Fluorescent probe for targeting mitochondrial ferric ions as well as preparation and application of fluorescent probe

Abstract

The invention discloses a fluorescent probe for targeting mitochondrial ferric ions as well as preparation and application of the fluorescent probe. The fluorescent probe is prepared by reacting cucurbit [7] uril with (E)-4-(4-(dimethylamino) styryl)-1-(3-(triphenylphosphonium) propyl) pyridine-1-onium bromide according to a molar ratio of 1: 1. The fluorescent probe can be used for detecting Fe < 3 + > ions, and has the characteristics of simple preparation method, convenience in operation, high sensitivity, high selectivity, low cost and rapidness in detection.

Description

TECHNICAL FIELD

[0001] The application relates to a fluorescent probe and preparation and application thereof, in particular to a fluorescent probe for targeting trivalent iron ions in mitochondria and preparation and application thereof. BACKGROUND

[0002] At present, the clinical diagnosis of Parkinson's disease (PD) mainly depends on imaging techniques such as MRI, SPECT and PET. These methods can only detect structural or functional abnormalities after the occurrence of neuronal death, thus delaying the opportunity for timely intervention.

[0003] In recent years, research has shown that iron metabolism disorder is a key molecular event in the occurrence and development of PD, and is closely related to mitochondrial iron homeostasis imbalance. The specific mechanism involves up-regulation of transferrin receptor 1 and divalent metal transporter 1 expression, down-regulation of iron transporter, leading to abnormal accumulation of Fe 3+ in mitochondria, and then promoting the generation of a large amount of active oxygen through the Fenton reaction, inducing lipid peroxidation, ferroptosis and abnormal aggregation of alpha-synuclein, and finally causing the degeneration of dopaminergic neurons.

[0004] The fluorescent probe technology shows great potential in monitoring iron metabolism disorders due to its high spatiotemporal resolution, molecular specificity and dynamic monitoring capability. However, the existing Fe 3+ fluorescent probes still cannot meet the multiple requirements of mitochondrial targeting, high selectivity, excellent light stability, good biocompatibility and blood-brain barrier penetration ability, which seriously limits their application in the early diagnosis of PD.

[0005] Therefore, it is of great significance to develop a new type of molecular probe that can monitor the dynamic state of mitochondrial Fe 3+ ions in real time and accurately, which can promote the early diagnosis and intervention of PD.

[0006] The application aims to provide a fluorescent probe for targeting trivalent iron ions in mitochondria and preparation and application thereof. The fluorescent probe of the application can detect Fe 3+ ions, and has the characteristics of simple preparation method, easy operation, high sensitivity, high selectivity, low cost and rapid detection.

[0007] The application provides a preparation method of a fluorescent probe for targeting trivalent iron ions in mitochondria, which is obtained by reacting a seven-membered cucurbituril with (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonium)propyl)pyridinium-1-olium bromide.

[0008] Preferably, the preparation method of the fluorescent probe for targeting trivalent iron ions in mitochondria is characterized in that the chemical structural formula of (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonium)propyl)pyridinium-1-olium bromide is as follows: .

[0009] Preferably, in the aforementioned method for preparing the fluorescent probe targeting mitochondrial ferric ions, the molar ratio of the seven-membered cucurbit ring to (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide is 1:1.

[0010] Preferably, the aforementioned method for preparing the fluorescent probe targeting mitochondrial ferric ions is as follows: (1) Prepare an aqueous solution A from the seven-membered cucurbitacin ring; (2) Prepare an aqueous solution B from (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide; (3) The probe solution can be prepared by mixing aqueous solution A and aqueous solution B and reacting at room temperature.

[0011] Preferably, in the aforementioned method for preparing the fluorescent probe targeting mitochondrial ferric ions, the concentration of the seven-membered cucurbitacin ring in the aqueous solution A is 1.0 × 10⁻⁶. -3 mol / L.

[0012] Preferably, in the aforementioned method for preparing the fluorescent probe targeting mitochondrial ferric ions, the concentration of (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide in the aqueous solution B is 1.0 × 10⁻⁶. -3 mol / L.

[0013] Preferably, in the aforementioned method for preparing the fluorescent probe targeting mitochondrial ferric ions, the concentration of the fluorescent probe in the probe solution is 1.0 × 10⁻⁶. -3 mol / L.

[0014] A fluorescent probe targeting mitochondrial ferric ions is provided, which is prepared according to the aforementioned method.

[0015] This invention provides an application of the aforementioned fluorescent probe in detecting ferric ions in aqueous solution.

[0016] This invention provides an application of the aforementioned fluorescent probe as a detection reagent for ferric ions in Parkinson's disease cells.

[0017] 1. The fluorescent probe of the present invention is a novel substance and its preparation method is simple.

[0018] 2. The fluorescent probe of this invention can be used to detect Fe. 3+ Ions are easy to operate, quick to detect, highly sensitive, and low in cost.

[0019] 3. The fluorescent probe of this invention is effective against Fe in water samples and Parkinson's disease cell models. 3+ Ions have high selectivity. Attached Figure Description

[0020] Figure 1 The 1H NMR spectrum of compound (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide.

[0021] Figure 2 The image shows the carbon NMR spectrum of compound (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide.

[0022] Figure 3 The mass spectrum of compound (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide.

[0023] Figure 4 The image shows the hydrogen NMR spectrum of a seven-membered cucurbit ring.

[0024] Figure 5 For different concentrations of Fe 3+ The concentration-fluorescence change value of ions in the fluorescent probe (hereinafter referred to as DTPP@Q[7]) (10μM) solution of the present invention is shown in the figure. Among them, (a) is the concentration-fluorescence change value of DTPP@Q[7] (10μM) and Fe in aqueous solution with different concentrations. 3+ (a) Fluorescence spectrum changes of coexisting ions; (b) Fe based on the fluorescence spectrum changes. 3+ Standard curve of ion concentration versus fluorescence intensity change ΔI.

[0025] Figure 6 The fluorescence spectra of various metal ions added to the aqueous solution of the DTPP@Q[7] fluorescent probe are shown.

[0026] Figure 7 For Fe 3+ Interference diagrams of different organic compounds in the presence of ions, fluorescence spectra of various metal ions after adding various metal ions to DTPP@Q[7](10μM)+FeCl3(100μM) aqueous solution, and bar charts of fluorescence intensity values ​​of different systems calculated based on fluorescence spectra.

[0027] Figure 8(a) Fluorescence image of PC12 cells after incubation with 10 μM DTPP@Q[7] for 3 h and co-staining with 100 nM mitochondrial tracer for 30 min; (b) Photostability image of PC12 cells after continuous irradiation with 10 μM DTPP@Q[7] and 100 nM mitochondrial tracer for 2 min under maximum laser power (100%) of confocal laser scanning microscope (CLSM).

[0028] Figure 9 DTPP@Q[7] on exogenous Fe in PC12 cells 3+ The dynamic response; (a) fluorescence images of PC12 cells pretreated with 50 μM FeCl3, 80 μM 6-OHDA or 80 μM 6-OHDA + 10-50 μM FeCl3 for 24 h and then incubated with 80 μM MTPP@Q[7] for 3 h; (b) quantitative analysis of the above PC12 cells; (c) iron content in PC12 cells under different treatment conditions measured using a cell iron content detection kit; (d) correlation curve between iron content and probe fluorescence quenching.

[0029] Figure 10 Mitochondrial Fe 3+ Induced fluorescence quenching was associated with ferroptosis markers in Parkinson's disease cells and could track pathological α-synuclein aggregation and changes in dopamine synthesis. PC12 cells were pretreated for 24 h with 50 μM MFeCl3, 80 μM 6-OHDA, or a combination of 80 μM 6-OHDA and 10-50 μM MFeCl3. The intracellular levels of (a) glutathione peroxidase 4 (GPX4) and (b) lipid peroxides (LPO) in PC12 cells were detected using a kit. (c) Correlation curves between GPX4 content, LPO level and probe fluorescence quenching were obtained. (d) Western blot analysis of protein immunoblotting of α-synuclein (α-syn) and tyrosine hydroxylase (TH) expression levels in PC12 cells were obtained. (e) Correlation curves between α-synuclein and tyrosine hydroxylase expression and probe fluorescence quenching were obtained. Detailed Implementation

[0030] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to various embodiments. It should be understood that the specific embodiments described herein are only for explaining the invention and are not intended to limit the invention; that is, the described embodiments are merely some embodiments of the invention, and not all embodiments.

[0031] The term "embodiment" used herein, as an example, is not necessarily to be construed as superior to or better than other embodiments. Performance testing in these embodiments, unless otherwise specified, employs conventional testing methods in the art. The terminology used in this invention is merely for describing particular implementations and is not intended to limit the scope of the disclosure.

[0032] Unless otherwise stated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; other raw materials, reagents, test methods and techniques not specifically mentioned herein refer to raw materials and reagents commonly used by one of ordinary skill in the art, as well as commonly employed test methods and techniques.

[0033] The compound used in this invention ( E )-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide (DTPP) is a new substance synthesized in this invention, and the seven-membered cucurbit ring (Q[7]) is a disclosed structure.

[0034] Compounds as guest molecules ( E )-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide (DTPP), its reaction formula is as follows: The specific synthesis steps are as follows: First, compound 1 was synthesized by dissolving 4-methylpyridine (4.7 g, 50.4 mmol) and potassium tert-butoxide (t-BuOK, 11.2 g, 99.8 mmol) in dimethylformamide (DMF, 20 mL); the mixture was stirred at 80 °C for 2 hours.

[0035] Subsequently, 4-dimethylaminobenzaldehyde (5.0 g, 33.5 mmol) was dissolved in 15 mL of DMF, and the solution was then added dropwise to the reaction flask. The reaction was carried out for 24 hours, and the reaction progress was monitored by TLC throughout. After the reaction was completed, the mixture was cooled to room temperature to obtain a pale yellow solution. The mixture was poured into ice water (2.5 L), and after standing, a precipitate was formed. The solid was collected by filtration and recrystallized from ethanol to obtain a pale yellow solid, namely compound 1.

[0036] Compound 1 (0.270 g, 1.20 mmol) and (3-bromopropyl)triphenylphosphonium bromide (1.156 g, 2.49 mmol) were dissolved in acetonitrile (20 mL) in a single-necked flask; the mixture was refluxed for 24 hours, and the reaction was monitored by TLC throughout; after the reaction was completed, the mixture was cooled and allowed to stand, and a red precipitate formed; the solid was collected by filtration, washed three times with dry acetonitrile (about 30 mL), and dried under vacuum to obtain compound (…). E )-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide (DTPP), yield 59.3%, the relevant spectra of the prepared DTPP are as follows. Figures 1-3 As shown.

[0037] The specific synthesis process of the seven-membered cucurbit ring (Q[7]) is as follows: 500g of urea, 200g of paraformaldehyde and 710mL of concentrated hydrochloric acid with a mass-volume concentration of 36%-38% were placed in a 3L three-necked flask and sonicated for 10 minutes to ensure thorough mixing of the solids. The flask was then placed in an oil bath, heated to 100℃ and refluxed for 3 hours. After the reaction was stopped, the mixture was allowed to cool naturally to room temperature, at which point a large amount of white precipitate was formed. The precipitate was collected to obtain a mixture of cucurbitacins containing Q[6] and Q[7]. First, the cucurbitacin mixture was initially separated based on differences in solubility, followed by fine separation using column chromatography: using silica gel G as the stationary phase and a water / acetic acid / formic acid mixed solution as the mobile phase, Q[6] and Q[7] were obtained by elution sequentially. During the separation process, thin-layer chromatography was used for monitoring, and each component was confirmed by spotting on a silica gel plate until clear and concentrated sample spots appeared. Finally, the filtrate of the target component was collected and treated by vacuum distillation. The 1H NMR spectrum of the obtained product is shown below. Figure 4 As shown, the above steps yielded high-purity Q[7].

[0038] The present application will now be described in detail with reference to specific embodiments and experimental data. Example 1

[0039] This embodiment prepared the probe solution for subsequent experiments and configured other related solutions, as detailed below: (1) Preparation of probe standard solution: Accurately weigh an appropriate amount of Q[7] and prepare a solution with a concentration of 1.0×10⁻⁶ using deionized water with pH=6.52. -3 A solution of Q[7] with a concentration of 1.0 × 10⁻⁶ mol / L was prepared by accurately weighing an appropriate amount of DTPP and preparing it with deionized water at pH 6.52. -3A 1 mol / L DTPP solution was prepared; the Q[7] solution and the DTPP solution were mixed in a volume ratio of 1:1 and reacted at room temperature for about 1 min. Then, deionized water with pH = 6.52 was added to dilute the concentration of Q[7] and DTPP to 1.0 × 10⁻⁶ mol / L. - 5 A probe standard solution can be obtained by using mol / L.

[0040] (2) Accurately weigh the required analytical grade ferric chloride standard, dissolve it in deionized water with pH = 6.52 to obtain a concentration of 1 × 10⁻⁶. -2 The standard test solution at mol / L. Example 2

[0041] In this embodiment, the probe standard solution prepared in Example 1 was used to detect the standard aqueous solution of ferric chloride and a standard curve was plotted. The specific steps are as follows: Fe 3+ Determination of the relationship between ion concentration and fluorescence change: Take a quartz fluorescence cuvette and add 3000 μL of a solution with a concentration of 1.0 × 10⁻⁶. -5 Add 0.3 μL of a 1.0 × 10⁻⁶ mol / L probe standard solution to the solution. -2 A 1.0 × 10⁻⁶ mol / L ferric chloride solution was mixed and its fluorescence emission spectrum was measured at a fixed excitation wavelength of 465 nm. Then, 0.3 μL of a 1.0 × 10⁻⁶ mol / L ferric chloride solution was added again. -2 Ferric chloride at a concentration of 1.0 × 10⁻⁶ mol / L was mixed thoroughly, and its fluorescence emission spectrum was measured at a fixed excitation wavelength of 465 nm. This process was repeated multiple times by adding 0.3 μL of a solution with a concentration of 1.0 × 10⁻⁶ mol / L. -2 The procedure of mixing ferric chloride (mol / L) and measuring its fluorescence emission spectrum at a fixed excitation wavelength of 465 nm was continued until the fluorescence intensity change tended to stabilize, as shown in the figure. Figure 5 The fluorescence spectrum change diagram shown ( Figure 5 a).

[0042] Plotting the concentration of ferric chloride on the x-axis and the initial emission intensity (I0) of the fluorescent probe at 602 nm against the values ​​of different concentrations of Fe, the results show the relationship between the concentration of ferric chloride and the concentration of Fe. 3+ The difference (ΔI) in fluorescence intensity after ionization in solution is used as the ordinate to obtain the Fe... 3+ Standard curve of ion concentration-fluorescence intensity change ΔI ( Figure 5 b), it can be seen that Fe 3+ There is a clear linear relationship between ion concentration and fluorescence intensity change ΔI. Based on the slope of the standard curve and the results of 11 standard solutions of the probe (fluorescent probe concentration 1.0 × 10⁻⁶),... -5 The mean of blank determination results (mol / L) was used to calculate Fe. 3+The detection limit for ionic solutions can reach 3.6 x 10⁻⁶. -7 mol / L. Example 3

[0043] This embodiment examines the detection of Fe using the probe standard solution prepared in Example 1. 3+ During the process of ion concentration, the probe targets Fe 3+ Ion selectivity. The specific experimental steps are as follows: (1) Take a quartz fluorescence cuvette and add 3000 μL of a solution with a concentration of 1.0 × 10⁻⁶. -5 Add 30 μL of a mol / L probe standard solution to a solution with a concentration of 1.0 × 10⁻⁶ mol / L. -2 Different metal ions at mol / L (interfering substances such as Figure 6 The solution (shown) was mixed thoroughly and its fluorescence emission spectrum was measured at a fixed excitation wavelength of 465 nm. The results are as follows. Figure 6 As shown, it contains only Fe 3+ The water sample containing ions showed a significant decrease in fluorescence intensity at 602 nm, while the water sample containing other metal ions did not show obvious fluorescence quenching at this wavelength, indicating that this fluorescent probe has high selectivity.

[0044] (2) Take a quartz fluorescence cuvette and add 3000 μL of a solution with a concentration of 1.0 × 10⁻⁶. -5 First, add 30 μL of a mol / L probe standard solution with a concentration of 1.0 × 10⁻⁶ mol / L. -2 A ferric chloride mixture solution of mol / L was then supplemented with 30 μL of a 1.0 × 10⁻⁶ mol / L solution. -2 Different metal ions at mol / L (metal ions such as...) Figure 7 The solution (shown) was mixed thoroughly and its fluorescence emission spectrum was measured at a fixed excitation wavelength of 465 nm. The results are as follows. Figure 10 As shown, other metal ions react with Fe. 3+ When ions coexist, it does not significantly affect the probe's sensitivity to Fe. 3+ Ion selectivity. Example 4

[0045] In this embodiment, the probe standard solution prepared in Example 1 was used to perform cell imaging experiments. The distribution of the probe in the cells was monitored using the commercial mitochondrial tracer Mito-Tracker, and the photostability of the probe and Mito-Tracker were further compared.

[0046] (1) PC12 cells were injected at a rate of 15 × 10⁻⁶ cells per milliliter. 4 Cells were seeded at a density of [number] cells per confocal culture dish and incubated at 37°C for 24 h. After incubation with the probe at 37°C for 3 h, Mito-Tracker (1.0 × 10⁻⁶ cells / mL) was added. -7(mol / L). After an incubation period of 30 min, the cells were washed three times with PBS and then observed using a confocal laser scanning microscope (CLSM). The excitation and emission wavelengths were as follows: excitation wavelength of the probe λex = 465 nm, emission wavelength λem = 580-620 nm; excitation wavelength of the mitochondrial tracer green fluorescence λex = 490 nm, emission wavelength λem = 500-550 nm. Analysis of the CLSM images using ImageJ software showed that the colocalization coefficient between the probe and the Mito-Tracker was as high as 0.95, indicating a high degree of colocalization and confirming the probe's excellent mitochondrial targeting ability. Figure 8 a).

[0047] (2) PC12 cells were injected at a rate of 15 × 10⁻⁶ cells per milliliter. 4 Cells were seeded at a density of [number] cells per confocal culture dish and incubated at 37°C for 24 h. After incubation with the probe at 37°C for 3 h, Mito-Tracker (1.0 × 10⁻⁶ cells / mL) was added. -7 (mol / L). After an incubation period of 30 min, the cells were washed three times with PBS, and then observed using a CLSM. Maintaining the same focal plane, the cells were continuously irradiated with the CLSM's built-in laser for 2 min, and CLSM images were acquired every 30 s. Analysis of the CLSM images using ImageJ software showed no significant attenuation of the probe's fluorescence signal, while Mito-TrackerGreen exhibited severe photobleaching within 30 seconds, with a signal intensity loss of nearly 95% (mol / L). Figure 8 b). This probe exhibits excellent photostability, making it ideal for mitochondrial localization. Example 5

[0048] In this embodiment, the probe standard solution prepared in Example 1 was used in the Fe... 3+ Content detection.

[0049] PC12 cells were injected at a rate of 15 × 10⁻⁶ per ml. 4 Cells were seeded at a density of [number] cells per confocal culture dish and incubated at 37°C for 24 h. To simulate iron metabolism disorder, cells were treated with FeCl3 (50 μM), 6-hydroxydopamine (6-OHDA) (80 μM), and combinations of 80 μM 6-OHDA with FeCl3 at concentrations of 10, 25, and 50 μM, respectively. After treatment, the culture medium was removed, and the cells were seeded with the probe (1.0 × 10⁻⁶ cells per 10⁻⁶). -5 Incubate with mol / L solution for 3 hours. Then wash cells three times with phosphate-buffered saline (PBS) and observe using CLSM. Fe concentration in each group was detected using commercial kits and Western blotting. 3+The content of ferroptosis markers (glutathione peroxidase 4, lipid peroxides), and the expression levels of α-synuclein and tyrosine hydroxylase were analyzed. ImageJ software was used to analyze CLSM images, and the degree of fluorescence quenching showed a dose-dependent linear correlation (R0) with Fe³⁺ concentration. 2 >0.93), demonstrating its ability to accurately trace Fe³⁺ overload within mitochondria ( Figure 9 Furthermore, in a Parkinson's disease cell model, mitochondrial Fe... 3+ Induced probe fluorescence quenching and downregulation of ferroptosis markers (glutathione peroxidase 4) Figure 10 a) Upregulation of lipid peroxides ( Figure 10 b) Related ( Figure 10 c), and can track pathological α-synuclein aggregation (upregulated expression) and dopamine synthesis (downregulated tyrosine hydroxylase) inhibition ( Figure 10 d), indicating that the probe can dynamically reflect the progression of neurodegenerative diseases ( Figure 10 e).

[0050] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a fluorescent probe targeting mitochondrial ferric ions, characterized in that: It is formed by the reaction of a seven-membered cucurbit ring with (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide.

2. The method for preparing a fluorescent probe targeting mitochondrial ferric ions according to claim 1, characterized in that, The chemical structural formula of the (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide is as follows: 。 3. The method for preparing a fluorescent probe targeting mitochondrial ferric ions according to claim 1, characterized in that: The molar ratio of the seven-membered cucurbit ring to (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide is 1:

1.

4. The method for preparing a fluorescent probe targeting mitochondrial ferric ions according to claim 1, characterized in that, The method is as follows: (1) Prepare an aqueous solution A from the seven-membered cucurbitacin ring; (2) Prepare an aqueous solution B from (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide; (3) The probe solution can be prepared by mixing aqueous solution A and aqueous solution B and reacting at room temperature.

5. The method for preparing a fluorescent probe targeting mitochondrial ferric ions according to claim 1, characterized in that: The concentration of the seven-membered cucurbitacin in the aqueous solution A is 1.0 × 10⁻⁶. -3 mol / L.

6. The method for preparing a fluorescent probe targeting mitochondrial ferric ions according to claim 1, characterized in that: The concentration of (E)-4-(4-(dimethylamino)styryl)-1-(3-(triphenylphosphonyl)propyl)pyridine-1-onium bromide in the aqueous solution B is 1.0 × 10⁻⁶. -3 mol / L.

7. The method for preparing a fluorescent probe targeting mitochondrial ferric ions according to claim 1, characterized in that: The concentration of the fluorescent probe in the probe solution is 1.0 × 10⁻⁶. -3 mol / L.

8. A fluorescent probe targeting mitochondrial ferric ions, characterized in that: Prepared by the method according to any one of claims 1-7.

9. The application of the fluorescent probe as described in claim 8 in the detection of ferric ions in aqueous solution.

10. The application of the fluorescent probe as described in claim 8 as a detection reagent for ferric ions in Parkinson's disease cells.

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