Rhodamine dye probe for mercury ion detection as well as synthesis method and application of rhodamine dye probe

Abstract

The invention discloses a rhodamine dye probe for mercury ion detection and a synthesis method and application thereof, and belongs to the technical field of fluorescent probe detection. The synthesis method of the rhodamine dye probe for mercury ion detection comprises the following steps: taking 2-amino thiol and 2, 4-dihydroxy benzaldehyde as raw materials, and reacting to obtain a precursor compound; taking the precursor compound and 4-diethylaminoketonic acid as raw materials, and reacting to obtain an intermediate product; taking the intermediate product and dimethyl sulfur carbamoyl chloride as raw materials, and reacting to obtain the rhodamine dye probe for mercury ion detection. The rhodamine dye probe synthesized by the invention can be efficiently combined with mercury ions, shows a remarkable fluorescence enhancement effect under low concentration, has high selectivity and high sensitivity, and can be widely applied to detection of mercury ions in environmental water samples and food and imaging of mercury ions in cells; and theoretical basis and technical support are provided for biological research and environmental monitoring of mercury ions.

Description

Technical Field

[0001] The present invention relates to the technical field of fluorescence probe detection, and particularly to a rhodamine dye probe for mercury ion detection, its synthesis method and application. Background Art

[0002] Mercury ion (Hg 2+ ) is a common and toxic heavy metal in environmental pollution, widely existing in water bodies, soil and food, and may cause health problems through bioaccumulation in organisms.

[0003] Existing mercury ion detection methods, such as atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS), have high precision, but these methods are complex to operate and the equipment is expensive, making it difficult to achieve on-site rapid detection. Therefore, the mercury ion detection technology based on fluorescence probes has gradually become a research hotspot. Rhodamine-based fluorescence probes have made remarkable progress in metal ion detection due to their good optical properties and structural tunability, but there is still room for improvement in selectivity, sensitivity and practical applications in the existing technology. Summary of the Invention

[0004] The purpose of the present invention is to provide a rhodamine dye probe for mercury ion detection, its synthesis method and application to solve the problems existing in the above-mentioned existing technology. The rhodamine derivative with an affinity coordination group (i.e., probe RB-HG) designed by the present invention can produce a significant fluorescence enhancement reaction after binding with mercury ions, and has high selectivity, high sensitivity and a low detection limit.

[0005] To achieve the above purpose, the present invention provides the following solutions:

[0006] One of the technical solutions of the present invention: A rhodamine dye probe for mercury ion detection (abbreviated as RB-HG), and the structural formula of the rhodamine dye probe for mercury ion detection is shown as follows:

[0007]

[0008] The probe RB-HG of the present invention can produce a significant fluorescence enhancement reaction after binding with mercury ions, realizing the sensitive detection of mercury ions. Specifically, the fluorescence intensity of RB-HG itself is very weak, but the acyl chloride group of RB-HG serves as the response site and will fall off after reacting with mercury ions. RB-HG will open the ring and turn into a keto structure (i.e., RB-II), and RB-II has a strong fluorescence intensity. Therefore, the probe RB-HG can produce a significant fluorescence enhancement reaction after binding with mercury ions, thereby realizing the sensitive detection of mercury ions.

[0009] The second technical solution of the present invention: The synthesis method of the rhodamine dye probe for mercury ion detection includes the following steps:

[0010] Using 2-aminobenzenethiol and 2,4-dihydroxybenzaldehyde as raw materials, reacting to obtain a precursor compound (abbreviated as RB-I);

[0011] Using the precursor compound and 4-diethylamino ketonic acid as raw materials, reacting to obtain an intermediate product (abbreviated as RB-II);

[0012] Using the intermediate product and dimethylthiocarbamyl chloride as raw materials, reacting to obtain the rhodamine dye probe for mercury ion detection;

[0013] The structural formula of the precursor compound is

[0014] The structural formula of the intermediate product is

[0015] Furthermore, the step of using 2-aminobenzenethiol and 2,4-dihydroxybenzaldehyde as raw materials to react to obtain the precursor compound includes:

[0016] Mixing the 2-aminobenzenethiol, 2,4-dihydroxybenzaldehyde with solvent 1 and catalyst 1, stirring and reacting for 8 - 12 h to obtain the precursor compound.

[0017] Furthermore, the solvent 1 includes methanol.

[0018] Furthermore, the catalyst 1 includes concentrated sulfuric acid.

[0019] Furthermore, the dosage ratio of the 2-aminobenzenethiol, 2,4-dihydroxybenzaldehyde and solvent 1 is 1 mmol:1 mmol:4 - 5 mL.

[0020] Furthermore, the concentration of the concentrated sulfuric acid is 18.4 mol / L.

[0021] Furthermore, the volume ratio of the catalyst 1 (concentrated sulfuric acid) to the solvent 1 is 1:8 - 10.

[0022] Furthermore, the step of using the precursor compound and 4-diethylamino ketonic acid as raw materials to react to obtain the intermediate product includes:

[0023] Mixing the precursor compound and 4-diethylamino ketonic acid with solvent 2, heating and reacting at 90 °C for 3 - 5 h to obtain the intermediate product.

[0024] Furthermore, the solvent 2 includes methanesulfonic acid (MSA), and methanesulfonic acid serves as both a solvent and a catalyst.

[0025] Further, the dosage ratio of the precursor compound, 4-diethylamino ketonic acid and solvent 2 is 1 mmol: 1 mmol: 3 - 5 mL.

[0026] Further, the reaction to obtain the rhodamine dye probe for mercury ion detection using the intermediate product and dimethylthiocarbamyl chloride as raw materials includes:

[0027] Mix the intermediate product, dimethylthiocarbamyl chloride with solvent 3 and catalyst 2, and stir and react for 1 - 2 h to obtain the rhodamine dye probe for mercury ion detection.

[0028] Further, the solvent 3 includes dichloromethane.

[0029] Further, the catalyst 2 includes triethylamine.

[0030] Further, the dosage ratio of the intermediate product, dimethylthiocarbamyl chloride and solvent 3 is 1 mmol: 1 - 2 mmol: 4 - 5 mL.

[0031] Further, the volume ratio of the catalyst 2 (triethylamine) to the solvent 3 is 1:8 - 10.

[0032] Technical solution three of the present invention: Application of the above-mentioned rhodamine dye probe for mercury ion detection in detecting mercury ions.

[0033] Technical solution four of the present invention: Application of the above-mentioned rhodamine dye probe for mercury ion detection in preparing a fluorescence detection product for fluorescence imaging of mercury ions in living cells.

[0034] The present invention discloses the following technical effects:

[0035] The present invention discloses a rhodamine dye probe RB - HG. Through experiments, it is found that the probe RB - HG has a high affinity for mercury ions and is a fluorescence probe with excellent performance, high stability and strong selectivity. The probe RB - HG can efficiently bind mercury ions, show a significant fluorescence enhancement effect at low concentrations, has high selectivity and high sensitivity, and can be widely applied to the detection of mercury ions in environmental water samples and foods and the imaging of mercury ions in cells, providing a theoretical basis and technical support for the biological research and environmental monitoring of mercury ions. Description of the Drawings

[0036] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0037] Figure 1 The ultraviolet absorption spectra of the probe RB-HG in PBS buffer solution with and without mercury ions.

[0038] Figure 2 The test results of the response performance of the probe RB-HG to mercury ions with different concentrations. Among them, A is the fluorescence spectrum of the probe RB-HG under mercury ions with different concentrations, and B is the linear relationship diagram of the probe RB-HG for mercury ion detection.

[0039] Figure 3 The schematic diagram of the principle for the fluorescence detection of mercury ions by the probe RB-HG.

[0040] Figure 4 For the product after the reaction of RB-HG and Hg 2+ (i.e., RB-HG+Hg 2+ ), as well as the 1H NMR spectra of the intermediate product RB-II prepared in step (2) of Example 1 and RB-HG prepared in step (3) of Example 1.

[0041] Figure 5 For the intermediate product RB-II prepared in step (2) of Example 1, RB-HG prepared in step (3) of Example 1, and the product after the reaction of RB-HG and Hg 2+ (i.e., RB-HG+Hg 2+ ), the mass spectra. Among them, A is the mass spectrum of RB-II, B is the mass spectrum of RB-HG, and C is the mass spectrum comparison of the product after the reaction of RB-HG and Hg 2+ and RB-HG.

[0042] Figure 6 The test results of the influence of pH value on the probe RB-HG.

[0043] Figure 7 The test results of the selectivity of the probe RB-HG. Among them, A is the maximum fluorescence intensity of the probe RB-HG under different ions, and B is the fluorescence spectrum of the probe RB-HG under different ions.

[0044] Figure 8 The test results of the anti-interference ability of the probe RB-HG.

[0045] Figure 9 The evaluation results of the cytotoxicity of the probe RB-HG.

[0046] Figure 10 The test results of the fluorescence imaging ability of the probe RB-HG for mercury ions in living cells. Detailed implementation manners

[0047] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0048] It should be understood that the terms used in the present invention are only for describing particular embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0049] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0050] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0051] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0052] It should be noted that those aspects of the present invention not described in detail are all conventional operating means in the art and are not the focus of the present invention.

[0053] As a first aspect of the present invention, the present invention provides a rhodamine-based dye probe for mercury ion detection (abbreviated as RB-HG), and the structural formula of the rhodamine-based dye probe for mercury ion detection is shown as follows:

[0054]

[0055] As a second aspect of the present invention, the present invention provides a synthesis method of the above rhodamine-based dye probe for mercury ion detection (i.e., probe RB-HG), including the following steps:

[0056] (1) Synthesis of precursor compound RB-I

[0057] The synthesis route is as follows:

[0058]

[0059] The synthesis steps are as follows: Mix 2-aminobenzenethiol, 2,4-dihydroxybenzaldehyde, solvent 1, and catalyst 1, and stir the reaction at room temperature for 8 - 12 h to obtain a precursor compound;

[0060] (2) Synthesis of intermediate product

[0061] The synthesis route is as follows:

[0062]

[0063] The synthesis steps are as follows: Mix the precursor compound and 4-diethylaminoketone acid with solvent 2, and heat the reaction at 90 °C for 3 - 5 h to obtain an intermediate product;

[0064] (3) Synthesis of probe RB-HG

[0065]

[0066] The synthesis steps are as follows: Mix the intermediate product, dimethylthiocarbamyl chloride, solvent 3, and catalyst 2, and stir the reaction for 1 - 2 h to obtain probe RB-HG.

[0067] As a preferred embodiment of the present invention, the solvent 1 includes methanol.

[0068] As a preferred embodiment of the present invention, the catalyst 1 includes concentrated sulfuric acid.

[0069] As a preferred embodiment of the present invention, the dosage ratio of 2-aminobenzenethiol, 2,4-dihydroxybenzaldehyde, and solvent 1 is 1 mmol:1 mmol:4 - 5 mL.

[0070] As a preferred embodiment of the present invention, the concentration of the concentrated sulfuric acid is 18.4 mol / L.

[0071] As a preferred embodiment of the present invention, the volume ratio of the catalyst 1 (concentrated sulfuric acid) to the solvent 1 is 1:8 - 10.

[0072] As a preferred embodiment of the present invention, the solvent 2 includes methanesulfonic acid (MSA).

[0073] As a preferred embodiment of the present invention, the dosage ratio of the precursor compound, 4-diethylaminoketone acid, and solvent 2 is 1 mmol:1 mmol:3 - 5 mL.

[0074] As a preferred embodiment of the present invention, the solvent 3 includes dichloromethane.

[0075] As a preferred embodiment of the present invention, the catalyst 2 includes triethylamine.

[0076] As a preferred embodiment of the present invention, the dosage ratio of the intermediate product, dimethylthiocarbamyl chloride and solvent 3 is 1 mmol: 1 - 2 mmol: 4 - 5 mL.

[0077] As a preferred embodiment of the present invention, the volume ratio of the catalyst 2 (triethylamine) to the solvent 3 is 1:8 - 10.

[0078] As the third aspect of the present invention, the present invention provides the application of the above-mentioned rhodamine dye probe for mercury ion detection in the detection of mercury ions.

[0079] As a preferred embodiment of the present invention, the steps of the application include:

[0080] (1) Mix RB-HG with mercury ion standard solutions of different concentrations, incubate for 90 min, then measure the fluorescence intensity at a wavelength of 580 nm, establish the quantitative relationship between the fluorescence intensity and the mercury ion concentration, draw a standard curve, and obtain a regression equation;

[0081] (2) Measure the fluorescence intensity of the sample determination solution according to the operation in step (1), substitute it into the regression equation, and calculate the concentration of mercury ions in the sample determination solution.

[0082] As the fourth aspect of the present invention, the present invention provides the application of the above-mentioned rhodamine dye probe for mercury ion detection in the preparation of a fluorescence detection product for fluorescence imaging of mercury ions in living cells.

[0083] The technical solution of the present invention will be further described below in conjunction with specific embodiments.

[0084] If room temperature or normal temperature is involved in the specific embodiments of the present invention, it specifically refers to 20 - 30 °C.

[0085] All raw materials used in the following examples and comparative examples of the present invention are ordinary commercially available products.

[0086] Example 1

[0087] A synthesis method of a rhodamine dye probe for mercury ion detection is as follows:

[0088] (1) Synthesis of the precursor compound RB-I

[0089] Add 125 mg of 2-aminobenzenethiol and 138 mg of 2,4-dihydroxybenzaldehyde to 5 mL of methanol under stirring, dropwise add 0.5 mL (18.4 mol / L) of concentrated sulfuric acid for catalysis, and stir at normal temperature for 10 h. After the reaction is completed, filter by suction to obtain a light yellow solid product (yield is about 50%), which is RB-I.

[0090] The prepared RB-I was subjected to nuclear magnetic resonance detection, and the results were as follows:

[0091] 1 H NMR(500MHz,DMSO)δ11.71(s,1H),10.22(s,1H),8.08(s,1H),7.96(s,1H),7.93(s,1H),7.50(s,1H),7.39(s,1H),6.47(s,2H).

[0092] 13 C NMR(126MHz,DMSO)δ166.82(s),162.08(s),158.75(s),151.95(s),133.68(s),130.52(s),126.82(s),125.01(s),122.31(s),121.87(s),110.62(s),108.95(s),103.15(s).

[0093] (2) Synthesis of intermediate RB-II

[0094] 243 mg of RB-I and 313 mg of 4-diethylaminoketone acid were dissolved in 3 mL of methanesulfonic acid and reacted at 90 °C for 3 h. After the reaction, it was neutralized and extracted with dichloromethane three times. The organic phase was collected and further evaporated. Then, using dichloromethane (DCM) and methanol (MA) (volume ratio 70:1) as the developing agent, thin-layer chromatography was used for separation to obtain a red solid (yield about 30%), which was RB-II.

[0095] The prepared RB-II was subjected to nuclear magnetic resonance detection, and the results were as follows:

[0096] 1 H NMR(500MHz,CDCl3)δ12.74(s,1H),8.01(d,J=7.4Hz,1H),7.85(d,J=8.1Hz,1H),7.69(d,J=7.9Hz,1H),7.61(dt,J=20.8,7.3Hz,2H),7.39(t,J=7.6Hz,1H),7.28(t,J=7.5Hz,1H),6.97(s,1H),6.86(s,1H),6.50(d,J=8.8Hz,1H),6.41(s,1H),6.30(d,J=8.6Hz,1H),3.30(q,J=6.8Hz,4H),1.11(t,J=6.9Hz,6H).

[0097] 1313C NMR (126 MHz, CDCl3) δ 169.58 (s), 168.17 (s), 159.68 (s), 154.98 (s), 152.74 (d, J = 11.3 Hz), 151.55 (s), 149.72 (s), 135.04 (s), 132.21 (s), 129.79 (s), 128.73 (d, J = 13.9 Hz), 127.18 (s), 126.76 (s), 125.59 (s), 125.08 (s), 124.08 (s), 122.04 (s), 121.44 (s), 114.01 (s), 112.38 (s), 108.61 (s), 104.63 (s), 97.80 (s), 83.85 (s), 44.54 (s), 12.53 (s).

[0098] MS (ESI) m / z: calcd for C 31 H 24 N2O4S [M+H] + m / z 521.15, found 521.56.

[0099] The corresponding 1H NMR spectrum is as shown in Figure 4 and the corresponding mass spectrum is as shown in Figure 5 .

[0100] (3) Synthesis of rhodamine-based dye probe RB-HG

[0101] Dissolve 200 mg of RB-II and 123.6 mg of dimethylthiocarbamyl chloride in 5 mL of dichloromethane, then add 0.5 mL of triethylamine, and stir the reaction at room temperature for 2 h. After the reaction is completed, first evaporate the solvent, then extract with dichloromethane and water three times, collect the organic phase, and further evaporate; then use dichloromethane (DCM) and methanol (MA) (volume ratio 100:1) as the developing solvent, and separate by thin layer chromatography to obtain a white solid (yield about 10%), which is RB-HG.

[0102] Perform nuclear magnetic resonance detection on the prepared RB-HG, and the results are as follows:

[0103] 11H NMR (500 MHz, CDCl3) δ 8.00 (d, J = 7.6 Hz, 1H), 7.85 (d, J = 8.1 Hz, 1H), 7.74 (d, J = 7.9 Hz, 1H), 7.69 - 7.51 (m, 3H), 7.35 (t, J = 7.6 Hz, 1H), 7.24 (dd, J = 15.5, 7.6 Hz, 2H), 7.04 (s, 1H), 6.55 - 6.25 (m, 3H), 3.43 (d, J = 13.4 Hz, 6H), 3.34 - 3.22 (m, 4H), 1.10 (t, J = 6.9 Hz, 6H).

[0104] 13 13C NMR (126 MHz, CDCl3) δ 186.00 (s), 169.67 (s), 161.35 (s), 153.40 (s), 152.58 (dd, J = 27.3, 13.8 Hz), 135.28 (d, J = 14.3 Hz), 130.26 (s), 129.87 (s), 128.84 (s), 126.15 (s), 125.15 (d, J = 9.6 Hz), 124.28 (s), 123.21 (s), 121.28 (s), 113.28 (s), 97.69 (s), 43.65 (s), 39.46 (s), 12.51 (s).

[0105] MS (ESI) m / z: calcd for C 34 H 29 N3O4S2 [M + H] + m / z 608.16, found 608.81.

[0106] The corresponding 1H NMR spectrum is as shown in Figure 4 and the corresponding mass spectrum is as shown in Figure 5 .

[0107] Example 2

[0108] Detect the UV response of the detection probe RB - HG in the presence and absence of mercury ions

[0109] Add RB - HG to PBS buffer at a concentration of 10 μM to obtain an RB - HG solution (pH = 7.4), and perform UV absorption spectroscopy on the RB - HG solution. Then add mercury ions (Hg 2+ ) with a final concentration of 500 μM to the RB - HG solution, incubate at 37 °C for 90 min, and then perform UV absorption spectroscopy again. In addition, add RB - II to PBS buffer at a concentration of 10 μM to obtain an RB - II solution (pH = 7.4), and perform UV absorption spectroscopy on the RB - II solution as a control.

[0110] The results are as Figure 1 shown (where RB-HG+Hg 2+ represents the presence of RB-HG and Hg in the solution 2+ , abbreviated as RB-HG+Hg 2+ solution). The ultraviolet absorption spectrum of the probe RB-HG in PBS buffer has a strong absorption peak at 350 nm. When mercury ions are added to the RB-HG solution, it can be seen that there is a certain red-shift process of the absorption peak, and the strongest absorption peak red-shifts to about 560 nm. This is because the probe RB-HG binds to mercury ions, and the molecule of the probe RB-HG changes back to RB-II, resulting in a red-shift of the ultraviolet absorption of the probe. The ultraviolet spectra of the RB-HG+Hg 2+ solution and the RB-II solution are highly similar, which can prove the above speculation about the reaction mechanism.

[0111] After testing the ultraviolet absorption spectra of the probe RB-HG before and after binding to mercury ions, the excitation wavelength of the probe RB-HG is obtained as 560 nm.

[0112] Example 3

[0113] Detect the response performance of the probe RB-HG to mercury ions with different concentrations. The specific steps are as follows:

[0114] First, prepare PBS solutions with different concentrations of mercury ions (0 μM to 100 μM, solution pH = 7.4) with a total volume of 1000 μL, then add the probe RB-HG (final concentration 5.0 μM) respectively and mix evenly, incubate at 37 °C for 90 min, and then perform fluorescence emission spectrum tests (excitation wavelength is 560 nm, the same below).

[0115] The fluorescence spectra of the probe RB-HG at different concentrations of mercury ions are as Figure 2 shown in A of. When the concentration of mercury ions is 0 μM, that is, when the probe RB-HG is alone in PBS buffer, its own fluorescence intensity is very weak. As the concentration of mercury ions increases continuously, the fluorescence intensity of the probe RB-HG at the emission wavelength of 580 nm also increases with the increase of the concentration of mercury ions. When the concentration of mercury ions increases to 80 μM, the fluorescence intensity of the probe RB-HG at 580 nm basically no longer increases with the increase of the concentration of mercury ions, indicating that the saturated binding concentration of the probe RB-HG to mercury ions is about 80 μM. It can be seen from the figure that when the probe RB-HG reaches the saturated response, the maximum fluorescence response of the probe RB-HG is about 15 times that of the control without mercury ions. The results show that the probe RB-HG responds very sensitively to mercury ions, indicating that the fluorescence signal of the probe RB-HG is activated by mercury ions. This may be due to the interaction between the probe RB-HG and mercury ions, which changes back to RB-II.

[0116] According to Figure 2 the fluorescence spectrum of A, with different concentrations of mercury ions as the abscissa and the strongest emission intensity of the probe RB-HG at the maximum emission wavelength of 580 nm as the ordinate, a linear relationship graph of the detection of mercury ions by the probe RB-HG was plotted (showing the standard curve and regression equation), as shown in B of Figure 2 . It can be found that the probe RB-HG has a very good linear relationship with mercury ions with a concentration of 0-80 μM, and its R 2 = 0.9927. The detection limit was calculated to be 1.16 μM by the formula LOD = 3σ / k (the ratio of the noise level (3σ) to the method sensitivity (k)).

[0117] From the above experimental results, it can be seen that the probe RB-HG is a fluorescent probe for mercury ions with excellent performance.

[0118] Test Example 1

[0119] The schematic diagram of the principle of fluorescence detection of mercury ions by the probe RB-HG in Examples 2-3 is as shown in Figure 3 . When there is only RB-HG in the solution, the fluorescence intensity of the probe RB-HG itself is very weak (visible from the fluorescence photo below the RB-HG structural formula in Figure 3 , that is, the fluorescence photo marked Non-florescence). However, after adding mercury ions to the solution, the acyl chloride group of RB-HG serves as the response site and will react with mercury ions and then fall off. RB-HG will open the ring to become a keto structure (i.e., RB-II), and RB-II has a strong fluorescence intensity (visible from the fluorescence photo below the RB-II structural formula in Figure 3 , that is, the fluorescence photo marked Non-florescence), emitting strong red fluorescence, thus realizing the fluorescence detection of mercury ions. To verify this principle, the following tests were carried out:

[0120] (1) Verification by hydrogen spectrum

[0121] The RB-HG+Hg 2+ solution after adding mercury ions and incubating in Example 2 was purified to obtain the product after the reaction of RB-HG and Hg 2+ (i.e., Figure 4 RB-HG+Hg in 2+ ). Its nuclear magnetic resonance hydrogen spectrum was detected and compared with the nuclear magnetic resonance hydrogen spectra of RB-II prepared in step (2) of Example 1 and RB-HG prepared in step (3) of Example 1. The results are as shown in Figure 4 . It can be seen that the RB-HG and Hg 2+ obtained by purification from the RB-HG+Hg 2+The 1H NMR spectrum of the product after the reaction was consistent with that of the intermediate RB-II prepared in step (2) of Example 1. The comparison of the 1H NMR spectra proved that RB-HG reacted with Hg 2+ to form RB-II, verifying the proposed mechanism is correct.

[0122] (2) Mass spectrometry verification

[0123] The RB-HG+Hg solution after adding mercury ions and incubating in Example 2 2+ was purified to obtain RB-HG and Hg 2+ The product after the reaction (i.e., Figure 5 RB-HG+Hg in C 2+ ) was subjected to mass spectrometry and compared with the mass spectra of RB-II prepared in step (2) of Example 1 and RB-HG prepared in step (3) of Example 1. The results are shown in Figure 5 Figure, where A is the mass spectrum of RB-II, B is the mass spectrum of RB-HG, and C is the comparison of the mass spectra of the product after the reaction of RB-HG and Hg 2+ and RB-HG. As shown in the figure, the mass spectrometry value of RB-HG is 608.81, that of RB-II is 521.56, and after RB-HG binds to Hg 2+ , a value of 521.66 appears in the mass spectrum, proving the formation of RB-II and further indicating that the proposed mechanism is correct.

[0124] Example 4

[0125] Detect the effect of pH on the probe RB-HG

[0126] Due to the complex intracellular environment, the pH values in different subcellular organelles vary significantly. Therefore, the effect of pH on the fluorescence emission of the probe RB-HG and when the probe RB-HG binds to mercury ions was investigated next. Specifically: The fluorescence intensity of a 5.0 μM probe RB-HG solution was measured in the pH range of 4.0 - 8.0 (i.e., RB-HG solutions with different pH values were prepared for testing) as a control; 50 μM mercury ions were added to the probe RB-HG solutions with different pH values, and the fluorescence intensity was detected after incubating at 37 °C for 90 min.

[0127] The results are shown in Figure 6 Figure (where Probe represents only RB-HG in the solution, Probe+50 μM Hg 2+ represents there is RB-HG and Hg in the solution 2+) When the pH value is in the range of 4.0 - 8.0, the activation of the probe RB-HG is not affected by pH because the probe RB-HG has almost no fluorescence in this pH range. However, the fluorescence intensity of the probe RB-HG after binding with mercury ions remains almost unchanged in the pH range of 6.0 - 8.0, indicating that the physiological pH has little effect on the response of the probe RB-HG to the binding of mercury ions. The decrease in fluorescence of the probe RB-HG at acidic pH values may be related to the reduced stability of the probe RB-HG. In summary, the above results show that the probe RB-HG can be used as a specific fluorescent probe for mercury ions, and the influence of pH changes can be ignored.

[0128] Example 5

[0129] Selectivity test of probe RB-HG

[0130] In this example, the specific selection of the probe RB-HG for mercury ions was confirmed by studying the reaction of the probe RB-HG with different ions. Specifically: Different ions with a final concentration of 50 μM were added to a 5.0 μM probe solution (the solvent was PBS buffer, and the solution pH = 7.4), and fluorescence tests were performed after incubation at 37 °C for 90 minutes.

[0131] The results are as Figure 7 shown. Among them, A is the maximum fluorescence intensity of the probe RB-HG under different ions (the gray column represents the fluorescence intensity when only the probe RB-HG is present, and the red column represents the fluorescence intensity after adding different ions to the probe RB-HG solution), and B is the fluorescence spectrum of the probe RB-HG under different ions. The results show that a significant increase in fluorescence was observed after the probe RB-HG bound to mercury ions, and in the presence of other ions, the fluorescence intensity of the probe RB-HG also increased. In contrast, after the probe RB-HG responded to other ions except mercury ions, the fluorescence enhancement of the probe RB-HG was very weak. That is, the probe RB-HG only showed a good response to mercury ions. This result indicates that the probe RB-HG can specifically interact with mercury ions and activate fluorescence, and has a very high specific selectivity response to mercury ions.

[0132] Example 6

[0133] Anti-interference ability test of probe RB-HG

[0134] In this example, the anti-interference ability of the probe RB-HG was confirmed by studying the response of the probe RB-HG to Hg 2+ in a mixed solution containing Hg 2+ and different other ions (including Ca 2+ 、Cd 3+ 、Al 2+ etc.). Specifically: Hg 2+ was prepared with PBS buffer as the solvent.A mixed solution with another kind of other ions (Hg 2+ and the concentration of another kind of other ions is 50 μM, and the pH of the solution is 7.4). By changing the types of other ions, multiple groups of solutions are obtained. Probe RB-HG with a final concentration of 5.0 μM is added respectively. After incubation at 30 °C for 90 minutes, fluorescence tests are carried out. And a Hg 2+ solution with a concentration of 50 μM is prepared. Probe RB-HG with a final concentration of 5.0 μM is added, and after incubation at 30 °C for 90 minutes, fluorescence tests are carried out, serving as a control for adding only mercury ions.

[0135] The results are as Figure 8 shown, indicating that after the probe RB-HG binds to mercury ions, a high fluorescence enhancement is observed. And under the condition of having mercury ions and adding another kind of other ions, the fluorescence intensity of the probe RB-HG also increases significantly. This result shows that the probe RB-HG has a very high selective response to mercury ions and strong anti-interference ability.

[0136] Example 7

[0137] Cytotoxicity evaluation

[0138] The WST-8 reagent is used to evaluate the cytotoxicity of the probe RB-HG to HeLa cells. The specific operation steps are as follows:

[0139] RB-HG solutions with different concentrations (0 μM - 40 μM) (the solvent is PBS buffer) are added to different wells of cells with a density of about 30%. After 24 h, they are washed 3 times with PBS, and then WST-8 reagent is added. After incubation for about 3 h, the absorbance at 580 nm is recorded with an enzyme-linked immunosorbent assay instrument to explore the cell viability.

[0140] The results are as Figure 9 shown, showing that after incubation with the probe RB-HG at different concentrations for 24 h, the survival rate of HeLa cells exceeds 95%, indicating that the probe TPEOP has good biocompatibility.

[0141] Example 8

[0142] Based on the excellent performance of the probe RB-HG in vitro, this example studies the ability of the probe RB-HG to perform fluorescence imaging of mercury ions in living cells. The specific operation steps are as follows:

[0143] A 5.0 μM probe RB-HG solution (the solvent is PBS buffer) and different concentrations of mercury ions are used to culture HeLa cells with a density of about 40%. Then, confocal fluorescence imaging is carried out, and confocal fluorescence images are obtained.

[0144] The results are as Figure 10As shown, in the case of different mercury ion concentrations (0, 20, 50 μM), the fluorescence intensity of the cells increased successively. This result indicates that the probe RB-HG can effectively penetrate cells and target mercury ions inside the cells.

[0145] The embodiments described above are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A rhodamine dye probe for mercury ion detection, characterized in that The structural formula of the rhodamine-based dye probe for mercury ion detection is as follows:

2. The synthesis method of the rhodamine dye probe for mercury ion detection according to claim 1, characterized in that, It includes the following steps: Using 2-aminobenzenethiol and 2,4-dihydroxybenzaldehyde as raw materials to react to obtain a precursor compound; Using the precursor compound and 4-diethylamino ketonic acid as raw materials to react to obtain an intermediate product; Using the intermediate product and dimethylthiocarbamyl chloride as raw materials to react to obtain the rhodamine-based dye probe for mercury ion detection; The structural formula of the precursor compound is The structural formula of the intermediate product is 3. The synthesis method according to claim 2, characterized in that, The step of using 2-aminobenzenethiol and 2,4-dihydroxybenzaldehyde as raw materials to react to obtain a precursor compound includes: Mixing the 2-aminobenzenethiol, 2,4-dihydroxybenzaldehyde with solvent 1 and catalyst 1, and stirring and reacting for 8 - 12 h to obtain the precursor compound.

4. The synthesis method according to claim 3, wherein The solvent 1 includes methanol; And / or, the catalyst 1 includes concentrated sulfuric acid; And / or, the dosage ratio of the 2-aminobenzenethiol, 2,4-dihydroxybenzaldehyde and solvent 1 is 1 mmol:1 mmol:4 - 5 mL.

5. The synthesis method according to claim 2, wherein The step of using the precursor compound and 4-diethylamino ketonic acid as raw materials to react to obtain an intermediate product includes: Mixing the precursor compound and 4-diethylamino ketonic acid with solvent 2, and heating and reacting at 90 °C for 3 - 5 h to obtain the intermediate product.

6. The synthesis method according to claim 5, characterized in that, The solvent 2 includes methanesulfonic acid; And / or, the dosage ratio of the precursor compound, 4-diethylamino ketonic acid and solvent 2 is 1 mmol:1 mmol:3 - 5 mL.

7. The synthesis method according to claim 2, characterized in that, The step of using the intermediate product and dimethylthiocarbamyl chloride as raw materials to react to obtain the rhodamine-based dye probe for mercury ion detection includes: Mixing the intermediate product and dimethylthiocarbamyl chloride with solvent 3 and catalyst 2, and stirring and reacting for 1 - 2 h to obtain the rhodamine-based dye probe for mercury ion detection.

8. The synthesis method according to claim 7, characterized in that, The solvent 3 includes dichloromethane; And / or, the catalyst 2 includes triethylamine; And / or, the dosage ratio of the intermediate product, dimethylthiocarbamyl chloride and solvent 3 is 1 mmol:1 - 2 mmol:4 - 5 mL.

9. Application of a rhodamine-based dye probe for mercury ion detection as described in claim 1 in detecting mercury ions.

10. Application of a rhodamine-based dye probe for mercury ion detection as described in claim 1 in preparing a fluorescence detection product for fluorescence imaging of mercury ions in living cells.

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