Preparation method and application of fluorescent probe for continuously detecting cadmium ions and glutathione

Abstract

The invention discloses 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 isophorone fluorescent probe. The method is used for solving the problem that two substances, namely cadmium ions and glutathione, cannot be continuously recognized under the same test condition by an existing fluorescent probe detection method. The preparation method of the fluorescent probe disclosed by the invention comprises the following step: carrying out condensation on (E)-2-(3-(3-formyl-4-hydroxystyryl)-5, 5-dimethylcyclohex-2-ene-1-subunit) malononitrile and quinoline-2-formylhydrazine, so as to obtain the fluorescent probe XL. The fluorescent probe prepared by the invention can quickly recognize cadmium ions with high selectivity, and meanwhile, a complex of the probe and the cadmium ions has good recognition selectivity on glutathione and is not interfered by other amino acids and anions. The detection method constructed by the invention is sensitive and efficient, expensive large instruments and professional experimenters are not needed, and glutathione in a water body environment can be detected.

Description

Technical Field

[0001] The invention relates to the field of cadmium ion and glutathione detection, and in particular to a method for preparing a fluorescent probe and application of the fluorescent probe in continuous detection of cadmium ions and glutathione. Background Art

[0002] Cadmium is one of the most toxic and harmful heavy metal elements. It can come from natural processes, such as volcanic eruptions and forest fires, or from various industrial production, including electroplating, alloy making, preparation of nickel-cadmium batteries, and military industry. Cadmium ions discharged from industrial production can cause soil and water pollution, bringing environmental risks. At the same time, cadmium ions are easily enriched in plants, which will damage the cell membranes and organelles of plants, which is not conducive to plant metabolism and causes plant death. In addition, when cadmium ions in the environment and plants enter the human body, they will cause renal tubular dysfunction and bone degeneration, gastrointestinal dysfunction and abnormal liver function, and will also increase the risk of cancer and threaten human health. Therefore, the detection of cadmium ions in the environment and organisms is of great significance.

[0003] Glutathione is a tripeptide substance containing active thiol groups composed of glutamic acid, cysteine ​​and glycine. As an important bioactive peptide in the body, reduced glutathione can be used as an antioxidant to remove free radicals in the human body, and can play a good role in anti-tumor and immune enhancement; as a heavy metal chelator, glutathione can form easily soluble and non-toxic complexes with metal ions to reduce the toxic effects of metal ions on the human body; in addition, glutathione can also alleviate the side effects produced during disease treatment and relieve the pain of patients. Therefore, in daily life, glutathione can be used as an additive and is widely used in the field of food processing. At the same time, it can also be used as an important ingredient in various health products to supplement the demand for glutathione in normal physiological activities of the human body. However, glutathione imbalance in the human body may lead to a variety of diseases, such as Alzheimer's disease, heart disease, cardiovascular disease, cancer and premature aging, so it is extremely important to monitor the glutathione content in food and organisms.

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

[0005] In recent years, fluorescent probes that can identify 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 invention designs and synthesizes a novel isophorone fluorescent probe, which can efficiently and quickly identify cadmium ions in a buffer solution, and the complex formed can also achieve specific recognition of glutathione, with the advantages of good selectivity, strong anti-interference and high sensitivity. The fluorescent probe prepared by the present invention can continuously identify cadmium ions and glutathione under the same test conditions, providing a preparation method and application of a multifunctional fluorescent probe. Summary of the invention

[0006] The invention aims to solve the problem that it is difficult to realize continuous detection of cadmium ions and glutathione under the same test conditions, and provides a preparation method and application of a fluorescent probe for continuous detection of cadmium ions and glutathione.

[0007] The present invention is a fluorescent probe for continuously detecting cadmium ions and glutathione, wherein the molecular structure of the fluorescent probe XL is:

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

[0009]

[0010] The method for preparing a fluorescent probe for continuously detecting cadmium ions and glutathione comprises the following steps: (E)-2-(3-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile and quinoline-2-carboxylic acid hydrazide are subjected to a condensation reaction to obtain a fluorescent probe XL.

[0011] The specific method of the present invention using fluorescent probe to continuously detect cadmium ions and glutathione is:

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

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

[0014] Furthermore, the complex XL-Cd 2+ The detection limit for glutathione was 7.8×10 -7 mol / L.

[0015] Furthermore, under fluorescent light, when the probe XL continuously detects cadmium ions and glutathione, the system solution will show a color change sequence of yellow-purple-yellow.

[0016] Furthermore, the complex XL-Cd 2+ It can realize the detection of glutathione in water.

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

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

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

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

[0021] Figure 2 IR spectrum of the fluorescent probe XL prepared by the present invention;

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

[0023] Figure 4 Fluorescent probe XL for Cd 2+ Job's plot curve;

[0024] Figure 5 Complex XL-Cd 2+ Relationship diagram of selective recognition of amino acids and anions;

[0025] Figure 6 Coexistence of amino acids and anion pairs in the complex XL-Cd 2+ Identify the GSH relationship graph;

[0026] Figure 7 Complex XL-Cd 2+ The fluorescence response relationship diagram to different concentrations of GSH;

[0027] Figure 8 Complex XL-Cd 2+ Job's plot curve for GSH;

[0028] Fig. 9 Under fluorescent light, the probe XL continuously detects the color change of cadmium ions and glutathione;

[0029] Fig.10 Fluorescence probe XL for Cd at 650nm wavelength 2+ Response time graph of

[0030] Fig.11 The complex XL-Cd at a wavelength of 565 nm 2+ Time plot of response to GSH. DETAILED DESCRIPTION

[0031] The technical solution of the present invention is not limited to the specific implementation modes listed below, but also includes any combination of the specific implementation modes.

[0032] Specific embodiment 1: The molecular structure of the fluorescent probe XL used for continuous detection of cadmium ions and glutathione in this embodiment is:

[0033] Specific implementation method 2: The preparation method of the fluorescent probe XL is as follows: (E)-2-(3-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile and quinoline-2-carboxylic acid hydrazide undergo condensation reaction to prepare the fluorescent probe XL.

[0034] Specific implementation method three: the application of the fluorescent probe XL in the continuous detection of cadmium ions and glutathione in this implementation method.

[0035] Specific implementation method four: This implementation method is different from specific implementation method three in that: the specific method of continuous detection of cadmium ions and glutathione by fluorescent probe XL is: under an excitation wavelength of 454nm, the fluorescent probe XL has a fluorescence spectrum at 565nm, and a cadmium ion aqueous solution is added to the probe solution, the fluorescence spectrum at 565nm disappears, and a new fluorescence spectrum appears at 650nm with significantly enhanced intensity, thereby realizing fluorescence detection of cadmium ions; glutathione solution is added to the above system, the fluorescence spectrum at 650nm disappears, and a fluorescence spectrum appears at 565nm, thereby realizing fluorescence detection of glutathione.

[0036] Specific embodiment 5: This embodiment differs from specific embodiment 4 in that: the response time of the fluorescent probe XL to cadmium ions, the complex XL-Cd 2+ The response time of glutathione is 3s. Other aspects are the same as those of the fourth embodiment.

[0037] Specific embodiment 6: This embodiment is different from specific embodiment 4 or 5 in that: the complex XL-Cd 2+ The detection limit for glutathione was 7.8×10 -7M. The rest is the same as the fourth or fifth specific implementation mode.

[0038] Specific embodiment 7: This embodiment is different from specific embodiment 4, 5 or 6 in that when the probe XL continuously detects cadmium ions and glutathione under a fluorescent lamp, the system solution will show a color change sequence of yellow-purple-yellow. Others are the same as specific embodiment 4, 5 or 6.

[0039] Specific embodiment eight: This embodiment differs from specific embodiments four to seven in that: the complex XL-Cd 2+ The detection of glutathione in water can be realized. Other specific implementation methods are the same as those of fourth to seventh.

[0040] When actually testing samples containing cadmium and glutathione, the samples can be pre-treated by centrifugation and filtration to remove solid particle impurities.

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

[0042] Example 1: The preparation method of the fluorescent probe XL in this example is carried out according to the following steps:

[0043] In a 25mL three-necked flask, (E)-2-(3-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile (0.3184g, 1mmol), quinoline-2-carboxylic acid hydrazide (0.2246g, 1.2mmol) and 10mL of anhydrous ethanol were placed, stirred and refluxed at 80°C for 4h, and monitored by thin layer chromatography until the reaction was completed. The reaction solution was cooled to room temperature, filtered, washed with anhydrous ethanol 3-4 times, and dried to obtain orange-red fluorescent probe XL with a yield of 90%. 1 H NMR (300MHz, DMSO-d6) δ12.66(s,1H),11.80(s,1H),8.94(s,1H),8.64(d,J=8.5 Hz,1H),8.25(d,J=3.6Hz,1H),8.22(d,J=3.6Hz,1H),8.13(d,J=8.2Hz,1H),7.9 5(d,J=7.4Hz,1H),7.90(d,J=6.2Hz,1H),7.81-7.73(m,2H),7.33(s,2H),7.02( d,J=8.6Hz,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 spectrum and infrared spectrum are shown in Figure 1 , 2 shown.

[0044] Example 2: The fluorescent probe XL in this example selectively recognizes cadmium ions according to the following steps:

[0045] Prepare Zn with a concentration of 0.01 mol / L 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+ The aqueous solution is ready for use.

[0046] Accurately weigh 4.88 mg of fluorescent probe XL and dissolve it in 1 mL of DMF solution to prepare 1.0×10 -2 mol / L solution A; take 100 μL of solution A and use the buffer solution system (V 四氢呋喃 :V 水 =95:5, HEPES 10mM, pH = 7.4) to a 100mL volumetric flask and prepared to 1.0×10 -5 mol / L solution B is set aside for use.

[0047] Take 3 mL of solution B in a cuvette each time, and 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 454nm excitation light, the fluorescence intensity of fluorescent probe XL was measured at 565nm; Cd was added to the fluorescent probe XL 2+ After that, the fluorescence spectrum at 565nm disappeared, and a new fluorescence spectrum appeared at 650nm with significantly enhanced intensity, and bright red fluorescence was emitted, and the color of the solution changed from yellow to purple-red. Except for the slight change in the fluorescence spectrum caused by the addition of zinc ions, the addition of other metal ions did not cause obvious fluorescence changes. The fluorescence probe XL was 2+ Showing good selectivity, the results are as follows Figure 3 shown.

[0048] Example 3: The complexation of the fluorescent probe XL with cadmium ions in this example is carried out according to the following steps:

[0049] Keeping the total concentration of probe and cadmium ions in the system at 10 μM, the fluorescence intensity was measured by changing the equivalent ratio of probe and cadmium ions, and the Job's Plot curve was drawn. The results are shown in Figure 4 As shown in the figure, Job's Plot shows that when the molar fraction of cadmium ions is 0.65, the fluorescence intensity has an inflection point, which indicates that the probe XL and Cd 2+ The complexation ratio is 1:2.

[0050] Example 4: Complex XL-Cd 2+ For selective recognition of glutathione, follow these steps:

[0051] Take 3mL of the solution with a concentration of 1.0×10 -5 2 eq. of cadmium ion solution was added to 10 mol / L solution B and incubated for 2 min to obtain XL-Cd 2+ system, add 3eq. of Arg, Val, Trp, Tyr, Lys, Gly, Ala, Phe, Leu, Iso, Met, Pro, Ser, Thr, Pry, SO3 2- 、S2O3 2- , GSH, under the action of 454nm excitation light, measure its fluorescence intensity, the results are as follows Figure 5 As shown in the data analysis, only when GSH is added to the system, the fluorescence spectrum at 650nm disappears, the fluorescence spectrum at 565nm appears, the fluorescence intensity returns to the probe level, the solution color changes from purple-red to yellow, and the red fluorescence returns to light pink. Other substances cannot cause this change. The complex XL-Cd 2+ Specific recognition of glutathione was achieved.

[0052] Example 5: Complex XL-Cd 2+To identify the anti-interference of glutathione, follow the steps below:

[0053] Take 3mL of the solution with a concentration of 1.0×10 -5 2 eq. of cadmium ion solution was added to 10 mol / L solution B and incubated for 2 min to obtain XL-Cd 2+ system, add 3eq. of Arg, Val, Trp, Tyr, Lys, Gly, Ala, Phe, Leu, Iso, Met, Pro, Ser, Thr, Pry, SO3 2- 、S2O3 2- , under the action of 454nm excitation light, record its fluorescence intensity, then add 3eq. of glutathione in sequence, observe and record the change of fluorescence intensity, the results are as follows Figure 6 From the data analysis, it can be seen that in the presence of other coexisting amino acids and anions, the fluorescence intensity and solution color change can still be achieved after adding glutathione. 2+ Recognizes glutathione without interference.

[0054] Example 6: Complex XL-Cd 2+ For the glutathione detection limit, follow these steps:

[0055] Take 3mL of the solution with a concentration of 1.0×10 -5 2 eq. of cadmium ion solution was added to 10 mol / L solution B and incubated for 2 min to obtain XL-Cd 2+ System C is ready for use. Add 3 μL of 1×10 -3 mol / L glutathione aqueous solution, measure the fluorescence intensity, the result is as follows Figure 7 As shown in the data analysis, within the range of 4μM-8μM, the fluorescence intensity at 565nm showed a good linear relationship with the addition of GSH, and the linear regression equation was Y=36.39X+46.12, R 2 =0.990, the detection limit is 7.8×10 -7 M, significantly lower than the normal intracellular concentration (1-2 mM). 2+ Trace detection of glutathione can be achieved.

[0056] Example 7: Complex XL-Cd 2+ For the glutathione action ratio, follow the steps below:

[0057] Keep XL-Cd in the system 2+ The total concentration of glutathione was kept constant at 10 μM. By changing the complex XL-Cd 2+The equivalent ratio of glutathione and fluorescence intensity was measured and Job's Plot was plotted. The results are shown in Figure 8 From the data analysis, it can be seen that when the molar fraction of glutathione is 0.57, the fluorescence intensity has an inflection point, which indicates that the complex XL-Cd 2+ The complexation ratio with glutathione is 1:1.

[0058] Example 8: Under fluorescent light, the probe XL qualitatively detects cadmium ions and glutathione according to the following steps:

[0059] Accurately measure 2 mL of the solution with a concentration of 1.0×10 -5 mol / L probe XL solution (1#), complex XL-Cd 2+ Solution C (2#) and complex XL-Cd containing glutathione 2+ The solution (3#) was tested. Fig. 9 As shown, under fluorescent light, when the probe XL continuously detects cadmium ions and glutathione, the solution will show a color change sequence from yellow, purple-red, to yellow, which is obvious to the naked eye. The probe XL can be used for qualitative visual continuous detection of cadmium ions and glutathione.

[0060] Example 9: Detection of Cadmium Ions and Complex XL-Cd by Probe XL 2+ To test the action time of glutathione, follow the steps below:

[0061] Add 3eq. of cadmium ions to solution B and measure the change in fluorescence intensity at 650nm. Fig.10 As shown in the figure, as time goes by, the fluorescence intensity at 650nm gradually increases, and after 3s, the fluorescence intensity gradually stabilizes and no longer increases. 3eq. of glutathione was added to solution C, and the change of fluorescence intensity at 565nm was measured, as shown in the figure. Fig.11 As shown in the figure, the fluorescence intensity at 565nm also stabilized after 3s and no longer increased. This shows that the probe XL can detect cadmium ions and the complex XL-Cd 2+ Glutathione detection can be completed in a short time, with a response time of 3s.

[0062] Example 10: The fluorescent sensor detects glutathione in an actual water sample according to the following steps:

[0063] In order to investigate the potential application of fluorescent probes in actual environments, domestic drinking water was selected and the water samples were pretreated: the water samples were centrifuged at 12000 rpm for 10 min, filtered with 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 in Example 42+ system, to the complex XL-Cd 2+ The above-mentioned glutathione aqueous solutions of different concentrations were added to the system. Under the action of 454nm excitation light, the peak intensity value of fluorescence emission was recorded and substituted into the following equation to calculate the concentration of the glutathione solution to be tested. The test results are shown in Table 1.

[0065] Y=36.39X+46.12

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

[0067] Table 1 Complex XL-Cd 2+ The system detects glutathione in real water samples

[0068] Actual water sample Addition amount (μM) Detection amount (μM) Recovery rate (%) 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] It can be seen from Table 1 that the recovery rate of glutathione in the actual water sample is 98.48% to 99.09%, the relative standard deviation is 0.33% to 2.29%, and the error between the measured glutathione concentration and the corresponding spiked concentration is very small. These results show that the fluorescent probe XL-Cd prepared by the present invention 2+ The system detects glutathione in actual water samples with good accuracy and can quantitatively detect glutathione in the range of 4 to 8 μM, showing good practical performance.

Claims

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

2. A fluorescent probe for continuously detecting cadmium ions and glutathione according to claim 1, characterized in that The preparation method of the fluorescent probe XL is as follows: (E)-2-(3-(3-formyl-4-hydroxyphenylvinyl)-5,5-dimethylcyclohex-2-ene-1-ylidene)malononitrile and quinoline-2-carboxylic acid hydrazide undergo condensation reaction to prepare the fluorescent probe XL.

3. Use of the fluorescent probe XL as claimed in claim 1 in the continuous detection of cadmium ions and glutathione.

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

5. The use according to claim 4, characterized in that Response time of fluorescent probe XL to cadmium ions and complex XL-Cd 2+ The response time to glutathione is 3 s.

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

7. The use according to claim 4, characterized in that Under fluorescent light, when the probe XL continuously detects cadmium ions and glutathione, the system solution will show a color change sequence of yellow-purple-yellow.

8. The use according to claim 3, wherein the complex XL-Cd 2+ It can realize the detection of glutathione in water.

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