N2O8P + NACYS, abbreviated as NACYS, has the structure shown in equation (I): 。 2. The fluorescent probe according to claim 1, characterized in that, The fluorescent probe is in the form of an oil or a solid.

3. A method for preparing a fluorescent probe according to claim 1 or 2, characterized in that, Includes the following steps: (1) 3-(2-(2-(2-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethoxy)ethoxy)ethoxy)propionic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole (HOBT), triethylamine and (2-aminoethyl)triphenylphosphine bromide were dissolved in N,N-dimethylformamide and reacted at room temperature for 3-6 hours to obtain (1-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium; (2) Dissolve the intermediate obtained in step (1) and triethylamine in dichloromethane, add acryloyl chloride dropwise under ice bath, and react for 0.5 to 1 hour to obtain (1-(5-(acryloyloxy)-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)-12-oxo-3,6,9-trioxa-13-azapentadecan-15-yl)triphenylphosphonium, which is the fluorescent probe.

4. The preparation method according to claim 3, characterized in that, In step (1), the molar ratio of 3-(2-(2-(2-(5-hydroxy-1,3-dioxo-1H-benzo[de]isoquinoline-2(3H)-yl)ethoxy)ethoxy)ethoxy)propionic acid, EDCI, HOBT, triethylamine and (2-aminoethyl)triphenylphosphine bromide is 1:1.5~2.5:1~2:4~6:1~1.5; the amount of N,N-dimethylformamide is 5~10 ml; and the reaction time is 3~6 hours.

5. The preparation method according to claim 3, characterized in that, In step (2), the molar ratio of the intermediate obtained in step (1), triethylamine, and acryloyl chloride is 1:1.5-2.5:1-1.5; the amount of dichloromethane used is 5-30 ml; the reaction temperature is 0-5℃; and the reaction time is 0.5-1 hour.

6. The preparation method according to claim 4, characterized in that, In step (1), the molar ratio is 1:2:1.5:5:1.2; the amount of N,N-dimethylformamide used is 5 ml; and the reaction time is 3 hours.

7. The preparation method according to claim 5, characterized in that, In step (2), the molar ratio is 1:1.5:1.2; the amount of dichloromethane used is 5 ml; the reaction temperature is 0℃; and the reaction time is 0.5 hours.

8. A method for detecting cysteine ​​content in a sample for non-diagnostic purposes using the fluorescent probe according to claim 1 or 2, characterized in that, Includes the following steps: (a) Prepare at least 5 standard solutions with different cysteine ​​concentrations by adding the same concentration of the compound shown in formula (I) to phosphate buffer solutions containing different concentrations of cysteine. The concentration of the compound represented by formula (I) in the standard solution is 100 nM to 10 μM; the concentration of cysteine ​​in the standard solution is 0.1 nM to 1 mM. (b) The fluorescence emission spectra of the standard solutions were measured respectively, with an excitation wavelength of 382 nm. The fluorescence emission peak intensity at 591 nm was plotted on the x-axis, with cysteine ​​concentration as the abscissa. 591 Establish a standard curve with the vertical axis as the ordinate; (c) Add the compound of formula (I) to the sample to be tested, so that its concentration is equal to that of the compound of formula (I) in the standard solution, measure the fluorescence emission spectrum of the sample to be tested at an excitation wavelength of 382 nm, and calculate the cysteine ​​content in the sample to be tested according to the standard curve.

9. The method according to claim 8, characterized in that, The phosphate buffer solution has a pH of 7.4 and a concentration of 10 mM.

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