Peptide-based real-time ratiometric fluorescence sensor with excellent methylmercury ion and mercury ion selectivity and method for detecting methylmercury and mercury ions using the same

Abstract

One embodiment of the present invention provides a peptide-based real-time proportional fluorescence sensor with excellent selectivity for methylmercury ions and mercury ions, and a method for detecting methylmercury and mercury ions using the same. According to one embodiment of the present invention, the peptide-based real-time proportional fluorescence sensor with excellent selectivity for methylmercury ions and mercury ions dissolves well in a pure aqueous solution (1% DMSO), is excited by visible light, and can recognize Hg(II) and methylmercury with high sensitivity through proportional fluorescence behavior. In particular, it exhibits a fast detection speed for methylmercury and has high water solubility, but this fluorescence sensor penetrates well into cells and can sensitize Hg(II) and methylmercury inside the cell through a change in the ratio of red and green fluorescence.

Description

Technology Field

[0001] The present invention relates to a peptide-based real-time proportional fluorescence sensor with excellent selectivity for methylmercury and mercury ions, and a method for detecting methylmercury and mercury ions using the same. Background Technology

[0002] The design and research of new sensors for major biological substances and ions have been actively pursued. Recently, the understanding and research of supramolecular chemistry have shown great potential for designing host compounds capable of selectively binding to ions or various other types of guest compounds. Furthermore, by linking these supramolecular compounds to fluorescent materials, this has greatly aided research on the development of fluorescent chemosensors, which allow for easier observation of selective binding to guest compounds using fluorescence changes.

[0003] Fluorescence is a photochemical phenomenon that occurs when a photon with a specific wavelength (excitation wavelength) collides with an indicator molecule, and as a result of the collision, an electron is excited to a high-energy level. Among various analytical methods, methods utilizing fluorescence are highly effective due to their excellent sensitivity, 10 -9 It has the significant advantage of being able to observe signals even at M concentrations. Recently, studies on fluorescent chemical sensors for cations, anions, and neutral organic molecules utilizing this property have been published.

[0004] Mercury (Hg) causes various environmental problems due to its high toxicity and presence in various water resources. In aquatic environments, mercury exists primarily in two forms: inorganic mercury (Hg(II)) and organic mercury, such as methylmercury (methylmercury(I)). Inorganic mercury is converted into methylmercury through the action of aquatic bacteria; methylmercury is the most harmful form among mercury compounds and is more dangerous than inorganic mercury because it easily penetrates cell membranes and accumulates in various organisms. Compared to other forms of mercury, methylmercury is easily absorbed by microorganisms, plants, and seafood, and can easily accumulate in humans through the food chain. Once absorbed into the body, mercury combines with various biomolecules to cause oxidative stress, protein denaturation, and DNA damage, exhibiting severe toxicity. Mercury emissions from natural causes and human activities are steadily increasing, and consequently, environmental pollution by methylmercury and Hg(II) is also on the rise. Therefore, there is a need to develop a system that can more easily and continuously monitor methylmercury and Hg(II) concentrations in water resources, environments, and cells.

[0005] In this case, traditional analytical methods such as atomic absorption spectroscopy (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) are generally used to detect Hg(II) and methylmercury. However, these techniques require complex sample pretreatment and are not suitable for real-time monitoring of mercury compounds in living cells or field samples.

[0006] In recent years, molecular fluorescence sensors have garnered attention due to their high sensitivity, fast detection speed, ease of use, and cost-effectiveness for Hg(II) detection. Some of these fluorescence sensors demonstrate excellent ability to detect Hg(II) in living cells and in aquatic environments.

[0007] However, most mercury-sensing molecular fluorescent sensors are unable to detect fluorescence due to the characteristic low charge, reduced sulfur affinity (thiophilicity), increased hydrophobicity, etc. of methylmercury, and while some fluorescent sensors can detect both Hg(II) and methylmercury, they require a long reaction time (e.g., more than 30 minutes) and a high concentration to obtain an interpretable fluorescence change when detecting methylmercury, which limits their practical application. Therefore, the development of a fluorescent sensor capable of rapidly recognizing both Hg(II) and methylmercury simultaneously remains an important challenge.

[0008] Generally, an ideal fluorescence sensor should possess characteristics such as good water solubility, excellent cell penetration ability, excitation by visible light, and fluorescence change at long wavelengths. Furthermore, it is difficult to perform accurate quantification with fluorescence-enhancing or fluorescence-decreasing sensors due to reasons such as the fluorescence extinction effect of mercury itself, environmental factors affecting fluorescence intensity (solvent polarity, viscosity, temperature, etc.), concentration correction of the fluorescence sensor, and efficiency correction of the fluorescence instrument.

[0009] To minimize these issues with existing fluorescence probes and achieve highly reliable quantification, a proportional fluorescence sensor utilizing two fluorescence bands is being developed. Among these, the proportional fluorescence sensor minimizes environmental factors affecting fluorescence and can compensate for the concentration of the fluorescence probe and the fluorescence efficiency of the fluorescence instrument, thereby offering distinct advantages in the quantitative analysis of mercury compounds in actual samples.

[0010] However, to date, no proportional fluorescence sensor capable of simultaneously detecting Hg(II) and methylmercury in an aquatic environment and in cells has been reported.

[0011] Therefore, there are still many challenges remaining in developing a proportional fluorescence sensor capable of simultaneously detecting Hg(II) and methylmercury in an aquatic environment and in cells. Prior art literature

[0012] Republic of Korea Published Patent No. 10-2024-0161357 The problem to be solved

[0013] The technical problem that the present invention aims to solve is to develop a proportional fluorescence sensor capable of simultaneously detecting Hg(II) and methylmercury by introducing a peptide receptor with self-assembly characteristics specific to Hg(II) and methylmercury and a fluorescence wavelength shifting upon aggregation, thereby detecting methylmercury and mercury ions present in an aqueous environment and within cells in real-time proportional fluorescence.

[0014] The technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned technical problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below. means of solving the problem

[0015] To achieve the above technical problem, one embodiment of the present invention provides a real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions.

[0016] A real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions according to one embodiment of the present invention is characterized by being represented by the following chemical formula 1.

[0017] [Chemical Formula 1]

[0018]

[0019] In the above chemical formula 1, substituent R is an amino acid compound, and R1 and R2 are C1 to C n It is a chain-type carbon compound, R3 is hydrogen (H), cyano group (CN), amide group (amide), or carboxyl group, and R4 is sulfur (S), amine group (NH), or oxygen (O).

[0020] In addition, according to one embodiment of the present invention, the mercury ion real-time proportional fluorescence detection sensor is water-soluble and can detect mercury ions present in a cell.

[0021] In addition, according to one embodiment of the present invention, a fluorescent compound may be bonded to the fluorescent dipeptide compound in Formula 1.

[0022] In addition, according to one embodiment of the present invention, a fluorescent compound is bound to the fluorescent dipeptide compound in Formula 1 to exhibit red fluorescence.

[0023] In addition, according to one embodiment of the present invention, the mercury ion (Hg 2+ ) and methyl mercury ions (CH3Hg + The real-time proportional fluorescence detection sensor of ) detects mercury ions or methylmercury ions (CH3Hg) at concentrations of 4.1 nM or higher + It can detect ).

[0024] To achieve the above technical problem, another embodiment of the present invention provides a method for detecting methylmercury ions and mercury ions.

[0025] A method for detecting methylmercury ions and mercury ions according to one embodiment of the present invention comprises the step of reacting a sample with a real-time proportional fluorescence detection sensor for methylmercury ions and mercury ions of claim 1 in order to detect mercury ions and methylmercury in a sample obtained from a living organism or environment; and

[0026] The method is characterized by including a step of analyzing the concentrations of mercury ions and methylmercury by observing whether there is a change in fluorescence after reacting the above-mentioned fluorescence detection sensor with a sample.

[0027] In addition, according to one embodiment of the present invention, in the step of reacting the fluorescence detection sensor with a sample, the sample may include a buffer solution.

[0028] In addition, according to one embodiment of the present invention, the buffer solution may have a pH between 6.2 and 8.2 to correspond to the pH concentration in vivo. Effects of the invention

[0029] According to an embodiment of the present invention, a peptide-based real-time proportional fluorescence sensor with excellent selectivity for methylmercury and mercury ions dissolves well in a pure aqueous solution (1% DMSO), is excited by visible light, and can recognize Hg(II) and methylmercury with high sensitivity through proportional fluorescence behavior. In particular, it exhibits a fast detection speed for methylmercury and has high water solubility, but this fluorescence sensor penetrates well into cells and can sensitize Hg(II) and methylmercury inside the cell through a change in the ratio of red and green fluorescence.

[0030] In addition, the method for detecting methylmercury and mercury ions using the peptide-based real-time proportional fluorescence sensor described above has the effect of exciting Hg(II) and methylmercury in visible light to detect them with high sensitivity through proportional fluorescence, and exhibiting characteristics such as high water solubility, high selectivity for mercury compounds, and a fast detection time (less than 30 seconds).

[0031] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description of the invention or the claims. Brief explanation of the drawing

[0032] Figure 1 shows the chemical structure of the peptide-based real-time proportional fluorescence sensor of the present invention. Figure 2 shows the results of the titration fluorescence spectra of mercury ions and methylmercury according to an embodiment of the peptide-based real-time proportional fluorescence sensor of the present invention. Figure 3 is a graph showing the binding constants of mercury ions and methylmercury for the peptide-based real-time proportional fluorescence sensor of the present invention, and an image showing the change in red fluorescence for mercury and methylmercury when irradiated with a fluorescent lamp. Figure 4 is a graph showing the results of measuring the fluorescence spectra of various metals using the peptide-based real-time proportional fluorescence sensor of the present invention in a buffer solution, and an image showing the change in red fluorescence when irradiated with a fluorescent lamp. Figure 5 is a graph showing the results of the fluorescence change with respect to the reaction rate of the peptide-based real-time proportional fluorescence sensor of the present invention for mercury ions and methylmercury in an aqueous solution of phosphoric acid (pH 7.4), and is a curve of the fluorescence change according to the pH of the aqueous solution for mercury ions and methylmercury. Figure 6 is a graph showing the results of fluorescence change for mercury ions and methylmercury in the presence of other metals in an aqueous solution of phosphoric acid (pH 7.4) of the peptide-based real-time proportional fluorescence sensor of the present invention, and is a calibration curve of proportional fluorescence for mercury ions and methylmercury in aqueous solution, groundwater, and tap water of the peptide-based real-time proportional fluorescence sensor of the present invention, and is a curve showing the limit of detection (LOD). Figure 7 is a confocal fluorescent cell image of the peptide-based real-time proportional fluorescence sensor of the present invention. Figure 8 is the result of a comparative analysis of the green and red fluorescence intensities of cells according to the concentrations of Hg(II) and CH₃Hg(I) of the peptide-based real-time proportional fluorescence sensor of the present invention. Specific details for implementing the invention

[0033] The present invention will be described below with reference to the attached drawings. However, the present invention may be implemented in various different forms and is therefore not limited to the embodiments described herein. Furthermore, in order to clearly explain the present invention in the drawings, parts unrelated to the explanation have been omitted, and similar parts throughout the specification have been given similar reference numerals.

[0034] Throughout the specification, when it is stated that a part is "connected (connected, in contact, combined)" with another part, this includes not only cases where they are "directly connected," but also cases where they are "indirectly connected" with other members interposed between them. Furthermore, when it is stated that a part "includes" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but rather allows for the inclusion of additional components.

[0035] The terms used herein are merely for describing specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as “comprising” or “having” are intended to indicate the presence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0036] Embodiments of the present invention will be described in detail below with reference to the attached drawings.

[0038] A real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions according to one embodiment of the present invention is described.

[0039] A real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions according to one embodiment of the present invention is,

[0040] It is characterized by being represented by the following chemical formula 1.

[0041] [Chemical Formula 1]

[0042]

[0043] In the above chemical formula 1, substituent R is an amino acid compound, and R1 and R2 are C1 to C n It is a chain-type carbon compound, R3 is hydrogen (H), cyano group (CN), amide group (amide), or carboxyl group, and R4 is sulfur (S), amine group (NH), or oxygen (O).

[0044] The above-mentioned real-time fluorescence detection sensor for methyl mercury ions and mercury ions is, mercury ions (Hg 2+ ) and methyl mercury ions (CH3Hg + It is characterized by simultaneously detecting ).

[0045] The above chemical formula 1 is characterized by the fact that a fluorescent compound is bound to the fluorescent dipeptide compound, and the fluorescent compound is bound to the fluorescent dipeptide compound in the above chemical formula 1 to exhibit red fluorescence.

[0046] The real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions of the present invention is Ag + , Al 3+ , Ca 2+ , Cd 2+ , Co 3+ , Cr 3+ , Cu 2+ , K + , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Zn 2+ , Pb 2+ It does not exhibit a red fluorescence change for other metal ions such as mercury ions (Hg 2+ It exhibits high selectivity and sensitivity only to ) and methylmercury.

[0047] At this time, in the real-time proportional fluorescence detection sensor of the methyl mercury ion and mercury ion, only mercury ions (Hg 2+ The reason it exhibits high selectivity only for ) and methylmercury is that the dipeptide functional group and the fluorescent portion of the fluorescent sensor of Chemical Formula 1 above are mercury ions (Hg 2+ This is because detection is possible by utilizing the shift in the fluorescence wavelength caused by the restriction of rotation within the fluorescent molecule as self-assembly proceeds by binding with ) and methylmercury.

[0048] The selectivity and sensitivity of the real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions will be demonstrated in the following experimental examples.

[0049] In addition, the real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions of the present invention is capable of detecting mercury ions at the nanomolar level, and mercury ions or methyl mercury ions (CH3Hg) at concentrations of 4.1 Nm or higher + It can detect ).

[0050] In addition, the methyl mercury ion and mercury ion real-time proportional fluorescence detection sensor of the present invention is water-soluble and can detect mercury ions present in cells.

[0051] At this time, the detection of methyl mercury ions and mercury ions by the real-time proportional fluorescence detection sensor of the present invention can be performed in an aqueous solution.

[0052] In addition, the fluorescent peptide sensor of the present invention can be easily synthesized using a solid-phase synthesis method, etc., and a detailed manufacturing method is described in the following examples.

[0054] A method for detecting methylmercury ions and mercury ions according to one embodiment of the present invention is described.

[0055] A method for detecting methylmercury ions and mercury ions according to one embodiment of the present invention may include the step of reacting a real-time proportional fluorescence detection sensor for methylmercury ions and mercury ions of claim 1 with a sample to detect mercury ions and methylmercury in a sample obtained from a living organism or environment; and the step of analyzing the concentration of mercury ions and methylmercury by observing whether there is a change in fluorescence after reacting the fluorescence detection sensor with the sample.

[0056] The presence or absence of mercury ions and methylmercury ions in a sample obtained from a living organism or the environment can be easily detected by reacting the sample with a peptide-based fluorescent sensor represented by Chemical Formula 1 and observing whether there is a change in fluorescence, and since the degree of fluorescence change increases in dependence on the concentration of mercury ions and methylmercury ions, quantitative analysis of mercury ions and methylmercury in the sample is also possible.

[0057] In order to detect using the fluorescent peptide sensor for detecting mercury ions and methylmercury ions, the sample reacted with the fluorescent peptide sensor represented by Chemical Formula 1 may further include a buffer solution.

[0058] At this time, the type and concentration of the buffer solution are not particularly limited, but can be appropriately changed according to the application of the water temperature ion detection sensor.

[0059] For example, when the water temperature ion detection sensor of the present invention is to be used for detecting mercury ions in the body, it is preferable that the buffer solution has a buffering range corresponding to the pH concentration in the body, such as pH 6.2 to 8.2.

[0061] Thus, the peptide-based real-time proportional fluorescence sensor, which has excellent selectivity for methylmercury and mercury ions, dissolves well in a pure aqueous solution (1% DMSO), is excited by visible light, and can recognize Hg(II) and methylmercury with high sensitivity through proportional fluorescence behavior. In particular, it shows a fast detection speed for methylmercury and has high water solubility, but this fluorescence sensor penetrates well into cells and can sensitize Hg(II) and methylmercury inside the cell through changes in the ratio of red and green fluorescence.

[0063] The present invention will be explained in more detail below through manufacturing examples and experimental examples. However, the present invention is not limited to the following manufacturing examples and experimental examples.

[0065] Example 1: Fabrication of a Real-time Proportional Fluorescence Detection Sensor for Methylmercury and Mercury Ions

[0066] In Example 1, the following chemical reaction scheme 1 is performed to produce a peptide-based real-time proportional fluorescence sensor with excellent selectivity for methyl mercury and mercury ions, and the produced material is named Compound 1 below.

[0067] In Example 1, His amino acid and Phe amino acid are bound to a resin, then a synthesized fluorescent agent is introduced into a dipeptide bound to the resin, and then removed from the resin to obtain Compound 1.

[0068] Compounds 2 and 3 can be synthesized by performing the same method as in Example 1, but by replacing the phenylalanine amino acid with Gly (glycine) and Trp (tryptophan); the fluorescent sensor with Gly (glycine) as the reactant can be represented as Compound 2, and the fluorescent sensor with Trp (tryptophan) as the reactant can be represented as Compound 3.

[0069] Compounds 1-3 synthesized in the solid phase were purified to high purity using HPLC (High-Performance Liquid Chromatography), the molecular weight of the synthesized fluorescent sensor was confirmed using a high-resolution mass spectrometer, and its structure was confirmed using NMR.

[0070] [Chemical reaction equation]

[0071]

[0072] The following are the results of analyzing compounds 1 to 3 using an NMR analyzer.

[0073] 화합물 1 : Yield 76%; red solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.92 (1H, s), 8.49 (1H, s), 8.39 (1H, d , J = 8.0 Hz), 8.31 (1H, d, J = 8.0 Hz), 8.22 (1H, d, J = 9.2 Hz), 8.01 (1H, d, J = 8.4 Hz), 7.91 (1H, d, J = 8.0 Hz), 7.48 (1H, t, J = 8.0 Hz), 7.38 (2H, t, J = 15.2 Hz), 6.53 (1H, d, J = 11.2 Hz), 6.08 (1H, s), 4.77-4.66 (2H, m), 4.52-4.51 (1H, m), 4.35-4.31 (1H, m), 3.44-3.40 (4H, m), 3.15-3.09 (1H, m), 2.99-2.95 (1H, m), 1.21 (3H, t, J = 18.0 Hz), 1.11 (6H, t, J = 24 Hz); 13C NMR (100 MHz, DMSO-d6) δ: 172.4, 167.7, 165.8, 160.4, 153.6, 141.2, 134.1, 129.9, 127.1, 125.6, 122.9, 118.7, 117.5, 109.1, 106.1, 94.6, 72.6, 70.1, 67.7, 51.4, 48.5, 44.7, 26.4, 24.1, 22.8, 18.7, 12.8 ; HR-MS (m / z): [M + H+]+ calculated for C 31 H 34 N7O5S: 616.2337, observed: 616.2336.

[0075] 화합물 2 : Yield 77%; red solid; 1H NMR (400 MHz, DMSO-d6) δ: 8.93 (1H, s), 8.59 (1H, d, J = 8.0 Hz), 8.47 (1H, s), 8.30 (1H, d, J = 8.0 Hz), 8.20 (1H, d, J = 8.0 Hz), 8.01 (1H, d, J = 8.0 Hz), 7.90 (1H, d, J = 8.0 Hz), 7.49 (1H, t, J = 20 Hz), 7.36 (2H, t, J = 28 Hz), 7.16-7.08 (5H, m), 6.50(1H, d, J = 12 Hz), 5.93 (1H, s), 4.73-4.51 (4H, m), 3.41-3.29 (4H, m), 3.17-3.10 (1H, m), 3.03-2.94 (2H, m), 2.86-2.78 (1H, m), 1.02 (6H, t, J = 16 Hz, ); 13C NMR (100 MHz DMSO-d6) δ: 172.2, 171.4, 167.8, 165.7, 160.3, 158.7, 153.8, 153.1, 141.3, 137.7, 134.4, 129.7, 128.4, 127.3, 126.7, 125.5, 122.5, 119.4, 117.4, 109.1, 106.4, 94.6, 67.6, 54.3, 51.7, 44.7, 26.6, 12.9 ; HR-MS (m / z): [M + H+]+ calculated for C 37 H 38 N7O5S: 692.2650 observed: 692.2655.

[0077] Compound 3 : Yield 71%; red solid; 1H NMR (400 MHz, DMSO-d6) δ: 10.70 (1H, s), 8.92 (1H, s), 8.60 (1H, d, J = 8.0 Hz), 8.46 (1H, s), 8.21 (2H, t, J = 20.4 Hz), 7.96 (1H, d, J = 7.6 Hz), 7.88 (1H, d, J = 14 Hz), 7.54 (1H, d, J = 8.0 Hz), 7.46 (2H, t, J = 16.8 Hz), 7.36 (1H, t, J = 14 Hz), 7.26 (1H, t, J = 15.2 Hz), 7.08 (1H, s), 6.99 (1H, t, J = 14.4 Hz), 6.90 (1H, t, J = 14 Hz), 6.50 (1H, d, J = 11.2 Hz), 5.94 (1H, s), 4.64-4.53 (4H, m), 3.37-3.22 (4H, m), 3.18-3.10 (2H, m), 3.01-2.94 (2H, m), 1.00 (6H, t, J = 8.0 Hz); 13C NMR (100 MHz DMSO-d6) δ: 172.1, 167.7, 165.7, 160.2, 158.8, 153.8, 153.1, 144.1, 141.1, 136.6, 134.3, 131.2, 129.7, 128.5, 127.5, 125.6, 124.1, 122.5, 121.2, 118.8, 117.5, 115.1, 111.7, 110.1, 109.1, 106.3, 94.6, 67.5, 53.7, 51.5, 44.7, 28.3, 26.8, 12.8 ; HR-MS (m / z): [M + H+]+ calculated for C 39 H 39 N8O5S: 731.2759 observed: 731.2764.

[0079] Example 2: Method for analyzing fluorescence changes using a real-time proportional fluorescence detection sensor for methyl mercury and mercury ions

[0080] To evaluate the metal ion binding characteristics of Compound 2 (10 μM) prepared according to Example 1 above, Ag+ , Al 3+ , Ca 2+ , Cd 2+ , Co 3+ , Cr 3+ , Cu 2+ , K + , Hg 2+ , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Zn 2+ , Pb 2+ Ions were used. A solution containing the above metal ions was prepared first.

[0081] Ag in aqueous solution + , Al 3+ , Ca 2+ , Cd 2+ , Co 3+ , Cr 3+ , Cu 2+ , K + , Hg 2+ , Mg 2+ , Mn 2+ , Na + , Ni 2+ , Zn 2+ , Pb 2+ A perchlorate salt stock solution (10 mM) of the ions was prepared.

[0082] And a stock solution (1 mM) of compound 2 was also prepared using water and an organic solvent.

[0083] The stock solution of the above compound 2 was transferred to a test tube, an appropriate amount of the stock solution of each metal ion was added, and the solution was diluted to 2 mL with 20 mM phosphate buffer (pH 7.4) and water to prepare a test solution.

[0084] Fluorescence spectra were measured for the above-mentioned test solutions. When measuring the fluorescence spectrum, the excitation wavelength was generally 440 nm.

[0086] Experimental Example 1

[0087] Hereinafter, with reference to FIGS. 2 to 8, the fabrication verification and fluorescence characteristics of the peptide-based real-time proportional fluorescence sensor of the present invention will be described.

[0088] Figure 2 shows the results of the titration fluorescence spectra of mercury ions and methylmercury according to an embodiment of the peptide-based real-time proportional fluorescence sensor of the present invention.

[0089] Figures 2(a) and 2(b) are graphs showing the titration fluorescence spectra of mercury ions and methylmercury in compound 1 (4 μM) in an aqueous solution of 10 mM phosphoric acid (pH 7.4).

[0090] Figures 2(c) and 2(d) are graphs showing the titration fluorescence spectra of mercury ions and methylmercury in compound 2 (4 μM) in an aqueous solution of 10 mM phosphoric acid (pH 7.4).

[0091] Figures 2(c) and 2(d) are graphs showing the titration fluorescence spectra of mercury ions and methylmercury in compound 3 (1 μM) in an aqueous solution of 10 mM phosphoric acid (pH 7.4).

[0092] Referring to Fig. 2, it can be confirmed that compounds 1-3 can detect mercury ions with proportional fluorescence behavior.

[0094] Figure 3 is a graph showing the binding constants of mercury ions and methylmercury for the peptide-based real-time proportional fluorescence sensor of the present invention, and an image showing the change in red fluorescence for mercury and methylmercury when irradiated with a fluorescent lamp.

[0095] Table 1 below summarizes information on the fluorescence recognition behavior, concentration, and binding constant for mercury ions and methylmercury for each compound in Figures 2 and 3.

[0096] Probe Fluorescence change behavior Hg(II) / CH3Hg(I) Operating concentration (saturation equiv.) coupling constant Hg(II) CH3Hg(I) Hg(II) CH3Hg(I) 1 Proportional fluorescence / fluorescence reduction 0-7 μM (1.25 equiv.) 0-380 μM (60 equiv.) 75.6 nM 176.3 μM 2 Proportional fluorescence / Proportional fluorescence 0-8 μM (1 equiv.) 0-220 μM (50 equiv.) 8.53 nM 106.4 μM 3 Proportional Fluorescence / Proportional Fluorescence 0-2.2 μM(2 equiv.) 0-80 μM (70 equiv.) 309. nM 46.0 μM

[0097] Referring to Table 1 above, it can be seen that compounds 1-3 have a high binding affinity for mercury ions and methylmercury in aqueous solution.

[0099] Figure 4 is a graph showing the results of measuring the fluorescence spectra of various metals using the peptide-based real-time proportional fluorescence sensor of the present invention in a buffer solution, and an image showing the change in red fluorescence when irradiated with a fluorescent lamp.

[0100] Figures 4(a) and 4(b) are graphs showing the results of measuring the fluorescence spectra of compounds 2 and 3 for various metals in an aqueous solution of 10 mM HEPES buffer (pH 7.4). Figure 4(c) is an image file showing the red fluorescence change of compound 2 for mercury, methylmercury, and other metals (1 mM for Group I and II metals, 10 μM for heavy metals) when irradiated with a fluorescent lamp. Through Figure 4, it can be confirmed that the compounds are selective for mercury ions and methylmercury based on the proportional fluorescence change.

[0102] Figure 5 is a graph showing the results of the fluorescence change with respect to the reaction rate of the peptide-based real-time proportional fluorescence sensor of the present invention for mercury ions and methylmercury in an aqueous solution of phosphoric acid (pH 7.4), and is a curve of the fluorescence change according to the pH of the aqueous solution for mercury ions and methylmercury.

[0103] Through Figure 5, it can be confirmed that mercury ions and methylmercury can be detected from neutral pH to pH 11.

[0105] Figure 6 is a graph showing the results of fluorescence change for mercury ions and methylmercury in the presence of other metals in an aqueous solution of phosphoric acid (pH 7.4) of the peptide-based real-time proportional fluorescence sensor of the present invention, and is a calibration curve of proportional fluorescence for mercury ions and methylmercury in aqueous solution, groundwater, and tap water of the peptide-based real-time proportional fluorescence sensor of the present invention, and is a curve showing the limit of detection (LOD).

[0106] Through Figure 6, it can be confirmed that mercury ions and methylmercury at the nanomolar level can be detected.

[0108] Figure 7 is a confocal fluorescent cell image of the peptide-based real-time proportional fluorescence sensor of the present invention.

[0109] Figure 7(a) shows the green and red fluorescence changes of cells according to the mercury ion concentration when Hg(II) is added, and Figure 7(b) shows the green and red fluorescence changes of cells according to the methylmercury concentration when methylmercury is added.

[0110] Referring to Figure 7, it can be confirmed that intracellular mercury or methylmercury can be detected by proportional fluorescence change.

[0112] Figure 8 is the result of a comparative analysis of the green and red fluorescence intensities of cells according to the concentrations of Hg(II) and CH₃Hg(I) of the peptide-based real-time proportional fluorescence sensor of the present invention.

[0113] Figure 8 shows the results of a comparative analysis of the green and red fluorescence intensities of cells according to the concentrations of Hg(II) and CH₃Hg(I), and the ratio of red / green fluorescence intensity of cells according to Hg(II) and CH₃Hg(I) treatment. The concentration of compound 2 used in this cell analysis is 10 μM.

[0114] Referring to Figure 8, it can be confirmed that intracellular mercury or methylmercury can be quantified by proportional fluorescence change.

[0116] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

[0117] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 Real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions, characterized by being represented by the following chemical formula 1: [Chemical Formula 1] In the above chemical formula 1, substituent R is an amino acid compound, and R1 and R2 are C1 to C n It is a chain-type carbon compound, R3 is hydrogen (H), cyano group (CN), amide group (amide), or carboxyl group, and R4 is sulfur (S), amine group (NH), or oxygen (O). Claim 2 In claim 1, the methyl mercury ion and mercury ion real-time fluorescence detection sensor comprises mercury ions (Hg 2+ ) and methyl mercury ions (CH3Hg + A real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions characterized by simultaneously detecting ). Claim 3 A real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions, characterized in that, in claim 1, a fluorescent compound is bound to a fluorescent dipeptide compound in the above chemical formula 1. Claim 4 A real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions, characterized in that, in claim 1, a fluorescent compound is bound to a fluorescent dipeptide compound in the above chemical formula 1 to exhibit red fluorescence. Claim 5 In claim 1, the mercury ion (Hg 2+ ) and methyl mercury ions (CH3Hg + The real-time proportional fluorescence detection sensor of ) detects mercury ions or methylmercury ions (CH3Hg) at concentrations of 4.1 Nm or higher + A real-time proportional fluorescence detection sensor for methyl mercury ions and mercury ions characterized by detecting ). Claim 6 In claim 1, the mercury ion real-time proportional fluorescence detection sensor is characterized by being water-soluble and detecting mercury ions present in a cell, and is a methyl mercury ion and mercury ion real-time proportional fluorescence detection sensor. Claim 7 A method for detecting methylmercury ions and mercury ions, characterized by comprising: a step of reacting a real-time proportional fluorescence detection sensor of methylmercury ions and mercury ions of claim 1 with a sample to detect mercury ions and methylmercury in a sample obtained from a living organism or environment; and a step of analyzing the concentration of mercury ions and methylmercury by observing whether there is a change in fluorescence after reacting the fluorescence detection sensor with the sample. Claim 8 A method for detecting methylmercury ions and mercury ions according to claim 7, characterized in that, in the step of reacting the fluorescence detection sensor with a sample, the sample comprises a buffer solution. Claim 9 A method for detecting methylmercury ions and mercury ions according to claim 8, characterized in that the buffer solution has a pH of 6.2 to 8.2 corresponding to the pH concentration in vivo.

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