Preparation method and application of a fluorescent probe for continuous detection of cadmium ions and glutathione

Abstract

The application relates to a preparation method and application of a fluorescent probe for continuously detecting cadmium ions and glutathione, and relates to the field of detection of cadmium ions and glutathione, in particular to a preparation method and application of an isoflurone fluorescent probe. The application solves the problem that the existing fluorescent probe detection method cannot realize continuous recognition of cadmium ions and glutathione under the same test condition. The preparation method of the probe is as follows: (E)-2-(3-(3-formyl-4-hydroxy styryl)-5,5-dimethylcyclohex-2-en-1-ylidene) propanedinitrile is condensed with quinoline-2-formylhydrazine to obtain the fluorescent probe XL. The fluorescent probe prepared in the application can highly selectively and quickly recognize cadmium ions, and the complex of the probe and the cadmium ions has good recognition selectivity for glutathione and is not interfered by other amino acids and anions. The detection method constructed in the application is sensitive and efficient, does not need expensive large-scale instruments and professional experimenters, and can realize detection of glutathione in a water body environment.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of cadmium ion and glutathione detection, and particularly relates to a preparation method of a fluorescent probe and application of the fluorescent probe in continuous detection of cadmium ion and glutathione. BACKGROUND

[0002] Cadmium is one of the most toxic and harmful heavy metal elements, which can be derived from natural processes such as volcanic eruptions and forest fires, and also can be derived from various industrial production, including electroplating, alloy making, preparation of nickel-cadmium batteries and military industry. The cadmium ions discharged in industrial production can cause soil and water pollution, bring environmental risks, and at the same time, the cadmium ions are easy to be enriched in plants, which can damage the cell membrane and organelles of plants, is not conducive to plant metabolism and causes plant death. In addition, the cadmium ions in the environment and plants entering the human body can cause renal tubular dysfunction and bone degeneration, gastrointestinal dysfunction and liver dysfunction, and also can increase the risk of cancer, threatening human health. Therefore, it is of great significance to detect cadmium ions in the environment and organisms.

[0003] Glutathione is a kind of tripeptide containing active sulfhydryl, which is composed of glutamic acid, cysteine and glycine. As an important bioactive peptide in the body, reduced glutathione can act as an antioxidant to scavenge free radicals in the body, and can well play the roles of anti-tumor and immune enhancement; as a heavy metal chelator, glutathione can form a complex with metal ions, which is easy to dissolve and non-toxic, and can reduce the toxic effects of metal ions on the human body; in addition, glutathione can also alleviate the side effects produced in the disease treatment process and relieve the pain of patients. Therefore, glutathione can be used as an additive in daily life and is widely used in the field of food processing, and also can be used as an important component in various health products to supplement the demand of normal physiological activities of the human body for glutathione. However, the imbalance of glutathione in the human body can cause a variety of diseases, such as Alzheimer's disease, heart disease, cardiovascular disease, cancer and premature aging, etc. Therefore, it is extremely important to monitor the content of glutathione in food and organisms.

[0004] At present, the traditional analysis techniques for detecting cadmium ions and glutathione include atomic absorption spectrometry, high performance liquid chromatography, inductively coupled plasma emission spectrometry, electrochemical method, etc. Compared with the traditional analysis techniques, the fluorescent detection method has great application potential due to its advantages of simple operation, high sensitivity, low detection limit, strong anti-interference ability and visualization, and has been widely studied and applied in the fields of environmental detection and biomedical detection.

[0005] In recent years, fluorescent probes for identifying cadmium ions or glutathione alone have been reported, but there are few reports on the use of the same fluorescent probe molecule to continuously identify cadmium ions and glutathione. The present application designs and synthesizes a novel isoflurone fluorescent probe, which can efficiently and quickly identify cadmium ions in a buffer solution, and the complex formed can realize specific identification of glutathione, and has the advantages of good selectivity, strong anti-interference and high sensitivity. The fluorescent probe prepared in the present application can continuously identify cadmium ions and glutathione under the same test conditions, and provides a preparation method and application of a multifunctional fluorescent probe. SUMMARY

[0006] The present application aims to solve the problem that cadmium ions and glutathione are difficult to be continuously detected under the same test conditions, and provides a preparation method and application of a fluorescent probe for continuously detecting cadmium ions and glutathione.

[0007] The fluorescent probe for continuously detecting cadmium ions and glutathione according to the present application has a molecular structure of:

[0008] The synthesis route of the above fluorescent probe is as follows:

[0009]

[0010] The preparation method of the fluorescent probe for continuously detecting cadmium ions and glutathione according to the present application is that (E)-2-(3-(3-formyl-4-hydroxy styryl)-5,5-dimethylcyclohex-2-en-1-ylidene) propanedinitrile and quinoline-2-carboxylic acid hydrazide are subjected to condensation reaction to obtain the fluorescent probe XL.

[0011] The specific method for continuously detecting cadmium ions and glutathione by using the fluorescent probe according to the present application is:

[0012] Under the excitation wavelength of 454 nm, the fluorescent probe XL has a fluorescence spectrum at 565 nm, the aqueous cadmium ion solution is added to the probe solution, the fluorescence spectrum at 565 nm disappears, a new fluorescence spectrum appears at 650 nm and the intensity is significantly enhanced, realizing the fluorescence detection of cadmium ions; the glutathione solution is added to the above system, the fluorescence spectrum at 650 nm disappears, and a fluorescence spectrum appears at 565 nm, realizing the fluorescence detection of glutathione.

[0013] Further, the response time of the fluorescent probe XL and cadmium ions, the complex XL-Cd 2+ The response time with glutathione is 3s.

[0014] Further, the complex XL-Cd 2+ The detection limit for glutathione is 7.8x10 -7 mol / L.

[0015] Further, under sunlight, when the probe XL continuously detects cadmium ions and glutathione, the system solution will present a color change sequence of yellow-violet red-yellow.

[0016] Further, the complex XL-Cd 2+ The glutathione in the water body can be detected.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] 1) The present application develops a multifunctional fluorescent probe, which can realize continuous detection of cadmium ions and glutathione under the same test condition, is simple to operate, and has good detection effect.

[0019] 2) The probe XL-Cd 2+ The complex has a large fluorescence emission wavelength (650 nm), can detect glutathione in the near-infrared region, maximally reduces background interference, and has a large Stokes shift (121 nm), which is beneficial to biological imaging. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 The present application prepares the fluorescent probe XL 1 H NMR spectrum;

[0021] Figure 2 The present application prepares the IR spectrum of the fluorescent probe XL;

[0022] Figure 3 The selectivity of the fluorescent probe XL in recognizing metal ions;

[0023] Figure 4 The Job's plot curve of the fluorescent probe XL for Cd 2+ ;

[0024] Figure 5 The complex XL-Cd 2+ The selectivity of the complex XL-Cd for amino acids and anions;

[0025] Figure 6 The complex XL-Cd 2+ The recognition relationship graph of the complex XL-Cd for GSH;

[0026] Figure 7 The complex XL-Cd 2+ The fluorescence response relationship graph of the complex XL-Cd for GSH of different concentrations;

[0027] Figure 8 The complex XL-Cd 2+ The Job's plot curve of the complex XL-Cd for GSH;

[0028] Figure 9 The color change chart of the probe XL for detecting cadmium ions and glutathione continuously under sunlight;

[0029] Figure 10 The response time chart of the fluorescent probe XL for Cd 2+ at 650nm wavelength;

[0030] Figure 11 The response time chart of the complex XL-Cd 2+ for GSH at 565nm wavelength. DETAILED DESCRIPTION

[0031] The technical solution of the present application is not limited to the following specific embodiments, but also includes any combination of the specific embodiments.

[0032] Specific embodiment one: the molecular structure of the fluorescent probe XL for continuously detecting cadmium ions and glutathione in this embodiment is as follows:

[0033] Specific embodiment two: the preparation method of the fluorescent probe XL is as follows: (E)-2-(3-(3-formyl-4-hydroxy styryl)-5,5-dimethylcyclohex-2-en-1-ylidene) propanedinitrile is condensed with quinoline-2-formylhydrazine to prepare.

[0034] Specific embodiment three: the application of the fluorescent probe XL in continuously detecting cadmium ions and glutathione.

[0035] Specific embodiment four: the difference between this embodiment and specific embodiment three is that the specific method of the fluorescent probe XL for continuously detecting cadmium ions and glutathione is as follows: under the excitation wavelength of 454nm, the fluorescent probe XL has a fluorescence spectrum at 565nm, cadmium ion aqueous solution is added into the probe solution, the fluorescence spectrum at 565nm disappears, a new fluorescence spectrum appears at 650nm and the intensity is significantly enhanced, thereby realizing the fluorescence detection of cadmium ions; glutathione solution is added into the above system, the fluorescence spectrum at 650nm disappears and a fluorescence spectrum appears at 565nm, thereby realizing the fluorescence detection of glutathione.

[0036] Specific embodiment five: the difference between this embodiment and specific embodiment four is that the response time of the fluorescent probe XL for cadmium ions, the response time of the complex XL-Cd 2+ for glutathione is 3s. The rest is the same as specific embodiment four.

[0037] Specific embodiment six: the difference between this embodiment and specific embodiment four or five is that the detection limit of the complex XL-Cd 2+ for glutathione is 7.8×10 -7M. The others are the same as embodiment four or five.

[0038] Embodiment seven: The embodiment is different from embodiment four or five or six in that the system solution presents a color change sequence of yellow-violet red-yellow when the probe XL continuously detects cadmium ions and glutathione under daylight. The others are the same as embodiment four or five or six.

[0039] Embodiment eight: The embodiment is different from embodiment four to seven in that the complex XL-Cd 2+ The detection of glutathione in water bodies can be realized. The others are the same as embodiment four to seven.

[0040] When actually detecting the sample containing cadmium and glutathione, the sample can be pretreated by centrifugation and filtration to remove solid particle impurities.

[0041] The following embodiments of the present application are described in detail, and the following embodiments are implemented on the premise of the technical scheme of the present application, and detailed implementation schemes and specific operation processes are given, but the protection scope of the present application is not limited to the following embodiments.

[0042] Embodiment 1: The preparation method of the fluorescent probe XL is as follows:

[0043] In a 25 mL three-necked flask, (E)-2-(3-(3-formyl-4-hydroxy styryl)-5,5-dimethylcyclohex-2-en-1-ylidene) malononitrile (0.3184 g, 1 mmol), quinoline-2-carbohydrazide (0.2246 g, 1.2 mmol) and 10 mL of anhydrous ethanol were placed, and stirred at 80°C under reflux for 4 h, and thin layer chromatography was used to monitor until the reaction was completed. The reaction liquid was cooled to room temperature, suction filtered, washed with anhydrous ethanol for 3-4 times, and dried to obtain orange fluorescent probe XL with a yield of 90%. 1 H NMR (300 MHz, DMSO-d6) δ 12.66 (s, 1H), 11.80 (s, 1H), 8.94 (s, 1H), 8.64 (d, J = 8.5 Hz, 1H), 8.25 (d, J = 3.6 Hz, 1H), 8.22 (d, J = 3.6 Hz, 1H), 8.13 (d, J = 8.2 Hz, 1H), 7.95 (d, J = 7.4 Hz, 1H), 7.90 (d, J = 6.2 Hz, 1H), 7.81-7.73 (m, 2H), 7.33 (s, 2H), 7.02 (d, J = 8.6 Hz, 1H), 6.86 (s, 1H), 2.62 (s, 2H), 2.57 (s, 2H), 1.03 (s, 6H) ppm. IR (KBr cm -1): 3430, 3260, 3210, 2960, 2920, 2370, 2220, 1700, 1630, 1560, 1350, 1160. Fluorescent probe XL 1 H NMR spectra, infrared spectra are shown in Figure 1 , 2 .

[0044] Example 2: This embodiment of fluorescent probe XL for selective recognition of cadmium ions, according to the following steps:

[0045] respectively prepared concentration of 0.01 mol / L of Zn 2+ , Cd 2+ , K + , Na + , Ca 2+ , Ba 2+ , Mg 2+ , Al 3+ , Li + , Hg 2+ , Cs + , Cr 3+ , Fe 3+ , Pb 2+ , Ag + , Cu 2+ , Bi 3+ aqueous solution for use.

[0046] accurately weighed 4.88 mg of fluorescent probe XL dissolved in 1 mL DMF solution, prepared into 1.0 x 10 -2 mol / L solution A; take 100 μL solution A with buffer solution system (V 四氢呋喃 :V 水 = 95:5, HEPES 10 mM, pH = 7.4) to 100 mL volumetric flask, prepared into 1.0 x 10 -5 mol / L solution B for use.

[0047] each take 3 mL solution B in a cuvette, add 3 eq. of Zn 2+ , Cd 2+ , K + , Na + , Ca 2+ , Ba 2+ , Mg 2+ , Al 3+ , Li + , Hg 2+ , Cs + , Cr 3+ , Fe 3+ , Pb 2+ , Ag + , Cu2+ , Bi 3+ aqueous solution. Under the action of 454 nm excitation light, the fluorescence intensity of the fluorescent probe XL at 565 nm was measured; after Cd 2+ was added into the fluorescent probe XL, the fluorescence spectrum at 565 nm disappeared, a new fluorescence spectrum appeared at 650 nm with significantly enhanced intensity, and bright red fluorescence was emitted, and the solution color changed from yellow to purple red. Except for zinc ions, the addition of other metal ions did not cause obvious fluorescence changes, and the fluorescent probe XL showed good selectivity for Cd 2+ , and the results are shown in Figure 3 .

[0048] Example 3: The complex ratio of the fluorescent probe XL and cadmium ions was determined according to the following steps:

[0049] The total concentration of the probe and cadmium ions in the system was kept at 10 μM, and the equivalent ratio of the probe and cadmium ions was changed to determine the fluorescence intensity, and a Job's Plot curve was drawn, and the results are shown in Figure 4 . According to the analysis of the Job's Plot curve, when the molar fraction of cadmium ions was 0.65, an inflection point appeared in the fluorescence intensity, which indicated that the complex ratio of the probe XL and Cd 2+ was 1:2.

[0050] Example 4: Selective recognition of glutathione by the complex XL-Cd 2+ , according to the following steps:

[0051] 3 mL of solution B with a concentration of 1.0 x 10 -5 mol / L was added with 2 eq. of cadmium ion solution, and after incubation for 2 min, the XL-Cd 2+ system was obtained, and 3 eq. of Arg, Val, Trp, Tyr, Lys, Gly, Ala, Phe, Leu, Iso, Met, Pro, Ser, Thr, Pry, SO3 2- , S2O3 2- , GSH was added in sequence, and under the action of 454 nm excitation light, the fluorescence intensity was measured, and the results are shown in Figure 5 . According to the data analysis, only when GSH was added in the system, the fluorescence spectrum at 650 nm disappeared, the fluorescence spectrum at 565 nm appeared, the fluorescence intensity returned to the level of the probe, the solution color changed from purple red to yellow, and the red fluorescence returned to light pink, and other substances could not cause such changes, and the complex XL-Cd 2+ achieved specific recognition of glutathione.

[0052] Example 5: The complex XL-Cd 2+Anti-interference of glutathione recognition was carried out according to the following steps:

[0053] Take 3 mL of solution B with a concentration of 1.0 x 10 -5 mol / L, add 2 eq. of cadmium ion solution, and incubate for 2 min to obtain XL-Cd 2+ System, sequentially add 3 eq. of Arg, Val, Trp, Tyr, Lys, Gly, Ala, Phe, Leu, Iso, Met, Pro, Ser, Thr, Pry, SO3 2- , S2O3 2- , under the action of 454 nm excitation light, record the fluorescence intensity, then sequentially add 3 eq. of glutathione, and observe and record the fluorescence intensity change, and the results are shown in Figure 6 From the data analysis, it can be seen that after adding glutathione, the fluorescence intensity and solution color change can still be realized in the presence of other coexisting amino acids and anions, and other compounds have no interference with the recognition of complex XL-Cd 2+ to glutathione.

[0054] Example 6: Complex XL-Cd 2+ Detection limit of glutathione, according to the following steps:

[0055] Take 3 mL of solution B with a concentration of 1.0 x 10 -5 mol / L, add 2 eq. of cadmium ion solution, and incubate for 2 min to obtain XL-Cd 2+ System C is ready for use, in 3 mL of solution C, add 3 μL of glutathione aqueous solution with a concentration of 1 x 10 -3 mol / L each time, and measure the fluorescence intensity, and the results are shown in Figure 7 From the data analysis, it can be seen that in the range of 4 μM-8 μM, the fluorescence intensity at 565 nm shows a good linear relationship with the addition of GSH, the linear regression equation is Y = 36.39X + 46.12, R 2 = 0.990, and the detection limit is 7.8 x 10 -7 M, which is significantly lower than the normal concentration in cells (1-2 mM). It shows that complex XL-Cd 2+ can realize trace detection of glutathione.

[0056] Example 7: Complex XL-Cd 2+ Action ratio of glutathione, according to the following steps:

[0057] Keep the total concentration of XL-Cd 2+ and glutathione in the system as 10 μM, and change the concentration of complex XL-Cd 2+The fluorescence intensity was measured by comparing the equivalence ratio with glutathione, and a Job's Plot curve was plotted. The results are as follows: Figure 8 As shown in the figure. Data analysis reveals that the fluorescence intensity reaches an inflection point when the molar fraction of glutathione is 0.57, indicating that the complex XL-Cd... 2+ The complexation ratio with glutathione is 1:1.

[0058] Example 8: Qualitative detection of cadmium ions and glutathione using probe XL under fluorescent light, performed according to the following steps:

[0059] Accurately measure 2 mL of each solution with a concentration of 1.0 × 10⁻⁶. -5 mol / L probe XL solution (1#), complex XL-Cd 2+ Solution C(2#) and the glutathione-containing complex XL-Cd 2+ The solution (3#) yielded the following results: Figure 9 As shown, when probe XL continuously detects cadmium ions and glutathione under fluorescent light, the solution exhibits a color change sequence of yellow, purplish-red, and back to yellow, which is clearly visible to the naked eye. Probe XL can be used for qualitative and visual continuous detection of cadmium ions and glutathione.

[0060] Example 9: Detection of cadmium ions and complex XL-Cd by probe XL 2+ To determine the duration of action of glutathione, follow these steps:

[0061] Add 3 eq. of cadmium ions to solution B and measure the change in fluorescence intensity at 650 nm. Figure 10 As shown, the fluorescence intensity at 650 nm gradually increased with time, and then stabilized after 3 seconds, ceasing to increase. The fluorescence intensity at 565 nm was measured after adding 3 eq. of glutathione to solution C. Figure 11 As shown, the fluorescence intensity at 565 nm stabilizes after 3 seconds and no longer increases. This indicates that probe XL can detect cadmium ions and the complex XL-Cd. 2+ The detection of glutathione can be completed in a short time, with a response time of 3 seconds.

[0062] Example 10: Detection of glutathione in actual water samples using a fluorescence sensor, following these steps:

[0063] To investigate the potential applications of fluorescent probes in real-world environments, domestic water samples were selected and pretreated: the water samples were centrifuged at 12,000 rpm for 10 min, filtered using a 0.45 μm filter, and glutathione aqueous solutions with concentrations of 4 μM, 6 μM, and 8 μM were prepared.

[0064] The complex XL-Cd prepared using Example 42+ System, to the complex XL-Cd 2+ The fluorescence emission peak intensity value of the system under the action of excitation light at 454 nm was recorded, and was brought into the following equation to calculate the concentration of the detected glutathione solution. The detection results are shown in Table 1.

[0065] Y = 36.39X + 46.12

[0066] Wherein, X is the concentration of glutathione, and Y is the fluorescence emission peak intensity value.

[0067] Table 1 Complex XL-Cd 2+ System for detecting glutathione in actual water samples

[0068] Actual water sample Amount added (μM) Detection limit (μM) Recovery (%) RSD (%) 1 (tap water) 4.00 3.94 98.48 2.29 2 (tap water) 6.00 5.95 99.09 0.33 3 (tap water) 8.00 7.89 98.66 1.03

[0069] As can be seen from Table 1, the recovery rate of glutathione in the actual water sample is 98.48% to 99.09%, and the relative standard deviation is 0.33% to 2.29%. The measured concentration of glutathione has a very small error with the corresponding standard concentration. These results show that the prepared fluorescent probe XL-Cd 2+ The system for detecting glutathione in actual water samples has good accuracy, can quantitatively detect glutathione in the range of 4 to 8 μM, and has good practical performance.

Claims

1. A fluorescent probe XL for continuous detection of cadmium ions and glutathione, characterized by The molecular structure of fluorescent probe XL is:

2. The preparation method of the fluorescent probe XL for continuous detection of cadmium ions and glutathione according to claim 1, characterized in that (E)-2-(3-(3-formyl-4-hydroxy styryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile is prepared by condensation reaction of (E)-2-(3-(3-formyl-4-hydroxy styryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile with quinoline-2-formylhydrazine.

3. The use of the fluorescent probe XL in the continuous detection of cadmium ions and glutathione according to claim 1 is for non-disease diagnosis and treatment purposes.

4. Use according to claim 3, characterized in that Under the excitation wavelength of 454 nm, the fluorescent probe XL has a fluorescence spectrum at 565 nm. When cadmium ion aqueous solution is added to the probe solution, the fluorescence spectrum at 565 nm disappears, a new fluorescence spectrum appears at 650 nm and the intensity is significantly enhanced, realizing the fluorescence detection of cadmium ions. When glutathione solution is added to the above system, the fluorescence spectrum at 650 nm disappears and a fluorescence spectrum appears at 565 nm, realizing the fluorescence detection of glutathione.

5. Use according to claim 4, characterized in that Response time of fluorescent probe XL to cadmium ion, complex XL-Cd 2+ Response time to glutathione is 3 s.

6. Use according to claim 4, characterized in that Complex XL-Cd 2+ The limit of detection for glutathione was 7.8 x 10 -7 mol / L.

7. Use according to claim 4, characterized in that Under the sunlight, when the probe XL continuously detects cadmium ions and glutathione, the system solution will present a color change sequence of yellow-violet red-yellow.

8. Use according to claim 4, complex XL-Cd 2+ The detection of glutathione in water bodies can be achieved.

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