Preparation method and application of fluorescent probe for series detection of copper ions and cysteine

Abstract

The invention discloses a preparation method and application of a fluorescent probe for tandem detection of copper ions and cysteine, relates to the field of analysis and detection of the copper ions and the cysteine, and in particular relates to a preparation method of a 1, 8-naphthalimide fluorescent probe L and tandem detection of the copper ions and the cysteine. The defects that an existing cysteine detection method is low in sensitivity and prone to being affected by glutathione can be overcome. The preparation method of the probe L provided by the invention comprises the following steps: carrying out a reflux reaction on 4-hydrazino-N-(2-ethoxyl) ethoxy-1, 8-naphthalimide and quinoline-2-formaldehyde in absolute ethyl alcohol to prepare the fluorescent probe L; the fluorescent probe L prepared by the invention can continuously realize specific fluorescence'on-off-on 'detection on copper ions and cysteine in a CH3CN / HEPES buffer solution system with the volume ratio of 3: 7, has good selectivity in the detection process, and can resist interference of various factors. The method can be used for qualitative and quantitative detection of cysteine in milk.

Description

Technical Field

[0001] The invention relates to the field of analysis and detection of copper ions and cysteine, and in particular to a preparation method of a 1,8-naphthalimide fluorescent probe L and application thereof in tandem detection of copper ions and cysteine. Background Art

[0002] Copper is the third most abundant transition metal ion in the human body and plays a vital role in redox reactions, signal transduction, hematopoiesis and other physiological processes. Abnormal copper ion content in the human body can lead to certain diseases, such as anemia, coronary heart disease, Menkes syndrome, etc. At the same time, the destruction of metal homeostasis by copper ions is also an important factor in causing cancer, cardiovascular disease and Parkinson's disease. In the ecological environment, excessive intake of copper ions can also lead to slow growth or even death of plants. Therefore, it is of great significance to dynamically monitor copper ions in the environment and organisms.

[0003] Cysteine ​​(Cys), as an indispensable amino acid in organisms, plays an important role in regulating physiological and pathological processes. It can directly participate in many physiological processes, such as protein synthesis, detoxification and cell metabolism, and is also related to the occurrence of many diseases. When cysteine ​​is deficient or excessive, it can lead to a series of diseases, including Alzheimer's disease, cancer, cardiovascular disease, diabetes and skin diseases. In daily life, its normal level in the body can be maintained by consuming foods rich in cysteine ​​or its precursors. High-protein foods such as beef, pork, and mutton, as well as dairy products such as eggs, milk, and cheese are good sources of cysteine. Therefore, the content of cysteine ​​in food has an important influence on its content in organisms. Sensitive and accurate detection of cysteine ​​content in food and biological samples is of great significance in clinical medicine and basic biochemistry.

[0004] 1,8-Naphthalimide is one of the excellent fluorophores. It is often used as the basis for the design of fluorescent probe molecules because of its advantages such as large conjugated system, large Stokes shift and easy modification. Its derivatives not only have high fluorescence quantum yield and excellent photostability, but also introduce strong electron-withdrawing groups and strong electron-donating groups at the 4th and 5th positions of the nitrogen atom and naphthalene ring of the 1,8-naphthalimide fragment, which is beneficial to electron transition, thereby making it have good compatibility and high selectivity, and therefore is widely used in the field of fluorescent probe preparation. In the present invention, the hydrophilicity of the probe is increased by introducing 2-(2-aminoethoxy)ethanol and hydrazine groups at the N and 4 positions of 1,8-naphthalimide, respectively, and the hydrazine structure is condensed with quinoline-2-carboxaldehyde to form a Schiff base, thereby constructing a recognition site. Based on the above ideas, a 1,8-naphthalimide fluorescent probe L was successfully designed and prepared. It can detect copper ions and cysteine ​​in series under the same test conditions. It belongs to the fluorescent "on-off-on" type detection. The detection process has good selectivity, strong anti-interference, high sensitivity and easy operation, and has good application prospects in the field of food monitoring. Summary of the invention

[0005] The present invention aims to solve the problem that cysteine ​​is easily interfered by common amino acids and bioactive peptide glutathione during the detection process, and provides a preparation method and application of a fluorescent probe for serial detection of copper ions and cysteine.

[0006] The fluorescent probe L used for serial detection of copper ions and cysteine ​​of the present invention has a molecular structure as follows:

[0007]

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

[0009]

[0010] The preparation method of the fluorescent probe L for serial detection of copper ions and cysteine ​​in the present invention is as follows: 4-hydrazino-N-(2-hydroxyethyl)ethoxy-1,8-naphthalimide and quinoline-2-carboxaldehyde are subjected to reflux reaction in anhydrous ethanol.

[0011] Application of the fluorescent probe L of the present invention in the quantitative and qualitative detection of copper ions and cysteine.

[0012] Preferably, probe L can realize continuous fluorescence "on-off-on" detection of copper ions and cysteine.

[0013] Preferably, the detection limit of probe L for copper ions is as low as 3.6×10 -8 mol / L, and the detection limit of cysteine ​​is as low as 4.2×10 -8 mol / L.

[0014] Preferably, the complex L-Cu 2+ In the process of detecting cysteine, it can resist the interference of common amino acids Phe, Phg, Ala, Gly, Glu, Tyr, Leu, Pro, Trp, Ser, Val, Ile, His and bioactive peptide Gsh.

[0015] Preferably, probe L can be used for qualitative and quantitative detection of cysteine ​​in milk.

[0016] Principle of the present invention:

[0017] The fluorescent probe L prepared by the present invention has a rigid plane and a large-scale π conjugated system in its structure, which can make the probe produce strong and stable fluorescence. The three nitrogen atoms at the 4-position of the probe L can form a complex with copper ions in a 1:1 ratio. The electron-donating ability of the nitrogen atoms in the group disappears, and the conjugation effect is blocked, resulting in fluorescence quenching, thereby realizing the detection of copper ions. 2+ After cysteine ​​is added to the system, cysteine ​​can capture copper ions from the complex and release the probe itself, so that the fluorescence is restored, and finally the detection of cysteine ​​is realized.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The fluorescent probe L prepared by the present invention can realize the tandem detection of copper ions and cysteine ​​under the same test conditions.

[0020] 2) The complex L-Cu 2+ During the detection of cysteine, there is no interference from other amino acids Phe, Phg, Ala, Gly, Glu, Tyr, Leu, Pro, Trp, Ser, Val, Ile, His and bioactive peptide Gsh. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The present invention prepares the fluorescent probe L 1 H NMR spectrum;

[0022] Figure 2 The FT-RT spectrum of the fluorescent probe L prepared by the present invention;

[0023] Figure 3 The selectivity of fluorescent probe L in recognizing metal ions;

[0024] Figure 4 Linear relationship diagram of fluorescence response of fluorescent probe L to different concentrations of copper ions;

[0025] Figure 5 Fluorescent probe L for Cu 2+Job's plot curve;

[0026] Figure 6 Complex L-Cu 2+ Selective recognition map for different amino acids;

[0027] Figure 7 Effect of coexisting amino acids and glutathione on the complex L-Cu 2+ Identify cysteine ​​influence maps;

[0028] Figure 8 Complex L-Cu 2+ Linear relationship diagram of fluorescence response to different concentrations of cysteine;

[0029] Fig. 9 Complex L-Cu 2+ Job's plot curve for Cys; DETAILED DESCRIPTION

[0030] 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.

[0031] Specific embodiment 1: The molecular structure of the fluorescent probe used for tandem detection of copper ions and cysteine ​​in this embodiment is:

[0032]

[0033] Specific implementation method 2: The preparation method of the fluorescent probe L comprises the following steps: 4-hydrazino-N-(2-hydroxyethyl)ethoxy-1,8-naphthalimide and quinoline-2-carboxaldehyde are refluxed in ethanol for 10 to 12 hours; the reaction product is filtered, washed and dried to obtain 1,8-naphthalimide fluorescent probe L.

[0034] Specific implementation method three: Application of the fluorescent probe L in the present implementation method in the tandem detection of copper ions and cysteine.

[0035] Specific embodiment 4: The specific method of the fluorescent probe L in this embodiment for serially detecting copper ions and cysteine ​​is:

[0036] 1. Dissolve the fluorescent probe L in a CH3CN / HEPES buffer solution with a volume ratio of 3:7 to prepare 1.0×10 -5 mol / L probe solution, under the action of 471nm excitation light, measure the fluorescence emission peak intensity of the fluorescent probe at 549nm; add copper nitrate aqueous solution to the probe solution, and measure the fluorescence emission peak intensity of the fluorescent probe at 549nm;

[0037] 2. The fluorescent probe L solution was incubated with copper nitrate for 10 to 12 seconds, and then cysteine ​​aqueous solution was added to the system. Under the action of 471 nm excitation light, the probe complex L-Cu 2+ The peak fluorescence emission intensity is at 549 nm.

[0038] Specific embodiment 5: This embodiment is different from specific embodiment 3 or 4 in that the fluorescent probe L can realize continuous fluorescence "on-off-on" detection of copper ions and cysteine. Other aspects are the same as specific embodiment 3 or 4.

[0039] Specific embodiment 6: This embodiment is different from specific embodiments 3 to 5 in that: the complex L-Cu 2+ The detection process of cysteine ​​can resist the interference of common amino acids Phe, Phg, Ala, Gly, Glu, Tyr, Leu, Pro, Trp, Ser, Val, Ile, His and bioactive peptide Gsh. Other aspects are the same as those of the third to fifth embodiments.

[0040] Specific embodiment 7: This embodiment is different from specific embodiments 3 to 6 in that the detection limit of probe L for copper ions is as low as 3.6×10 -8 mol / L and L-Cu 2+ The detection limit of cysteine ​​is as low as 4.2×10 -8 mol / L. The rest is the same as the specific embodiments three to six.

[0041] Specific embodiment eight: This embodiment differs from specific embodiments three to seven in that the probe L is used for qualitative and quantitative detection of cysteine ​​in milk. The recovery rate of cysteine ​​in milk samples is 93.38% to 106.25%, and the relative standard deviation is 1.11% to 2.19%. Other aspects are the same as specific embodiments three to seven.

[0042] When actually testing samples containing copper ions and cysteine, the samples can be pre-treated by centrifugation and filtration to remove solid particle impurities.

[0043] 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 plans and specific operation processes are given, but the protection scope of the present invention is not limited to the following embodiments.

[0044] Example 1: The preparation method of the fluorescent probe L in this example is carried out according to the following steps:

[0045] 4-Hydrazino-N-(2-hydroxyethyl)ethoxy-1,8-naphthalimide (0.2000 g, 0.63 mmol), quinoline-2-carboxaldehyde (0.1189 g, 0.76 mmol) and 15 mL of anhydrous ethanol were added to a 50 mL three-necked flask and refluxed for 10 h. After cooling to room temperature, the mixture was vacuum filtered and dried, and washed with ethyl acetate to obtain 1,8-naphthalimide fluorescent probe L (0.1821 g) with a yield of 63.60%. 1 HNMR (300MHz, DMSO-d6) δ11.78(s,1H),8.84(d,J=8.7Hz,1H),8.61(s,1H),8.51(d,J=8.7Hz,1H),8. 42(t,J=8.6Hz,2H),8.27(d,J=9.4Hz,1H),8.02(t,J=8.7Hz,2H),7.92–7.76(m,3H),7.62(t,J=6.0Hz ,1H),4.54(s,1H),4.23(t,J=6.7Hz,2H),3.66(t,J=6.4Hz,2H),3.48(s,4H)ppm.FT-IR(KBr):3407,3 257,2868,1680,1649,1575,1505,1428,1380,1277,1244,1277,1121,1059,850,827,778,757,620cm -1 . Probe L 1 H NMR spectrum, FT-RT spectrum such as Figure 1 , 2 shown.

[0046] Example 2: Preparation of probe L solution is carried out according to the following steps:

[0047] Accurately weigh 3.2 mg of probe L and prepare 1.0×10 -2 mol / L probe mother solution A; take 50 μL of probe mother solution A and dilute it to 50 mL with CH3CN / HEPES buffer solution with a volume ratio of 3:7 to prepare 1.0×10 -5 mol / L probe solution B is set aside.

[0048] Example 3: The probe L in this example selectively recognizes copper ions according to the following steps:

[0049] Take 3 mL of solution B in a cuvette each time, and add 3 eq. of Cu 2+ , Ba 2+ 、Cd 2+ 、Co 2+ 、Zn 2+ 、Ag + , Ca2+ Cr 3+ , K + 、Al 3+ , Fe 3+ Mg 2+ , Pb 2+ 、Na + 、Ni 2+ , Cs + 、Ce 3+ , Li + , Hg 2+ 、Bi 2+ Aqueous solution. Under an excitation wavelength of 471 nm, the peak fluorescence emission intensity of the fluorescent probe L was measured at 549 nm, and the slit width was 5 nm. The results are shown in Figure 3 As shown, Cu was added to the fluorescent probe L 2+ After addition, there is an obvious fluorescence quenching effect, while the addition of other metal ions does not cause obvious fluorescence changes, indicating that the fluorescent probe L has a strong effect on Cu 2+ Shows specific recognition effect.

[0050] Example 4: The detection limit of the fluorescent probe L for copper ions in this example is carried out according to the following steps:

[0051] Take 3mL of the solution with a concentration of 1.0×10 -5 mol / L solution B, add 3 μL each time to a concentration of 1×10 -3 mol / L copper ion aqueous solution, measure the fluorescence intensity. Plot the graph with copper ion concentration as the horizontal axis and fluorescence intensity as the vertical axis. The results are as follows Figure 4 As shown in the figure, when the copper ion concentration is within the range of 0 μM-10 μM, the fluorescence intensity decreases with the increase of copper ion concentration, and the fluorescence intensity shows a good linear relationship with the copper ion concentration. The fitting equation is y=-111.09x+1202.21, R 2 =0.994. According to the calculation formula of detection limit 3σ / k, the detection limit of probe L for copper ions is calculated to be 3.6×10 -8 mol / L, probe L can realize trace detection of copper ions.

[0052] Example 5: The complexation of probe L with copper ions in this example is carried out according to the following steps:

[0053] The concentration is 1.0×10 -5 mol / L solution B and 1×10 -3 mol / L copper ion aqueous solution, keeping the total concentration of probe L and copper ions in the system at 1×10 -5 mol / L remains unchanged, by changing the equivalent ratio of probe L and copper ions, the fluorescence intensity is measured respectively, and the Job's Plot curve is drawn. Figure 5As shown in the figure, when the molar fraction of copper ions is 0.51, the fluorescence intensity has an inflection point, which indicates that the probe L and Cu 2+ The complexation ratio is 1:1.

[0054] Example 6: Complex L-Cu 2+ For selective recognition of cysteine, follow these steps:

[0055] Take 3mL of the solution with a concentration of 1.0×10 -5 mol / L solution B was added with 1eq. of copper ion solution and incubated for 10s to obtain the complex L-Cu 2+ System solution, add 3eq. concentration of 1×10 -2 mol / L of Cys, Phe, Phg, Ala, Gly, Glu, Tyr, Leu, Pro, Trp, Ser, Val, Ile, His, Gsh aqueous solution, and measure its fluorescence intensity at an excitation wavelength of 471nm. The results are as follows Figure 6 As shown in the figure, after adding cysteine, the fluorescence of the system began to increase, while when other anions and amino acids were added, the fluorescence intensity did not change significantly. 2+ The system achieves specific recognition of cysteine.

[0056] Example 7: Complex L-Cu 2+ The anti-interference of cysteine ​​recognition is carried out according to the following steps:

[0057] Take 3mL of the solution with a concentration of 1.0×10 -5 mol / L solution B was added with 1eq. of copper ion solution and incubated for 10-12s to obtain the complex L-Cu 2+ system, and then add 3eq. of 1×10 -2 mol / L of Cys, Phe, Phg, Ala, Gly, Glu, Tyr, Leu, Pro, Trp, Ser, Val, Ile, His and Gsh aqueous solutions were added at an excitation wavelength of 471 nm and their fluorescence intensity was recorded. Then, 3 eq. of cysteine ​​aqueous solution were added respectively and the changes in fluorescence intensity were observed and recorded. The results are shown in Figure 7 As shown, in the presence of other amino acids and glutathione, the complex L-Cu 2+ The fluorescence of the system did not change significantly, but fluorescence enhancement was achieved after the addition of cysteine. Therefore, other amino acids and glutathione have a significant effect on the complex L-Cu 2+ The system recognizes cysteine ​​without interference.

[0058] Example 8: Complex L-Cu 2+ The detection limit of cysteine ​​was determined by the following steps:

[0059] The concentration is 1.0×10 -5 mol / L solution B was added with 1eq. of copper ion solution and incubated for 10-12s to obtain the complex L-Cu 2+ System solution C is set aside. Take 3 mL of solution C and add 3 μL of 1×10 -3 mol / L cysteine ​​aqueous solution, detect and record the fluorescence intensity each time. Figure 8 As shown in the figure, when the cysteine ​​concentration is within the range of 0 μM-20 μM, the fluorescence intensity of the complex system increases with the increase of cysteine ​​concentration, and the fluorescence intensity shows a good linear relationship with the cysteine ​​concentration. The fitting equation is y=45.56x+97.33, R 2 =0.993. According to the calculation formula of detection limit 3σ / k, the complex L-Cu 2+ The detection limit for cysteine ​​is 4.2×10 -8 mol / L. This shows that the complex L-Cu 2+ The system can realize trace detection of cysteine.

[0060] Example 9: The complex L-Cu 2+ To react with cysteine, follow these steps:

[0061] The concentration is 1.0×10 -5 mol / L solution C and 1×10 -3 mol / L cysteine ​​aqueous solution, keeping the total concentration of complex and copper ion in the system at 1×10 -5 mol / L remains unchanged, by changing the complex L-Cu 2+ The fluorescence intensity was measured and the Job's Plot curve was drawn. Fig. 9 As shown in the figure, when the molar fraction of cysteine ​​is 0.65, the fluorescence intensity has an inflection point, which indicates that the complex L-Cu 2+ The complexation ratio with cysteine ​​is 1:2.

[0062] Example 10: Complex L-Cu 2+ Quantitative determination of cysteine ​​in milk

[0063] In order to investigate the complex L-Cu 2+For potential application in milk, commercially available milk was selected as the test sample, and the milk was pretreated: diluted 70 times with double distilled water, 3 mL of cysteine ​​content in the determination solution was taken, and then different amounts of cysteine ​​were added to the milk sample to prepare cysteine ​​milk solutions with concentrations of 4 μmol / L, 8 μmol / L, and 12 μmol / L. Under the action of 471 nm excitation light, the peak intensity of the fluorescence emission of the fluorescent probe at 549 nm was measured, and it was substituted into the following equation to calculate the concentration of the cysteine ​​to be tested. The test results are shown in Table 1. Each sample was tested 3 times. Complex L-Cu 2+ The probe complex L-Cu prepared in Example 7 2+ System solution.

[0064] y=45.56x+97.33

[0065] Where x is the cysteine ​​concentration and y is the fluorescence emission peak intensity value.

[0066] Table 1 Complex L-Cu 2+ Detection of cysteine ​​in milk

[0067]

[0068] It can be seen from Table 1 that the recovery rate of cysteine ​​in milk samples is 93.38% to 106.25%, the relative standard deviation is 1.11% to 2.19%, and the error between the measured cysteine ​​concentration and the corresponding spiked concentration is very small. These results show that the probe complex L-Cu prepared by the present invention 2+ The system has good accuracy in detecting cysteine ​​in milk samples and can quantitatively detect cysteine ​​in the range of 0 to 20 μmol / L, with good practical performance.

Claims

1. A fluorescent probe L for tandem detection of copper ions and cysteine, characterized in that The molecular structure of probe L is:

2. A fluorescent probe L for tandem detection of copper ions and cysteine ​​according to claim 1, characterized in that The preparation method of probe L is as follows: 4-hydrazino-N-(2-hydroxyethyl)ethoxy-1,8-naphthalimide and quinoline-2-carboxaldehyde are refluxed in anhydrous ethanol.

3. Use of the fluorescent probe L as claimed in claim 1 in the quantitative and qualitative detection of copper ions and cysteine.

4. The use of the fluorescent probe L according to claim 3, characterized in that Probe L can realize continuous fluorescence "on-off-on" detection of copper ions and cysteine.

5. The use of the fluorescent probe L according to claim 3, characterized in that Complex L-Cu 2+ In the process of detecting cysteine, it can resist the interference of common amino acids Phe, Phg, Ala, Gly, Glu, Tyr, Leu, Pro, Trp, Ser, Val, Ile, His and bioactive peptide Gsh.

6. The use of the fluorescent probe L according to claim 3, characterized in that The detection limit of copper ions is as low as 3.6×10 - 8 mol / L, the detection limit of cysteine ​​is as low as 4.2×10 -8 mol / L.

7. The use of the fluorescent probe L according to claim 3, characterized in that Complex L-Cu 2+ Used for the detection of cysteine ​​in milk, the recovery rate of cysteine ​​in the samples is 93.38% to 106.25%, and the relative standard deviation is 1.11% to 2.19%.

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