(2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile and its preparation method and application

Abstract

The present invention provides a (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile and a preparation method and application thereof, belonging to the technical field of metal ion detection, and is used to solve the technical problems of fluorescent probes having a single detection element, a high detection limit, and a slow response speed. The structural formula of the (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile of the present invention is as follows: #imgabs0# The preparation method comprises adding 7-(N,N-diethylamino)-2H-chromene-3-aldehyde, 2,3-diamino-2-butenedinitrile, a catalyst, and a solvent to a reaction flask, stirring, and heating under reflux until the reaction is completed. The preparation method of the present invention has simple synthesis steps, mild reaction conditions, and simple post-processing operations, and can realize multiple detection (ClO ‑ 、Fe 3+ 、Cu 2+ ) and other advantages, for ClO ‑ 、Cu 2+ The linear range of response concentration is wide (0-1.7×10 ‑4 M(ClO ‑ ) and 0‑1.0×10 ‑4 M(Cu 2+ )), high sensitivity (detection limits were 60nM (ClO ‑ ) and 45nM (Cu 2+ )), fast response (response time is 30s (ClO ‑ ), 2min(Fe 3+ ), 20min(Cu 2+ )).

Description

Technical Field

[0001] The invention belongs to the technical field of metal ion detection, and in particular relates to (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile and a preparation method and application thereof. Background Art

[0002] Hypochlorite (ClO - ), the lowest valence oxygen-containing acid radical ion of chlorine, the strongest oxidizing agent among the oxygen-containing acid radicals of chlorine, ClO - Used in disinfectants, sterilizers and bleaches. ClO - It is an important active oxygen in the immune system and plays a key role in physiological activities. However, abnormal ClO - The concentration will affect its normal physiological function, so it is possible to detect ClO in organisms or the environment in real time and non-destructively. - It is of great significance.

[0003] Copper and iron are transition metal elements with high content on the earth and are widely used in human production and life. 2+ and Fe 3+ It participates in the growth and metabolism of organisms as a coenzyme or other auxiliary factor. 2+ and Fe 3+ It can cause metabolic disorders, organ dysfunction, and neurodegenerative diseases. 2+ and Fe 3+ Detection is also very important.

[0004] Fluorescent probes have the advantages of simple pretreatment, convenient operation, high sensitivity, fast response, low cost, non-destructive detection of biological samples, and in situ detection. They are important analytical methods for monitoring target analytes in organisms. Cell fluorescence imaging has attracted much attention as an efficient detection method in the fields of biology and pharmaceutical sciences. Fluorescent small molecules are used for intracellular imaging detection due to their good biocompatibility and superior photophysical properties. However, previous probes have problems such as a single detection element, high detection limit, and slow response speed, which limit the relevant application range of these probes. Therefore, there is an urgent need to design a fluorescent probe to overcome these shortcomings. Summary of the Invention

[0005] Aiming at the technical problems of fluorescent probes with single detection elements, high detection limit and slow response speed, the present invention proposes a (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile and its preparation method and application, which can simultaneously detect ClO - 、Fe 3+ and Cu 2+, and has the characteristics of wide response linear concentration range, high sensitivity, fast response speed and simple probe synthesis steps.

[0006] In order to achieve the above object, the technical solution of the present invention is achieved as follows:

[0007] Beneficial effects of the present invention:

[0008] A (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile, also known as ACAN, has the structural formula:

[0009] A method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile comprises the following steps: mixing 7-(N,N-diethylamino)-2H-chromene-3-aldehyde, 2,3-diamino-2-butenedinitrile, a catalyst, and a solvent, reacting the mixture, stirring the mixture, heating the mixture under reflux until the reaction is complete, cooling the reaction solution to generate a large amount of precipitate, filtering a solid crude product, and purifying the mixture through recrystallization to obtain a product with a yield of 60-87%.

[0010] The synthetic route is:

[0011]

[0012] The molar ratio of 7-(N,N-diethylamino)-2H-chromene-3-al to 2,3-diamino-2-butenedinitrile is 1:(1-2); preferably, the molar ratio is 1:1.3.

[0013] The molar ratio of the 7-(N,N-diethylamino)-2H-chromene-3-al to the catalyst is 1:(0.05-0.8); the preferred molar ratio is 1:0.4.

[0014] The ratio of the 7-(N,N-diethylamino)-2H-chromene-3-al to the solvent is 1:(10-30) mmoL / mL.

[0015] The catalyst is glacial acetic acid and ammonium acetate.

[0016] The solvent is any one of alcohol, tetrahydrofuran, dichloromethane, acetonitrile, acetone, 1,4-dioxane, ethyl acetate and N,N-dimethylformamide.

[0017] The reaction temperature is 60-140° C., and the reaction time is 6-18 hours.

[0018] Application of (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile as a fluorescent probe for detecting ClO - 、Fe3+ or Cu 2+ ion.

[0019] Beneficial effects:

[0020] The present invention is based on ClO - Oxidation of Cu 2+ The coordination effect of Fe 3+ A fluorescent probe named (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile (ACAN) was designed based on the paramagnetic properties of the molecule. The probe reacted with ClO in a buffer solution. - Oxidation reaction occurs with Cu 2+ The coordination reaction with Fe 3+ Electron transfer occurs, enabling multiple recognition of ClO by one probe - 、Cu 2+ and Fe 3+ ACAN itself has weak red fluorescence, with the peak at 582nm. - As an oxidant, it attacks the C=N double bond of ACAN and generates 7-(N,N-diethylamino)-2H-chromen-3-aldehyde after hydrolysis, which emits bright yellow-green fluorescence with a fluorescence peak at 530nm. The recognition mechanism has been verified by nuclear magnetic resonance. - The nuclear magnetic resonance signal of 7-(N,N-diethylamino)-2H-chromen-3-aldehyde was detected in the nuclear magnetic resonance tube after the reaction. 2+ The complex has a coordination effect with the probe ACAN, and the complex produces an enhanced fluorescence peak at 500nm, emitting bright blue-green fluorescence, thereby achieving the detection of Cu 2+ Ratio detection of Fe 3+ As a paramagnetic ion, it undergoes electron transfer with the probe ACAN, quenching the weak fluorescence of ACAN at 582nm, and no new fluorescence peak appears. Therefore, the probe ACAN is used to react with ClO - 、Cu 2+ and Fe 3+ The difference in fluorescence spectrum after the reaction can realize the effect of ClO - 、Fe 3+ 、Cu 2+ Qualitative detection and ClO - 、Cu 2+ Quantitative detection of .

[0021] The probe molecule of the present invention is prepared by the condensation reaction of 7-(diethylamino)-2H-chromene-3-aldehyde and 2,3-diamino-2-butenedinitrile. The reaction has simple synthesis steps, mild reaction conditions, and simple post-processing operations, and can realize multiple detection (ClO - 、Fe3+ 、Cu 2+ ) and other advantages, for ClO - 、Cu 2+ The linear range of response concentration was wide (0-1.7×10 -4 M(ClO - ) and 0-1.0×10 -4 M(Cu 2+ )), high sensitivity (detection limits were 60nM (ClO - ) and 45nM (Cu 2+ )), fast response (response time is 30s (ClO - ), 2min(Fe 3+ ), 20min(Cu 2+ )). BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 ACAN prepared in Example 1 1 H NMR spectrum (solvent CD3OD).

[0024] Figure 2 ACAN prepared in Example 1 13 C NMR spectrum (solvent CD3SOCD3).

[0025] Figure 3 The probe ACAN (10 μM) prepared in Example 1 and its reaction with 100 μM anions and active oxygen species (1: blank, 2: ClO3 - ,3:NO3 - ,4:AcO - ,5:ClO - ,6:F - ,7:Cl - ,8:Br - ,9:I - ,10:SO3 2- ,11:S2O3 2- ,12:S 2- ,13:SO4 2- ,14:CO3 2- ,15:PO4 3- ,16:P2O74- ,17:t-BuO·,18:·OH,19: 1 Fluorescence spectra of the probe + ClO - The excitation wavelength of the probe and the rest of the analytes is 420 nm, and the excitation wavelength of the probe and the rest of the analytes is 480 nm.

[0026] Figure 4 ACAN (10 μM) prepared in Example 1 was exposed to 10 times the analyte (blank, ClO3 - ,NO3 - ,AcO - ,ClO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4- ,t-BuO·,·OH, 1 Fluorescence intensity at 530nm in the presence of O2, ONOO, NO, H2O2 (orange); 10 times ClO was added to - Fluorescence intensity of ACAN at 530 nm (green).

[0027] Figure 5 (a) ACAN prepared in Example 1 and ClO - Fluorescence spectra of ACAN and ClO with concentration changes, excitation wavelength is 420nm; (b) - Partial concentration linear relationship diagram.

[0028] Figure 6 ACAN (10 μM) + ClO prepared in Example 1 - (100 μM) fluorescence intensity and time response, excitation wavelength 420 nm.

[0029] Figure 7 ACAN and ACAN+ClO prepared in Example 1 - (5eq) in CD3OD 1 HNMR spectrum.

[0030] Figure 8 The probe ACAN (10 μM) prepared in Example 1 and its + ,Pb2+ , Al 3+ ,Ca 2+ ,Co 2+ ,K + ,Zn 2+ ,Hg 2+ ,Cr 3+ ,Ni 2+ ,Mn 2+ ,Cd 2+ ,Fe 3+ ,Cu 2+ ) in the presence of fluorescence spectra, probe + Cu 2+ The excitation wavelength of the probe and the rest of the analytes is 480 nm.

[0031] Figure 9 ACAN (10 μM) prepared in Example 1 was 10 times the amount of cationic (Ag + ,Pb 2+ , Al 3+ ,Ca 2+ ,Co 2+ ,K + ,Zn 2 + ,Hg 2+ ,Cr 3+ ,Ni 2+ ,Mn 2+ ,Cd 2+ ,Fe 3+ ,Cu 2+ ) in the presence of fluorescence peak (orange); ACAN (10 μM) plus 10 times Cu 2+ After that, 10 times of cations (Ag + ,Pb 2+ , Al 3+ ,Ca 2+ ,Co 2+ ,K + ,Zn 2+ ,Hg 2+ ,Cr 3+ ,Ni 2+ ,Mn 2+ ,Cd 2 + ,Fe 3+ ), the fluorescence intensity of the solution at the fluorescence peak (green); ACAN (10 μM) plus 10 times Fe 3+ After that, 10 times of cations (Ag + ,Pb 2+ , Al 3+ ,Ca 2+ ,Co 2+ ,K +,Zn 2+ ,Hg 2+ ,Cr 3+ ,Ni 2+ ,Mn 2+ ,Cd 2+ ,Cu 2+ ), the fluorescence intensity of the solution at the fluorescence peak (purple).

[0032] Figure 10 (a) Different Cu were added to the ACAN prepared in Example 1. 2+ Fluorescence spectrum of the concentration, excitation wavelength is 400nm, (b) Fluorescence intensity of ACAN at 500nm and Cu 2+ Linear relationship with concentration.

[0033] Figure 11 (a) ACAN and Cu 2+ Working curve of the effect (total concentration is 10 μM, the vertical axis is the fluorescence intensity at 500 nm), (b) ACAN-Cu 2+ Benesi–Hildebrand diagram.

[0034] Figure 12 Different Fe 3+ Fluorescence spectrum of the concentration (excitation wavelength is 480 nm).

[0035] Figure 13 (a) ACAN (10 μM) prepared in Example 1 and Cu 2+ (b) Fluorescence intensity and time relationship of the response of ACAN (100 μM) with an excitation wavelength of 400 nm; (c) Fluorescence intensity and time relationship of the response of ACAN (10 μM) with Fe 3+ (100 μM) fluorescence intensity versus time response, excitation wavelength 480 nm.

[0036] Figure 14 No. 1-4 are probes ACAN, ACAN+ClO - , probe ACAN+Fe 3+ , probe ACAN+Cu 2+ Photos taken under natural light and 365nm illumination. DETAILED DESCRIPTION

[0037] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0038] Example 1

[0039] A method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile comprises the following steps: adding 7-(diethylamino)-2H-chromene-3-aldehyde (231 mg, 1 mmol), 2,3-diamino-2-butenediconitrile (119 mg, 1.1 mmol), acetonitrile (20 ml), and glacial acetic acid (22 μl, 0.4 mmol) to a 100 ml round-bottom flask. The mixture is heated to 100°C with magnetic stirring and refluxed for 8 hours. After the reaction is complete, the reaction solution is cooled, the precipitate is separated, and filtered. The solid is washed twice with 1 ml of acetonitrile and recrystallized from anhydrous ethanol to obtain 193 mg of a reddish-brown solid with a yield of 60%. Characterization confirmed the solid to be 7-(N,N-diethylamino)-2H-chromene-3-aldehyde 2,3-diamino-2-butenediconitrile (ACAN).

[0040] Nuclear magnetic resonance measurement: 1 HNMR(400MHz,MeOD)δ7.95(s,1H),7.00(d,J=8.4Hz,2H),6.31(dd,J=8.7,2.4Hz,1H ), 6.13 (d, J = 2.3Hz, 1H), 5.08 (s, 2H), 3.40 (q, J = 7.0Hz, 4H), 1.17 (t, J = 7.1Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 157.43, 153.86, 151.00, 135.38, 130.50, 125.05, 124.95, 115.32, 114.27, 110.80, 105.97, 104.43, 97.97, 64.61, 44.38, 13.02. The H NMR spectrum is shown in the attached figure. Figure 1 As shown in the attached carbon NMR spectrum Figure 2 shown.

[0041] Example 2

[0042] A method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile comprises the following steps: adding 7-(diethylamino)-2H-chromene-3-aldehyde (231 mg, 1 mmol), 2,3-diamino-2-butenedinitrile (140 mg, 1.3 mmol), anhydrous ethanol (20 ml), and glacial acetic acid (22 μl, 0.4 mmol) to a 100 ml round-bottom flask. The mixture is heated to 100°C with magnetic stirring and refluxed for 12 hours. After the reaction is complete, the reaction solution is cooled, the precipitate is separated, and filtered. The solid is washed twice with 1 ml of ethanol and recrystallized from anhydrous ethanol to obtain 273 mg of a reddish-brown solid with a yield of 85%. Characterization confirmed the solid to be ACAN.

[0043] Example 3

[0044] A method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile comprises the following steps: adding 7-(diethylamino)-2H-chromene-3-aldehyde (231 mg, 1 mmol), 2,3-diamino-2-butenedinitrile (140 mg, 1.3 mmol), anhydrous ethanol (20 ml), and ammonium acetate (30 mg, 0.4 mmol) to a 100 ml round-bottom flask. The mixture is heated to 100°C with magnetic stirring and refluxed for 12 hours. After the reaction is complete, the reaction solution is cooled, the precipitate is separated, and filtered. The solid is washed twice with 1 ml of ethanol and recrystallized from anhydrous ethanol to obtain 280 mg of a reddish-brown solid with a yield of 87%. Characterization confirmed the solid to be ACAN.

[0045] Example 4

[0046] A method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile comprises the following steps: adding 7-(diethylamino)-2H-chromene-3-aldehyde (231 mg, 1 mmol), 2,3-diamino-2-butenedinitrile (140 mg, 1.3 mmol), dichloromethane (20 ml), and ammonium acetate (30 mg, 0.4 mmol) into a 100 ml round-bottom flask; heating to 60° C. under magnetic stirring; and reflux for 15 hours. After completion of the reaction, cooling the reaction solution, separating the precipitate, filtering it, washing it twice with 1 ml of ethanol, and recrystallizing it from anhydrous ethanol to obtain 241 mg of a reddish-brown solid with a yield of 75%. The solid was characterized and confirmed to be ACAN.

[0047] Example 5

[0048] A method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile comprises the following steps: adding 7-(diethylamino)-2H-chromene-3-aldehyde (231 mg, 1 mmol), 2,3-diamino-2-butenedinitrile (108 mg, 1 mmol), anhydrous ethanol (10 ml), and ammonium acetate (3.75 mg, 0.05 mmol) to a 100 ml round-bottom flask. The mixture is heated to 60°C with magnetic stirring and refluxed for 18 hours. After the reaction is complete, the reaction solution is cooled, the precipitate is separated, and filtered. The solid is washed twice with 1 ml of ethanol and recrystallized from anhydrous ethanol to obtain 215 mg of a reddish-brown solid with a yield of 67%. Characterization confirmed the solid to be ACAN.

[0049] Example 6

[0050] A method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile comprises the following steps: adding 7-(diethylamino)-2H-chromene-3-aldehyde (231 mg, 1 mmol), 2,3-diamino-2-butenedinitrile (215 mg, 2 mmol), acetone (30 ml), and ammonium acetate (60 mg, 0.8 mmol) into a 100 ml round-bottom flask; heating the mixture to 140° C. under magnetic stirring; and reflux for 6 hours. After the reaction is completed, the reaction solution is cooled to separate the precipitate, which is filtered, washed twice with 1 ml of ethanol, and recrystallized from anhydrous ethanol to obtain 275 mg of a reddish-brown solid with a yield of 86%. The solid is characterized and confirmed to be ACAN.

[0051] Implementation Effect

[0052] Preparation of 1 mM probe (ACAN) solution: Accurately weigh the probe (ACAN) prepared in Example 1 and dissolve it in ethanol (EtOH) to prepare a 1 mM solution for later use.

[0053] Separately add anion sodium salt or potassium salt: ClO3 - ,NO3 - ,AcO - ,ClO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4- , active oxygen species include t-BuO·,·OH, 1O2, ONOO, NO, and H2O2 were prepared in distilled water with a concentration of 10 mM and stored in a refrigerator for future use.

[0054] The cation hydrochloride or nitrate: Ag + ,Pb 2+ , Al 3+ ,Ca 2+ ,Co 2+ ,K + ,Zn 2+ ,Hg 2+ ,Cr 3+ ,Ni 2+ ,Mn 2+ ,Cd 2+ ,Fe 3+ ,Cu 2+ , prepared in distilled water with a concentration of 10mM, stored in the refrigerator for future use.

[0055] Probe pair ClO - Fluorescence response selectivity

[0056] The probe (ACAN, 10 μM) was dissolved in ethanol and water (v / v = 9:1, v / v) and 10 times the amount of anions and active oxygen was added. The effect of the probe ACAN on ClO was investigated by fluorescence spectroscopy. - Selectivity. Figure 3 As shown in the figure, it can be seen that the probe (blank) has a weak fluorescence peak at 582nm. After adding the above 21 kinds of anions and active oxygen, except for the addition of ClO - In addition to causing the probe solution to have an obvious fluorescence peak at 530nm, the addition of 20 other anions ClO3 - ,NO3 - ,AcO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4- 、Active oxygen (t-BuO·,·OH, 1 O2,·ONOO,NO,H2O2) did not cause significant changes in probe fluorescence. This indicates that the probe is sensitive to ClO - Has good fluorescence selectivity.

[0057] Probe pair ClO - Fluorescence response anti-interference

[0058] To the probe (ACAN, 10 μM) solution in ethanol-water (v / v=9:1, v / v), 10 times the amount of ClO - Then add 10 times the amount of anions and active oxygen. Use fluorescence spectroscopy to test the probe's recognition of ClO - Anti-interference ability to common anions and active oxygen. Figure 4 It can be seen that in the probe ACAN (10 μM), the - ,NO3 - ,AcO - ,F - ,Cl - ,Br - ,I - ,SO3 2- ,S2O3 2- ,S 2- ,SO4 2- ,CO3 2- ,PO4 3- ,P2O7 4- 、Active oxygen (t-BuO·,·OH, 1 In the presence of O2, ONOO, NO, H2O2) 100 μM ClO - With the addition of 100 μM ClO - The fluorescence intensity of the probe solution obtained when the probe was ACAN was basically the same, indicating that the probe ACAN was effective for ClO - The detection has strong anti-interference ability.

[0059] Probe pair ClO - The linear range and detection limit of fluorescence response

[0060] The width of the linear response range and the sensitivity are important criteria for judging whether a probe molecule has application value. - The probe (ACAN, 10 μM) was titrated in ethanol-water (v / v=9:1, v / v) solution to determine the probe's response to different concentrations of ClO - The fluorescence spectrum of the response is Figure 5 a It can be seen that the fluorescence of probe ACAN (10 μM) at 530 nm is very weak. - With the continuous addition of ClO, the probe ACAN showed a fluorescence peak at 530nm, and the intensity increased with the increase of concentration. - When the concentration reached 100 μM, the fluorescence intensity increased 30 times. - The concentration range was 0-170 μM. Figure 5b) The linear equation is I = -16.3475 + 5.2412 × 10 6 C (I is the fluorescence intensity, C is ClO - concentration), linear correlation coefficient R 2 =0.9972, according to the formula LOD = 3σ / k, it is calculated that the probe molecule has a strong affinity for ClO - The detection limit LOD was 60 nM, indicating that the probe was sensitive to ClO - The response concentration linear range is wide and the sensitivity is high.

[0061] Probe pair ClO - Speed ​​of fluorescence response

[0062] The response speed is also an important basis for judging whether a probe molecule has application value. Figure 6 It can be seen that when ClO was added to the probe ACAN (10 μM), - After 100 μM, the fluorescence intensity of the probe solution at 530 nm almost reached its maximum at 30 s. The fluorescence intensity hardly changed significantly after the time was prolonged. - The response speed is very fast, which is beneficial to the ClO - Real-time detection.

[0063] Probe pair ClO - Mechanism of fluorescence response

[0064] ACAN, ACAN+ClO - (5eq) in CD3OD 1 HNMR spectra were compared and analyzed, and ClO - Then a new NMR signal peak appeared (see attached Figure 7 As shown in the figure, indicated by arrows), where δ9.29 can be attributed to the peak of aldehyde hydrogen -CHO, from which it can be concluded that ACAN has a strong affinity for ClO - Mechanism of fluorescence response: ClO - As an oxidant, it attacks the C=N double bond of ACAN and hydrolyzes to generate 7-(N,N-diethylamino)-2H-chromene-3-aldehyde, which emits bright yellow-green fluorescence.

[0065] Probe to Cu 2+ 、Fe 3+ Fluorescence response selectivity

[0066] The probe (ACAN, 10 μM) was added with 10 times the amount of cations in ethanol-water (v / v=9:1, v / v) solution. 2+ 、Fe 3+ The fluorescence response selectivity of Figure 8 As shown in the figure, it can be seen that the probe ACAN has a weak fluorescence peak at 582nm. After adding cations, Cu 2+ The probe solution showed a significantly enhanced fluorescence peak at 500 nm. 3+ The weak fluorescence of the probe solution at 582 nm is quenched, and the addition of other cations Ag + , Pb 2+ , Al 3+ , Ca 2+ ,Co 2+ , K + , Zn 2+ , Hg 2+ , Cr 3+ , Ni 2+ , Mn 2+ , Cd 2+ It did not cause obvious changes in the probe fluorescence. 2+ 、Fe 3+ Has good fluorescence selectivity.

[0067] Probe to Cu 2+ or Fe 3+ Fluorescence response cation anti-interference

[0068] Testing the probe Cu by fluorescence spectroscopy 2+ or Fe 3+ The fluorescence response is anti-interference. Figure 9 It can be seen that the probe ACAN (10 μM) is respectively + , Pb 2+ , Al 3+ , Ca 2+ ,Co 2+ , K + , Zn 2+ ,Hg 2+ ,Cr 3+ , Ni 2+ , Mn 2+ , Cd 2+ , Fe 3+ In the presence of 100 μM Cu 2+ 100 μM Cu was added to the probe ACAN (10 μM) 2+ The fluorescence intensity of the probe solution obtained was basically the same. Similarly, the probe ACAN (10 μM) was respectively + , Pb 2+ , Al 3+ , Ca 2+ ,Co 2+ , K + , Zn 2+,Hg 2+ ,Cr 3+ , Ni 2+ , Mn 2+ , Cd 2+ , Cu 2+ In the presence of 100 μM Fe 3+ 100 μM Fe was added to the probe ACAN (10 μM) 3+ The fluorescence intensity of the probe solution obtained was basically the same when 2+ or Fe 3+ The detection has strong anti-interference ability of cations.

[0069] Probe to Cu 2+ The linear response range and detection limit of fluorescence response

[0070] With different concentrations of Cu 2+ The probe (ACAN, 10 μM) was titrated in ethanol-water (v / v=9:1, v / v) solution to determine the probe's effect on different concentrations of Cu. 2+ The fluorescence spectrum of the response is Figure 10 a It can be seen that the fluorescence intensity of the probe ACAN (10 μM) at 500 nm is very low. 2+ With the continuous addition of Cu, the fluorescence peak intensity of the probe ACAN at 500nm increased with the increase of concentration. 2+ When the concentration reached 100 μM, the fluorescence intensity increased 12 times. 2+ The concentration range is 0-100 μM, as shown in the attached figure. Figure 10 b, the linear equation is I = 87.32 + 7.22 × 10 6 C (I is the fluorescence intensity, C is Cu 2+ concentration), linear correlation coefficient R 2 =0.9943, the detection limit LOD is 45nM. Compared with the limit of copper ions in drinking water (20μM) stipulated by the U.S. Environmental Protection Agency (EPA), the minimum detection limit is much lower than the concentration stipulated in the standard. 2+ It has the characteristics of wide linear response concentration range and high sensitivity.

[0071] Probe to Cu 2+ Fluorescence response binding ratio and binding constant

[0072] Working curves are often used to determine the stoichiometry of complexes. 2+ A series of solutions of Cu ions were prepared with a total concentration of 10 μM. The fluorescence intensity of these solutions at 500 nm was plotted as Cu 2+ The relationship diagram of the scores is as follows Figure 11As shown in a, when the fluorescence intensity is maximum, Cu 2+ The score is 0.5, indicating that ACAN and Cu 2+ The stoichiometric ratio of ACAN and Cu is 1:1. 2+ The complex constant (K a ), according to the equation: 1 / (F-F0)=[1 / K a (F max -F0)[Cu 2+ ]+1 / (F max -F0), where F and F0 are respectively 2+ Concentration and Cu-free 2+ The fluorescence intensity at 500 nm, F max It is in Cu 2+ The maximum fluorescence intensity at 500 nm in the presence of 2+ ] function to plot the Benesi-Hildebrand plot, showing a linear relationship with a correlation coefficient of 0.99 ( Figure 11 b). It further confirmed that ACAN and Cu 2+ The complexation constant K a 7.49×10 3 M -1 , indicating that ACAN and Cu 2+ The ion binding is stable and has a high affinity.

[0073] With Fe 3+ With the increase of concentration, the weak fluorescence of the probe at 582 nm was gradually quenched ( Figure 12 ), and its fluorescence peak was blue-shifted. 3+ It can completely quench the fluorescence of the probe, but no new fluorescence peaks are observed when changing the excitation wavelength.

[0074] Probe to Cu 2+ or Fe 3+ Fluorescence response speed

[0075] Cu was added to the probe ACAN (10 μM) 2+ After 100 μM, the fluorescence intensity of the probe solution at 500 nm almost reached its maximum at 20 min. The fluorescence intensity hardly changed significantly after the time was extended (see Appendix Figure 13 a), indicating that ACAN can inhibit the growth of Cu 2+ The response speed is relatively fast. Add Fe to the probe ACAN (10μM) 3+ (100μM), the fluorescence intensity of the probe solution at 580nm almost dropped to the lowest at 2min. If the time is extended, the fluorescence intensity will hardly change significantly ( Figure 13 b), indicating that ACAN can inhibit the growth of Fe 3+ The response speed is very fast, which is conducive to real-time detection in the environment or biological systems.

[0076] Probe pair ClO - 、Cu 2+ 、Fe 3+ Fluorescent naked eye identification

[0077] The probe solution is yellow-brown under natural light. - After the solution turned yellow, Fe 3+ After the solution turned colorless, Cu 2+ The solution then turned slightly yellow and the probe emitted weak fluorescence under 365nm light. - The solution then emits a strong yellow-green fluorescence, and the addition of Fe 3+ After that, the solution was almost non-fluorescent. 2+ The solution then emits a blue-green fluorescence ( Figure 14 ). Under natural light or 365nm illumination, ClO can be identified by naked eyes using the probe ACAN. - , Fe 3+ , Cu 2+ .

[0078] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile, characterized in that Its structural formula is:

2. The method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile according to claim 1, characterized in that: The following steps are involved: 7-(N,N-diethylamino)-2H-chromene-3-al, 2,3-diamino-2-butenedinitrile, a catalyst, and a solvent are mixed and reacted to prepare (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile.

3. The method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile according to claim 2, wherein: The molar ratio of the 7-(N,N-diethylamino)-2H-chromene-3-al to 2,3-diamino-2-butenedinitrile is 1:(1-2).

4. The method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile according to claim 3, wherein The molar ratio of the 7-(N,N-diethylamino)-2H-chromene-3-al to the catalyst is 1:(0.05-0.8).

5. The method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile according to claim 4, wherein The ratio of the 7-(N,N-diethylamino)-2H-chromene-3-al to the solvent is 1:(10-30) mmoL / mL.

6. The method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile according to claim 5, wherein The catalyst is glacial acetic acid or ammonium acetate.

7. The method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile according to claim 6, wherein The solvent is any one of alcohol, tetrahydrofuran, dichloromethane, acetonitrile, acetone, 1,4-dioxane, ethyl acetate and N,N-dimethylformamide.

8. The method for preparing (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile according to claim 7, wherein The reaction temperature is 60-140° C., and the reaction time is 6-18 hours.

9. Use of the (2-(7-(N,N-diethylamino)-2H-chromene-3-)methyleneamino)-3-aminomaleonitrile according to claim 1 as a fluorescent probe, characterized in that For detection of ClO - 、Fe 3+ or Cu 2+ ion.

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