Phenanthrenequinone-containing rhodamine 6G fluorescent probe for acid-base detection, and preparation method and application of phenanthrenequinone-containing rhodamine 6G fluorescent probe

Abstract

The invention provides a phenanthrenequinone-containing rhodamine 6G fluorescent probe for acid-base detection as well as a preparation method and application of the phenanthrenequinone-containing rhodamine 6G fluorescent probe. The structure of the phenanthrenequinone-containing rhodamine 6G fluorescent probe is shown as follows: the # imgabs0 # phenanthrenequinone-containing rhodamine 6G fluorescent probe has high molar absorption coefficient and quantum yield, so that the # imgabs0 # phenanthrenequinone-containing rhodamine 6G fluorescent probe can realize low-detection-limit and high-sensitivity pH value detection, shows good light stability and is suitable for being applied to long-term experiments and complex environments.

Description

Technical Field

[0001] The present application relates to the field of chemical analysis technology, and in particular to a phenanthroquinone rhodamine 6G fluorescent probe for acid-base detection, a preparation method and an application thereof. Background Art

[0002] At present, fluorescent probe technology is an important research direction in the field of molecular and ion recognition. This type of probe has characteristic fluorescence in the ultraviolet-visible-near infrared region, and its fluorescence properties change sensitively with environmental changes. Rhodamine 6G, as a fluorescent dye, has the advantages of high molar absorption coefficient and good photostability, so it has been widely used in the design of fluorescent probes.

[0003] In existing studies, rhodamine 6G has been used to synthesize a variety of fluorescent probes. For example, some studies have synthesized rhodamine 6G-based probe molecules TrisRh and R6G-Flu, which can be used for specific metal ions (such as Co 2+ and Al 3+ ) with high selectivity and high sensitivity. Existing Rhodamine 6G is prone to photobleaching under long-term illumination, resulting in a decrease in its fluorescence intensity, limiting its application in continuous monitoring and long-term experiments. The selectivity of existing Rhodamine 6G probes may be affected in complex biological samples, resulting in false positive or false negative test results. In some detection applications, the sensitivity of existing Rhodamine 6G probes is not sufficient to detect low concentrations of target substances, limiting their application in certain high-precision detection fields. The fluorescence lifetime and quantum yield of existing Rhodamine 6G are often affected by the polarity of the solvent and the rigidity of the alkyl group on the N atom in the molecular structure. These factors may lead to unstable fluorescence performance in different application environments. Summary of the invention

[0004] The present application is proposed to solve the above technical problems existing in the prior art. The present application aims to provide a fluorescent probe containing phenanthroquinone rhodamine 6G for acid-base detection, a preparation method and application thereof, which can quickly and accurately measure the pH value of a solution and can be used as an efficient pH detection tool in the fields of biomedicine, environmental monitoring and industrial process control.

[0005] According to the first scheme of the present application, a phenanthroquinone rhodamine 6G-containing fluorescent probe for acid-base detection is provided, and the structure of the phenanthroquinone rhodamine 6G-containing fluorescent probe is as follows:

[0006]

[0007] The phenanthroquinone rhodamine 6G fluorescent probe has a high molar absorption coefficient and quantum yield, can detect small changes in pH at low concentrations, can achieve low detection limits and high sensitivity pH detection, and ensure that its reaction in acid-base environments is highly selective, can be widely used under different pH values, and meet the needs of different pH detection. In addition, the phenanthroquinone rhodamine 6G fluorescent probe has high stability and reliability in different acid-base environments, and can ensure accuracy and repeatability under long-term use.

[0008] According to the second embodiment of the present application, a method for preparing a phenanthroquinone rhodamine 6G fluorescent probe is provided, and the preparation method comprises:

[0009] Mixing a first mass of rhodamine 6G, a first volume of hydrazine hydrate, and a second volume of methanol to perform a first reaction;

[0010] Performing a first purification treatment on the product obtained after the first reaction to obtain a reaction intermediate;

[0011] mixing a second mass of the reaction intermediate, a third mass of phenanthrenequinone, a fourth mass of ammonium acetate, and a third volume of methanol to perform a second reaction;

[0012] A second purification treatment is performed on the product obtained after the second reaction to obtain a target product, which is a phenanthroquinone rhodamine 6G fluorescent probe.

[0013] Wherein, the first mass is 3-8 g, preferably, the first mass is 5 g. The first volume is 4-9 ml, preferably, the first volume is 6.1 ml, and the hydrazine hydrate is 85% hydrazine hydrate. In some other embodiments, the second volume is 15-30 ml, preferably 20 ml.

[0014] Specifically, during the first reaction, by controlling the dosage of hydrazine hydrate and methanol, the first reaction can be stably carried out, and it can be ensured that the obtained reaction intermediate will not agglomerate, thereby facilitating the subsequent reaction.

[0015] In some other embodiments, the first reaction specifically includes adding a first mass of rhodamine 6G, a first volume of hydrazine hydrate, and a second volume of methanol to a round-bottom flask in sequence, and reacting at room temperature for 1-3 hours. The volume of the round-bottom flask can be 100 ml, and preferably, the reaction time at room temperature is 2 hours.

[0016] After the first reaction is completed, continue to add an appropriate amount of deionized water to the round-bottom flask, wash thoroughly and filter to dryness. The specific washing and filtering process is not limited as long as the impurities attached to the reaction intermediate can be removed.

[0017] The first purification treatment can be performed by column chromatography, wherein the adsorbent can be, for example, silica gel, alumina, activated carbon, etc., and the eluent can be a mixture of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 20-40:0.5-2, preferably, the volume ratio is 30:1, so that efficient extraction of the reaction intermediates can be achieved and the purity of the reaction intermediates can be guaranteed.

[0018] Specifically, there is no limitation on the choice of column, for example, it can be a glass column or a stainless steel column, and the diameter and length of the column are determined according to the amount of the mixture to be separated and the difficulty of separation.

[0019] During the first purification process, the mixture containing the reaction intermediate to be separated is dissolved in the eluent to prepare a sample solution. The concentration of the sample solution should not be too high to avoid overloading the column. Open the valve at the bottom of the column and let the eluent flow out slowly until the liquid level approaches the surface of the adsorbent. Use a dropper or syringe to slowly add the sample solution to the column, taking care not to damage the surface of the adsorbent. A small amount of quartz sand or glass wool can be added to the sample solution to prevent the sample solution from splashing. When all the sample solution is added, close the valve at the bottom of the column, rinse the inner wall of the column with a small amount of eluent, and rinse the remaining sample solution to the surface of the adsorbent.

[0020] Elution can be performed by isocratic elution or gradient elution. Isocratic elution means using the same eluent composition throughout the elution process; gradient elution means gradually changing the eluent composition during the elution process to improve the separation effect.

[0021] The effluent from the column is collected into different fractions based on the color, UV absorption or other characteristics of the compounds in the effluent. A fractionation funnel, test tube or other container can be used for collection. To ensure that the collected fractions are pure, thin layer chromatography or other detection methods can be performed during the collection process to monitor the purity of the fractions. If the fractions contain impurities, they can be further purified or re-separated by column chromatography.

[0022] This is merely an illustrative description and does not constitute a limitation on the specific first purification treatment method.

[0023] In some other embodiments of the present application, the second mass is 3-5g, preferably, the second mass is 4.28g. The third mass is 1-3g, preferably, the third mass is 2.08g. The fourth mass is 1-5g, preferably, the fourth mass is 3.2g, and the third volume is 8-20ml, preferably 15ml. By controlling the amount of the reaction intermediate, phenanthrenequinone, ammonium acetate and methanol, it can be ensured that the second reaction can be carried out stably, so that the second reaction is complete and has fewer impurities.

[0024] Specifically, the second mass of the reaction intermediate, the third mass of phenanthrenequinone, the fourth mass of ammonium acetate and the third volume of methanol can be added to a round-bottom flask, and heated and stirred until the reaction is completed. The round-bottom flask can have a capacity of 50 ml or other capacities, which is not limited.

[0025] Furthermore, the heating temperature is between 100 and 140° C.; preferably, the heating temperature is 120° C., and the mixture is heated and stirred until the reaction is completed. The reaction time of the second reaction is about 2 to 4 hours.

[0026] After the second reaction is completed, water is continued to be added to the round-bottom flask for mixing, for example, 100 nl of deionized water can be added, suction filtration is performed, and the precipitate is repeatedly washed and dried, and a second purification treatment is performed by column chromatography to obtain the target product. For example, a column chromatography method is used for the second purification treatment, and the eluent in the second purification treatment is a mixture of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 20-40:0.5-2, preferably, 30:1, so that the target product can be fully extracted and the purity of the target product is ensured.

[0027] In some embodiments of the present application, a phenanthroquinone rhodamine 6G-containing fluorescent probe is provided, which is prepared according to the preparation method of the phenanthroquinone rhodamine 6G-containing fluorescent probe described in each embodiment of the present application. The phenanthroquinone rhodamine 6G-containing fluorescent probe prepared by the preparation method is expected to provide an efficient and sensitive method for accurate measurement of pH, and further expand the application scope of fluorescent probe technology in the fields of chemical analysis and biological detection.

[0028] In some embodiments of the present application, the phenanthroquinone rhodamine 6G fluorescent probe described in various embodiments of the present application is used in biomedicine, environmental monitoring or industrial process control. In biomedical research, the phenanthroquinone rhodamine 6G fluorescent probe can be used to monitor the acid-base balance inside and outside cells, which is crucial for understanding biological processes such as cell metabolism, signal transduction and disease development.

[0029] In the field of environmental monitoring, the phenanthroquinone rhodamine 6G fluorescent probe can be used to detect the pH of environmental samples such as water and soil, and plays an important role in environmental protection and maintenance of ecological balance.

[0030] Compared with the prior art, the beneficial effects of the embodiments of the present application are:

[0031] The phenanthroquinone rhodamine 6G fluorescent probe for acid-base detection provided in the embodiments of the present application has a unique spirocyclic structure. This structure has no fluorescence emission when it does not interact with the target substance, but opens the spirocyclic structure after interaction to produce fluorescence emission. This "open ring" form is conducive to specific detection. The phenanthroquinone rhodamine 6G fluorescent probe has a high molar absorption coefficient and quantum yield, which enables it to achieve low detection limits and high sensitivity pH detection, so as to achieve accurate and rapid measurement of pH. The phenanthroquinone rhodamine 6G fluorescent dye shows good photostability and is suitable for long-term experiments and applications in complex environments.

[0032] Many existing fluorescent probes are easily interfered under certain conditions, especially in biological samples. The phenanthroquinone rhodamine 6G-containing fluorescent probe for acid-base detection provided in the embodiments of the present application is highly selective and stable. In addition, the interaction of the phenanthroquinone rhodamine 6G-containing fluorescent probe with the target substance, i.e., different acidic and alkaline substances, can be converted into easily detectable ultraviolet and fluorescent signals, which facilitates detection and expands its application range.

[0033] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above description and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In the drawings, which are not necessarily drawn to scale, the same reference numerals may describe similar parts in different views. Similar reference numerals with letter suffixes or different letter suffixes may represent different examples of similar components. The accompanying drawings generally illustrate various embodiments by way of example and not by way of limitation, and together with the description and claims, serve to illustrate the disclosed embodiments. Such embodiments are illustrative and exemplary and are not intended to be exhaustive or exclusive embodiments of the present method.

[0035] Figure 1 A schematic diagram of the synthesis of intermediate A according to an example of the present application is shown.

[0036] Figure 2 A schematic diagram of the synthesis of a phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application is shown.

[0037] Figure 3 The 1H NMR of Intermediate A according to the Examples of the present application is shown.

[0038] Figure 4 The IR spectrum of Intermediate A according to an example of the present application is shown.

[0039] Figure 5The UV spectrum of Intermediate A according to an example of the present application is shown.

[0040] Figure 6 An ORTEP diagram of Intermediate A according to an example of the present application is shown.

[0041] Figure 7 The 1H NMR of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application is shown.

[0042] Figure 8 The IR spectrum of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application is shown.

[0043] Fig. 9 The UV spectrum of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application is shown.

[0044] Fig.10 An ORTEP diagram of a phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application is shown.

[0045] Fig.11 The UV spectra corresponding to the phenanthroquinone rhodamine 6G fluorescent probe in different solvents according to an embodiment of the present application are shown.

[0046] Fig.12 The UV spectra (0.5h) corresponding to the phenanthroquinone rhodamine 6G fluorescent probe in different ratios of methanol: water solvents according to the embodiment of the present application are shown, wherein: Fig.12 (a) shows the UV spectra of the phenanthroquinone rhodamine 6G fluorescent probe in different ratios of methanol (less): water (more) solvents. Fig.12 (b) shows the UV spectra of the phenanthroquinone rhodamine 6G fluorescent probe in solvents with different ratios of methanol (more) and water (less).

[0047] Fig.13 The UV spectra (8h) corresponding to the phenanthrenequinone rhodamine 6G fluorescent probe in different ratios of methanol: water solvent according to the embodiment of the present application are shown, wherein: Fig.13 (a) shows the UV spectra of the phenanthroquinone rhodamine 6G fluorescent probe in different ratios of methanol (more): water (less). Fig.13 (b) shows the corresponding UV spectra of the phenanthroquinone rhodamine 6G fluorescent probe in solvents with different ratios of methanol (less): water (more).

[0048] Fig.14 The UV spectra (24h) corresponding to the phenanthroquinone rhodamine 6G fluorescent probe according to the embodiment of the present application in different ratios of methanol: water solvents are shown.

[0049] Fig.15The fluorescence spectra (0.5 h) corresponding to the phenanthroquinone rhodamine 6G fluorescent probe in different ratios of methanol: water solvents according to an embodiment of the present application are shown.

[0050] Fig.16 The fluorescence spectra (24 h) corresponding to the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in different ratios of methanol: water solvents are shown.

[0051] Fig.17 The fluorescence spectra (36h) corresponding to the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in different ratios of methanol: water solvents are shown.

[0052] Fig.18 The UV spectrum (0.5h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to acid in a solvent with a methanol: water ratio of 20:1 is shown.

[0053] Fig.19 The UV spectrum (7h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application responding to acid in a solvent with a methanol: water ratio of 20:1 is shown.

[0054] Fig. 20 The fluorescence spectrum (0.5 h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to acid in a solvent with a methanol: water ratio of 20:1 is shown.

[0055] Fig.21 The fluorescence spectrum (22h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to acid in a solvent with a methanol: water ratio of 20:1 is shown.

[0056] Fig. 22 The UV graph (0.5h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application responding to a base in a solvent with a methanol: water ratio of 20:1 is shown.

[0057] Fig.23 The UV image (7h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application responding to a base in a solvent with a methanol: water ratio of 20:1 is shown.

[0058] Fig.24 A fluorescence detection graph (0.5 h) of the phenanthroquinone rhodamine 6G fluorescent probe in accordance with an embodiment of the present application in response to a base in a solvent having a methanol: water ratio of 20:1 is shown.

[0059] Fig.25 A fluorescence detection diagram (23h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application responding to a base in a solvent with a methanol: water ratio of 20:1 is shown.

[0060] Fig.26 The UV spectrum (0.5h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to acid in a solvent with a methanol: water ratio of 1:1 is shown.

[0061] Fig. 27 The UV spectrum (7h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application responding to acid in a solvent with a methanol: water ratio of 1:1 is shown.

[0062] Fig.28 The fluorescence spectrum (0.5 h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to acid in a solvent with a methanol: water ratio of 1:1 is shown.

[0063] Fig.29 The UV spectrum (24h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to acid in a solvent with a methanol: water ratio of 1:1 is shown.

[0064] Fig.30 The UV spectrum (0.5h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to a base in a solvent with a methanol: water ratio of 1:1 is shown.

[0065] Fig.31 The UV spectrum (16h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to a base in a solvent with a methanol: water ratio of 1:1 is shown.

[0066] Fig.32 The fluorescence spectrum (0.5 h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to a base in a solvent with a methanol: water ratio of 1:1 is shown.

[0067] Fig.33 The fluorescence spectrum (24 h) of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to a base in a solvent with a methanol: water ratio of 1:1 is shown.

[0068] Fig.34 The UV spectra (0.5h) corresponding to the phenanthroquinone rhodamine 6G fluorescent probe according to the embodiment of the present application in different ratios of THF: water solvents are shown.

[0069] Fig.35 The fluorescence spectra (0.5 h) corresponding to the phenanthroquinone rhodamine 6G fluorescent probe in different ratios of THF: water solvents according to an embodiment of the present application are shown.

[0070] Fig.36 The UV spectrum of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to acid in a solvent with a THF: water ratio of 1:2 is shown. The upper figure is 0.5 hours after the probe is added.

[0071] Fig.37 The UV spectrum of the phenanthroquinone rhodamine 6G fluorescent probe according to an embodiment of the present application in response to acid in a solvent with a THF: water ratio of 1:2 is shown. The upper figure is 11 hours after the probe is added. DETAILED DESCRIPTION

[0072] In order to enable those skilled in the art to better understand the technical solution of the present application, the present application is described in detail below in conjunction with the accompanying drawings and specific implementation examples. The embodiments of the present application are further described in detail below in conjunction with the accompanying drawings and specific implementation examples, but are not intended to limit the present application.

[0073] The words "first", "second" and similar words used in this application do not indicate any order, quantity or importance, but are only used to distinguish. The words "include" or "comprises" and similar words used in this application mean that the elements before the word include the elements listed after the word, and do not exclude the possibility of covering other elements. In this application, the arrows shown in the figures of each step are only examples of the execution order, not limitations. The technical solution of this application is not limited to the execution order described in the embodiments. The various steps in the execution order can be combined, decomposed, or the order can be changed, as long as the logical relationship of the execution content is not affected.

[0074] All terms (including technical terms or scientific terms) used in this application have the same meaning as those understood by ordinary technicians in the field to which this application belongs, unless otherwise specifically defined. It should also be understood that terms defined in general dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and should not be interpreted in an idealized or extremely formal sense, unless explicitly defined here. Techniques and equipment known to ordinary technicians in the relevant field may not be discussed in detail, but where appropriate, the techniques and equipment should be considered as part of the specification.

[0075] Example:

[0076] (I) Preparation method:

[0077] 5 g of rhodamine 6G (0.01 mol), 6.1 mL of 85% hydrazine hydrate (0.10 mol), and 20 mL of methanol were added to a 100 mL round-bottom flask in sequence and stirred at room temperature for 2 h. The reaction principle is as follows: Figure 1 As shown. The reaction was tracked by TLC. After the reaction was completed, an appropriate amount of deionized water was added, the mixture was washed thoroughly and filtered to dryness. Column chromatography was used for purification, and the eluent was dichloromethane: methanol (DCM: MeOH) with a volume ratio of 30:1. The intermediate A was obtained by rotary evaporation as a white powder (5.41 g, 52.94%). 1H NMR (400MHz, DMSO-d6) δ7.76 (s, 1H), 7.46 (s, 2H), 6.93 (s, 1H), 6.18 (d, J = 66.1Hz, 4H), 5.01(s, 2H), 4.48–4.05(m, 2H), 3.13(s, 4H), 1.87(s, 6H), 1.47–0.96(m, 6H).

[0078] Add 4.28g intermediate A (0.01mol), 2.08g phenanthrenequinone (0.01mol), 3.2g ammonium acetate, and 15mL methanol into a 50mL round-bottom flask, heat and stir at 120℃, track the reaction by TLC, and cool to room temperature after the reaction is completed. Figure 2 As shown. 100 mL of deionized water was added to a round-bottom flask, and the precipitate was filtered and washed repeatedly, and then dried. Column chromatography was used for purification, and the eluent was dichloromethane: methanol with a volume ratio of 30:1. The obtained product (containing phenanthroquinone rhodamine 6G fluorescent probe) was a red powder with a yield of 2.14 g and a yield of 33.64%. 1 H NMR (400MHz, DMSO-d6) δ8.21 (d, 1H), 8.14 (d, 1H), 7.99–7.94 (m, 1H), 7.90 (d, 1H), 7.78 (d, 1H), 7.69–7.49 (m, 5H), 7 .33 (s, 1H), 7.16 (d, 1H), 6.58 (s, 1H), 6.36 (s, 2H), 6.00 (s, 1H), 5.07 (s, 2H), 3.15 (s, 4H), 1.84 (s, 6H), 1.22 (m, 6H).

[0079] (II) Structural analysis:

[0080] The NMR spectrum of intermediate A is as follows Figure 3 As shown, 1 H NMR (400MHz, DMSO-d6) δ7.76 (s, 1H), 7.46 (s, 2H), 6.93 (s, 1H), 6.18 (d, J=66.1Hz, 4H), 5.01(s, 2H), 4.48–4.05(m, 2H), 3.13(s, 4H), 1.87(s, 6H), 1.47–0.96(m, 6H).

[0081] like Figure 3As shown, the multiple peaks of chemical shift δ = 1.46-0.96 ppm are attributed to hydrogen at position a. The single peak of chemical shift δ = 3.13 ppm corresponds to hydrogen at position b; the single peak of chemical shift δ = 1.87 ppm corresponds to hydrogen at position c; the single peak of chemical shift δ = 5.01 ppm corresponds to two hydrogens on the secondary amino group; the multiple peaks of chemical shift δ = 4.48-4.05 ppm correspond to hydrogen at position e on the amino group; the double peak of chemical shift δ = 6.18 ppm corresponds to hydrogen at positions g and f on the benzene ring of intermediate A; the single peak of chemical shift δ = 6.93 ppm corresponds to hydrogen at position h on the benzene ring of intermediate A; the single peak of chemical shift δ = 7.76 ppm corresponds to hydrogen at position k on the benzene ring of intermediate A; the single peak of chemical shift δ = 7.46 ppm corresponds to hydrogen at positions i and j on the benzene ring of intermediate A.

[0082] The infrared spectrum of intermediate A is as follows Figure 4 , at 739cm -1 The absorption peak at 1206cm -1 and 690cm -1 The absorption peak corresponds to CO bending vibration; at 1621cm -1 and 1508cm -1 The strong absorption peak at 1681cm -1 The strong absorption peak corresponds to the stretching vibration of C=O; at 2965cm -1 and 2841cm -1 The characteristic absorption peak of intermediate A corresponds to the stretching vibration of the saturated CC single bond in intermediate A; intermediate A at 3424cm -1 and 3341cm -1 The characteristic absorption peaks correspond to the stretching vibrations of the primary amine and secondary amine of intermediate A.

[0083] Depend on Figure 5 Analysis shows that intermediate A produces a strong absorption peak. The peak at 319nm comes from the conjugated π bond formed by the benzene ring and the carbonyl group. Therefore, when the valence electrons undergo π→π* transition, the absorption wavelength red-shifts and becomes longer due to the larger conjugated system.

[0084] Single crystal structure analysis shows that ( Figure 6 )N(2)…C(21) bond length is The N(2)—C(24) bond length is The bond length of N(3)…C(12) is The N(3)—C(17) bond length is The C(17)—O(2) bond length is The C(9)—O(1) ​​bond length is There are 4 intermediates A in one unit cell.

[0085] Figure 7 The 1H NMR of the phenanthroquinone rhodamine 6G fluorescent probe according to the embodiment of the present application is shown. The peak with a chemical shift of δ = 1.22 ppm corresponds to the hydrogen at position 6. The peak with a chemical shift of δ = 1.84 ppm corresponds to the hydrogen at position 5; the peak with a chemical shift of δ = 3.15 ppm corresponds to the hydrogen at position 7; the peak with a chemical shift of δ = 5.07 ppm corresponds to the two hydrogens at position 8 on the secondary amino group; the peaks with chemical shifts of δ = 6.00, 6.36, and 6.58 ppm correspond to the hydrogens at positions 4 and 9 on the benzene ring; the peaks with a chemical shift of δ = 7.16-8.21 ppm correspond to the hydrogens on the remaining benzene rings.

[0086] like Figure 8 As shown, intermediate A is at 3422 cm -1 and 3329cm -1 The characteristic absorption peak comes from the stretching vibration of the secondary amine in the product; at 2962cm -1 and 2867cm - The characteristic absorption peak of 1 comes from the characteristic absorption peak produced by the stretching vibration of the saturated CC single bond in the product; at 3176cm -1 The characteristic absorption peak of comes from the characteristic absorption peak produced by C=N stretching vibration; and the remaining peaks are compared with the spectrum of intermediate A, and it can be seen that the two are basically the same.

[0087] Depend on Fig. 9 Analysis shows that the UV spectrum of the product has two absorption peaks. Compared with intermediate A, a larger conjugated structure is formed, so the wavelength is generally red-shifted.

[0088] Depend on Fig.10 Analysis shows that the bond length of N(1)…N(2) in the product is The C(22)—N(1) bond length is The C(54)—O(3) bond length is The angle between the five-membered ring composed of C50, N2, C48, C43, C42 and the six-membered ring composed of C65, C63, C64, C55, C54, C53 is 52.268°. The angle between the five-membered ring composed of N2, C50, C48, C43, C42 and the six-membered ring composed of C41, C32, O1, C29, C28, C42 is 49.301°, and one unit cell contains 2 structural units.

[0089] (III) Analysis of the response of the phenanthroquinone rhodamine 6G fluorescent probe to acid and base

[0090] (1)Solvent selection:

[0091] Add the phenanthroquinone rhodamine 6G fluorescent probe to a mixed solvent of methanol, tetrahydrofuran (THF), ethyl acetate, toluene, dichloromethane, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and water in a certain proportion, observe and record the color change and make an ultraviolet spectrum.

[0092] By comparing the responses of the phenanthrenequinone rhodamine 6G fluorescent probe in different solvents, it was found that the phenanthrenequinone rhodamine 6G fluorescent probe was well soluble in methanol and tetrahydrofuran solvents and the color changed significantly. Fig.11 In the UV spectrum, the absorption peaks of methanol and THF with water in a ratio of 1:1 at 536nm and 538nm respectively are relatively obvious, indicating that the phenanthroquinone rhodamine 6G fluorescent probe responds well in methanol and tetrahydrofuran solvents. Therefore, methanol and tetrahydrofuran solvents are selected as solvents for subsequent acid-base detection.

[0093] (2) Response in protic solvent (methanol)

[0094] According to different volume ratios, a mixed solvent of methanol and water in a certain proportion was prepared, and a fluorescent probe containing phenanthroquinone rhodamine 6G was added to observe and record the color changes and make an ultraviolet spectrum.

[0095] go through Fig.12 , 13 Compared with 14, it can be seen that as the proportion of methanol decreases, the absorption peak intensity of the phenanthroquinone rhodamine 6G fluorescent probe decreases. The phenanthroquinone rhodamine 6G fluorescent probe has the first significant absorption peak at 285nm, the second significant absorption peak at 355nm, and the third significant absorption peak at 535nm. Among them, after 8 hours, the first absorption peak at 294nm and the second absorption peak at 352nm of the 1:1 group disappeared, the third absorption peak at 537nm was blue 527nm, and the peak value decreased, and there was no obvious change after 24 hours. After 8 hours, the three peaks of the 1:1.5 group were significantly reduced, and the third peak blue-shifted from the 540nm absorption peak to 535nm, and there was no obvious change after 24 hours.

[0096] A series of buffer solutions containing different concentrations of methanol were prepared, and then a trace amount of rhodamine 6G DMF stock solution (40 μl) was added to a volumetric flask (specification: 5 ml) to make the test concentration of the probe 4×10 -5 mol / L, and then perform fluorescence test after stirring evenly. The experimental results are as follows Fig.15 , 16 , 17. It can be seen that as the methanol concentration of the system decreases, the fluorescence intensity of the probe gradually weakens; and the maximum emission wavelength of the fluorescence red-shifts over time. For example, for methanol: water 20:1, after 24 hours, the maximum emission wavelength gradually red-shifts from 556nm to 602nm, and red-shifts to 607nm at 36 hours.

[0097] Add fluorescent probe and acid-base solution with a certain concentration gradient (acid is hydrochloric acid and base is sodium hydroxide) to a solvent with a methanol: water ratio of 20:1, observe and record the color change and make a UV spectrum.

[0098] In a solvent with a methanol: water ratio of 20:1, in response to an acid with a certain concentration gradient, the experimental phenomena showed that the solutions of each concentration gradient showed significantly different color changes from the blank group, which indicates that the probe can effectively indicate in an acidic environment.

[0099] like Fig.18 , 19 , the UV spectrum shows that the probe has a significant absorption peak at 535nm. As the acid ratio increases, the absorption peak intensity of the probe decreases. The probe has the first significant absorption peak at 295nm, the second significant absorption peak at 350nm, and the third significant absorption peak at 535nm. After 8h, the three absorption peaks basically do not shift, but the peak value decreases as a whole. The UV graph shows that the solutions of each concentration gradient are significantly different from the blank group, which indicates that the probe can effectively indicate in an acidic environment.

[0100] like Fig. 20 , 21 The fluorescence spectrum shows that increasing the H + concentration, the fluorescence intensity of the probe gradually weakened, and the maximum emission wavelength of the fluorescence gradually red-shifted from 558nm to 562nm. + =1:50 (c:c), the fluorescence intensity of the probe reached the lowest value. After 22 hours, the maximum emission wavelength of each ratio was red-shifted. Under 365nm ultraviolet light, the naked eye can also observe the orange-red fluorescence of the probe rhodamine 6G in H + It is quickly quenched under the action of

[0101] In a solvent with a methanol: water ratio of 20:1, the experimental phenomena showed that the solution phenomena of each concentration gradient were different from those of the blank group, indicating that the probe can effectively indicate in an alkaline environment; and the color gradually became lighter as the alkali ratio increased, and obvious fluorescence appeared after being placed for 7 hours, indicating that the probe was very sensitive to changes in alkali concentration.

[0102] like Fig. 22 , 23, as the proportion of alkali decreases, the absorption peak intensity of the probe weakens. The probe shows the first significant absorption peak at 288nm, the second significant absorption peak at 350nm, and the third significant absorption peak at 530nm. After 8h, the three absorption peaks basically did not shift, but the peak value decreased as a whole. Among them, the peak value of the 1:30 group at 525nm increased instead of decreased. The UV graph shows that the solutions of each concentration gradient are significantly different from the blank group, which indicates that the probe can effectively indicate in an alkaline environment.

[0103] like Fig.24 , 25 , increase the OH - concentration, the fluorescence intensity of the probe gradually weakened, and the maximum emission wavelength of the fluorescence also red-shifted. - =1:50 (c:c), the fluorescence intensity of the probe reached the highest value. - =1:30 (v:v) significantly red-shifted from 551nm to 625nm. And under 365nm ultraviolet light, the naked eye can also observe the probe rhodamine 6G in OH - Yellow fluorescence is rapidly produced under the action of

[0104] Add fluorescent probe and acid-base solution with a certain concentration gradient (acid is hydrochloric acid, base is sodium hydroxide) to a solvent with a methanol: water ratio of 1:1, observe and record the color change and make a UV spectrum.

[0105] In a solvent with a methanol: water ratio of 1:1, in response to an acid with a certain concentration gradient, the experimental phenomena showed that the solutions of each concentration gradient showed significantly different color changes from the blank group, which indicates that the probe can effectively indicate in an acidic environment.

[0106] UV spectrum shows ( Fig.26 , 27 ), the probe has a significant absorption peak at 532nm, and the absorption peak intensity of the probe increases with the increase of the acid ratio. The probe has the first significant absorption peak at 295nm, the second significant absorption peak at 355nm, and the third significant absorption peak at 540nm. After 7h, the three absorption peaks are basically not offset, among which the absorption peaks of 540nm in the blank group, 1:50 and 1:3 groups are reduced. As shown in the UV graph, the solutions of each concentration gradient are significantly different from the blank group, which indicates that the probe can effectively indicate in an alkaline environment.

[0107] like Fig.28 , 29 , increase the H + concentration, the fluorescence intensity of the probe gradually weakened, reducing the H +concentration, the maximum emission wavelength of the probe also gradually increases. + =1:1 (c:c), the fluorescence intensity of the probe reached the highest value. After 24h, the probe:H + =1:1 (c:c) significantly red-shifted from 551nm to 625nm. Under 365nm ultraviolet light, the naked eye can also observe the orange-red fluorescence of the probe rhodamine 6G in H + It is quickly quenched under the action of

[0108] like Fig.30 , 31 , the UV spectrum shows that as the proportion of alkali increases, the absorption peak intensity of the probe gradually decreases. The probe has the first significant absorption peak at 290nm, the second significant absorption peak at 350nm, and the third significant absorption peak at 530nm. After 16h, the three absorption peaks basically did not shift, and the intensity of the third absorption peak at 530nm increased. The UV graph shows that the solutions of each concentration gradient are significantly different from the blank group, which indicates that the probe can effectively indicate in an alkaline environment.

[0109] like Fig.32 , 33 , it can be seen from the fluorescence spectrum that increasing the OH - The fluorescence intensity of the probe gradually weakened with increasing concentration. After 24 hours, it significantly red-shifted from 550nm to 570nm. Under a 365nm ultraviolet lamp, the naked eye can also observe the orange-red fluorescence of the probe Rhodamine 6G in H + It is quickly quenched under the action of

[0110] like Fig.34 , 35 , by observing the UV spectrum, we can find that three peaks appear at 0.5h, namely: the first significant absorption peak is around 290nm, the second absorption peak is around 360nm, and the third significant absorption peak is around 540nm. In each ratio, the 1:2 peak is in the range of 0.8-1.2, which is the ideal peak. At 15h, the change pattern remains unchanged, and three peaks still appear, namely: the first significant absorption peak is around 280nm, the second absorption peak is not very obvious at around 360, and the third significant absorption peak is around 540nm. Comparing the peak changes of 0.5h and 15h, the first peak blue-shifts from around 290 to around 280, the second small peak basically does not shift, but the peak value decreases, the third peak slightly red-shifts, and the peak value decreases slightly. Overall, the displacement change is not obvious, but the peak value decreases more obviously. By comparison, it can be found that the peak of 1:2 has no obvious change and stable color development, the peak of 1:3-1:5 decreases significantly, and the peak and displacement decreases of the other ratio peaks do not change significantly.

[0111] A series of THF buffers with different concentrations were prepared, and then a trace amount of rhodamine 6G DMF stock solution (40 μl) was added to a volumetric flask (specification: 5 ml) to make the test concentration of the probe 4×10 -5 mol / L, stirred evenly and then conducted fluorescence test. The experimental results showed that at 0.5h and 15h of reaction, as the concentration of THF in the system increased, the fluorescence intensity of the probe showed regular changes, and as the reaction time prolonged, the maximum emission wavelength of the fluorescence red-shifted from 548nm to about 570nm. By comparing the fluorescence spectra of 0.5h and 15h of reaction, the red shift was most obvious when the THF: water ratio was 1:1, 1:2, 1:3, and 1.1:1. After 15h of reaction, the maximum emission wavelength basically red-shifted from 550nm to above 570nm, and the red shift of the other ratios was not obvious, but the peak value changes were extremely obvious.

[0112] Add fluorescent probe and acid-base solution with a certain concentration gradient (acid is hydrochloric acid and base is sodium hydroxide) to a solvent with a THF: water ratio of 20:1, observe and record the color change and make a UV spectrum.

[0113] In a solvent with a THF: water ratio of 20:1, in response to an acid with a certain concentration gradient, the experimental phenomena showed that the solutions of each concentration gradient showed significantly different color changes from the blank group, which indicates that the probe can effectively indicate in an acidic environment with a THF: water ratio of 20:1.

[0114] The experimental results show that at 0.5h of reaction, with the increase of acid concentration, the absorption peak of the probe gradually becomes stronger, and three absorption peaks appear, namely: the first significant absorption peak is around 295nm, the second absorption peak is around 355nm, and the third absorption peak is around 538nm, of which the first and third are more significant. After 10h of reaction, the rule remains unchanged, and there are still three absorption peaks, namely: the first is around 288nm, the second is around 355nm, and the third is around 537nm. By comparing the reaction of 0.5h and 10h, it can be found that the first absorption peak blue-shifts from 295nm to 288nm, the second absorption peak does not change, and the third absorption peak has a very small displacement change that can be ignored. Overall, the peak values ​​of the three absorption peaks are all increased, but at 537nm, the peak values ​​of 1:1.5, 1:2, 1:2.5, and 1:3 have a significant decrease, and the decrease value is about 1.

[0115] The experimental results show that when the reaction time is 0.5h and 20h, increasing the H +The fluorescence intensity of the probe gradually increased and then weakened with the increase of reaction time. As the reaction time increased, the maximum emission wavelength of the fluorescence of some proportions gradually red-shifted from 555nm to 570nm, while some proportions blue-shifted from 570nm to around 555nm. By comparing the reaction time of 0.5h and 10h, it can be seen that the fluorescence of each ratio of 1:1.5-1:8 red-shifted from 555nm to around 570nm and the peak value increased significantly, and the fluorescence of 1:10 and 1:30 blue-shifted from 575nm to around 560nm and the peak value decreased significantly. At 0.5h, when the probe: H + =1:5 (c:c), the fluorescence intensity of the probe reached the highest value. + When the ratio of c:c is 1:10, the fluorescence intensity of the probe reaches the highest value.

[0116] In a solvent with a THF: water ratio of 20:1, the experimental phenomena showed that the solutions of each concentration gradient had no significantly different color changes from the blank group in response to a certain concentration gradient of alkali, indicating that the probe was very insensitive to changes in alkali concentration in a solvent with a THF: water ratio of 20:1 (no fluorescence change).

[0117] Add fluorescent probe and acid-base solution with a certain concentration gradient (acid is hydrochloric acid, base is sodium hydroxide) to a solvent with a THF: water ratio of 1:2, observe and record the color change and make a UV spectrum.

[0118] In a solvent with a THF: water ratio of 1:2, the experimental phenomena showed that the solutions of each concentration gradient had no significantly different color changes from the blank group in response to a certain acid concentration gradient, indicating that the probe was very insensitive to changes in acid concentration in a solvent with a THF: water ratio of 1:2.

[0119] like Fig.36 , 37 The UV spectrum shows that at 0.5h of reaction, with the increase of acid concentration, the absorption peak of the probe gradually becomes stronger, and three absorption peaks appear, namely: the first absorption peak appears at about 294nm, the second absorption peak appears at about 370nm, and the third absorption peak appears at about 541nm. At 11h of reaction, the intensity change law remains unchanged, and there are still three absorption peaks, namely: the first at about 294nm, the second at about 370nm, and the third at about 541nm. By comparing the reaction time of 0.5h and 11h, it can be seen that after 11h, the three absorption peaks basically do not shift, and the peak value increases slightly as a whole, but the ratios of 1:0.5, 1:1.5, and 1:2 have dropped significantly.

[0120] Based on the fluorescence graphs (0.5h) and (20h) of the fluorescent probe's response to acid, it can be seen that at 0.5h and 20h of reaction, increasing the H+ The fluorescence intensity of the probe gradually weakened with the increase of reaction time. As the reaction time increased, the maximum emission wavelength of the fluorescence of some proportions gradually red-shifted from 551nm to about 582nm, and the fluorescence of some proportions blue-shifted from 570nm to about 550nm. By comparing the reaction time of 0.5h and 11h, it can be found that the ratio of 1:0.5 was red-shifted from 561nm to about 575nm, and most of the ratios were blue-shifted from 568nm-574nm to about 549nm-566nm, while the ratio of 1:2.5 remained basically unchanged and was very stable. The peak value rose as a whole. At 0.5h of reaction, when the probe:H+=1:0.5 (c:c), the fluorescence intensity of the probe reached the highest value, and at 11h of reaction, the fluorescence intensity of 1:0.5 was still the highest.

[0121] The above description is intended to be illustrative rather than restrictive. For example, the above examples (or one or more of them) can be used in combination with each other. For example, those of ordinary skill in the art can use other embodiments when reading the above description. In addition, in the above-mentioned specific embodiments, various features can be grouped together to simplify the application. This should not be interpreted as an intention that a disclosed feature that does not require protection is necessary for any claim. On the contrary, the subject matter of the present application may be less than all the features of a specific disclosed embodiment. Thus, the claims are incorporated into the specific embodiments as examples or embodiments, wherein each claim is independently used as a separate embodiment, and it is considered that these embodiments can be combined with each other in various combinations or arrangements. The scope of the present application should be determined with reference to the full scope of the equivalent forms of the attached claims and these claims.

[0122] The above embodiments are only exemplary embodiments of the present application and are not intended to limit the present application. The protection scope of the present application is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present application within the essence and protection scope of the present application, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present application.

Claims

1. A phenanthroquinone rhodamine 6G fluorescent probe for acid-base detection, characterized in that: The structure of the phenanthroquinone rhodamine 6G fluorescent probe is as follows: 。 2. A method for preparing a phenanthroquinone rhodamine 6G fluorescent probe, characterized in that: The preparation method comprises: Mixing a first mass of rhodamine 6G, a first volume of hydrazine hydrate, and a second volume of methanol to perform a first reaction; Performing a first purification treatment on the product obtained after the first reaction to obtain a reaction intermediate; mixing a second mass of the reaction intermediate, a third mass of phenanthrenequinone, a fourth mass of ammonium acetate, and a third volume of methanol to perform a second reaction; A second purification treatment is performed on the product obtained after the second reaction to obtain a target product, which is a phenanthroquinone rhodamine 6G fluorescent probe.

3. The preparation method according to claim 2, characterized in that: The eluent in the first purification treatment is a mixture of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 20-40:0.5-2.

4. The preparation method according to claim 2, characterized in that: The eluent in the second purification treatment is a mixture of dichloromethane and methanol, wherein the volume ratio of dichloromethane to methanol is 20-40:0.5-2.

5. The preparation method according to claim 2, characterized in that: The first mass is 3-8 g, the first volume is 4-9 ml, and the second volume is 15-30 ml; The second mass is 3-5 g, the third mass is 1-3 g, the fourth mass is 1-5 g, and the third volume is 8-20 ml.

6. The preparation method according to claim 2, characterized in that: The first reaction specifically includes: adding a first mass of rhodamine 6G, a first volume of hydrazine hydrate and a second volume of methanol into a round-bottom flask in sequence, and reacting at room temperature for 1-3 hours.

7. The preparation method according to claim 2, characterized in that: The second reaction specifically includes: adding a second mass of the reaction intermediate, a third mass of phenanthrenequinone, a fourth mass of ammonium acetate and a third volume of methanol into a round-bottom flask, and heating and stirring until the reaction is completed.

8. The preparation method according to claim 7, characterized in that: The heating temperature is between 100-140°C; After the second reaction is completed, water is continuously added to the round-bottom flask for mixing, and the precipitate is filtered and repeatedly washed. After drying, a second purification treatment is performed using column chromatography to obtain the target product.

9. A phenanthroquinone rhodamine 6G fluorescent probe, characterized in that: The method for preparing the phenanthroquinone rhodamine 6G fluorescent probe according to any one of claims 2 to 8 is used.

10. Use of the phenanthroquinone rhodamine 6G fluorescent probe according to claim 9 in biomedicine, environmental monitoring or industrial process control.

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