FAM-GGH polypeptide fluorescent probe and application

Abstract

The invention discloses an FAM-GGH polypeptide fluorescent probe and application, and relates to the technical field of biochemical sensors, and the key point of the technical scheme is as follows: the probe is composed of 5-FAM labeled tripeptide FAM-Gly-Gly-His-NH2; the probe is coordinated with Cu < 2 + > ions to form FH-Cu < 2 + > with high affinity, the FH-Cu < 2 + > shows high sensitivity to S < 2->, and the detection limit is 13.7 nM. The novel fluorescence sensor FAM-GGH based on 5-FAM is constructed and can be used for detecting Cu < 2 + > and S < 2-> in an HEPES solution system, fluorescence, ultraviolet-visible and theoretical simulation calculation analysis prove that a sensing mechanism and the FAM-GGH are suitable for detecting Cu < 2 + > in an actual sample, the recovery rate is good, it is indicated that the FAM-GGH has actual applicability, and in addition, the FAM-GGH can be used for detecting Cu < 2 + > and S < 2-> in an HEPES solution system. The FAM-GGH also has good biocompatibility, which indicates that the FAM-GGH has potential application value in the biological field.

Description

Technical Field

[0001] The invention relates to the technical field of biochemical sensors, and more specifically to a FAM-GGH polypeptide fluorescent probe and application thereof. Background Art

[0002] Copper plays a key role in many metabolic processes. Copper is an important micronutrient in many physiological processes, including metabolism, enzyme activity and immune function. Copper deficiency in the body can lead to vitiligo, gastrointestinal diseases, skeletal abnormalities, anemia and mental retardation. Copper is one of the earliest metals used by humans and is now widely used in various manufacturing industries. As a result, a large amount of Cu 2+ It is difficult to degrade when released into the environment, so it is easy to accumulate in the human food chain. Long-term exposure to high levels of Cu 2+ It may cause liver damage, metabolic disorders, and neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In Alzheimer's disease, trace copper promotes the precipitation of amyloid beta, participates in the formation of amyloid plaques, and leads to increased oxidative damage. Reducing the amount of copper ions in the body can achieve the purpose of anti-angiogenesis, anti-fibrosis and anti-inflammation. The World Health Organization stipulates that Cu 2+ The content in drinking water cannot exceed 2 mg / L; therefore, it is necessary to develop an effective, convenient and sensitive method to measure and monitor Cu 2+ Concentration is essential.

[0003] Hydrogen sulfide is considered a toxic gas with a bad odor; long-term exposure to this gas can damage the eyes and respiratory system. However, the existence of endogenous hydrogen sulfide in the human body was demonstrated in the 1990s. Since then, this molecule has gradually been recognized as an important gaseous signal transmitter like nitric oxide (NO) and carbon monoxide (CO). Hydrogen sulfide is reported to be involved in a variety of biological processes such as metabolism, inflammation, signal transduction and apoptosis. Due to the strong nucleophilicity and redox reactivity of sulfhydryl groups, thiols are indispensable functional molecules in biological systems. Thiols are also ideal nucleophiles in enzyme functions, excellent sensors and mild buffer molecules for maintaining the redox state of cells. Fluctuations in thiol concentrations can indicate It has been shown that it may even lead to various cancers, liver damage, Alzheimer's disease, cardiovascular disease and neurodegenerative diseases. The selectivity of peptide-based sensors mainly depends on the metal chelating ability of the receptor amino acid residues. According to the analysis of the Protein Data Bank (PDB), some special amino acid residues are easy to chelate with metal ions in biological systems, such as the carboxyl group of glutamic acid (Glu) and aspartic acid (Asp), the imidazole group of histidine (His) and the sulfhydryl group of cysteine ​​(Cys). The imidazole group of histidine and the indole group of tryptophan can be used as ligands in the chelation of active metal ions. Intracellular thiols, including Cys, Hcy and GSH, play a vital role in cell function and metabolism.

[0004] Fluorescence detection technology can be used for qualitative and quantitative analysis of metal ions. Fluorescence sensors have become a new strategy for the analysis of thiols and hydrogen sulfide due to their high sensitivity, low cost, and ability to detect and image thiols in biological samples. Compared with many chemical sensors, peptide fluorescent sensors can selectively bind to the measured ions and can be used to detect copper ions and sulfur ions in environmental and biological systems; however, the currently reported Cu 2+ The probe has limitations, such as Cu 2+ Low selectivity, long response time and specific reaction conditions (such as high temperature, acidic or alkaline environment) are required.

[0005] Therefore, the present invention aims to provide a FAM-GGH polypeptide fluorescent probe and application to solve the above problems. Summary of the invention

[0006] The purpose of the present invention is to provide a FAM-GGH polypeptide fluorescent probe and its application. The present invention constructs a new type of 5-FAM-based fluorescent sensor FAM-GGH, which can be used to detect Cu in HEPES solution system. 2+ and S 2- The sensing mechanism was demonstrated by fluorescence, UV-visible, and theoretical simulation analysis. FAM-GGH is suitable for detecting Cu in actual samples. 2+ , the recovery rate was good, indicating that FAM-GGH has practical applicability. In addition, FAM-GGH also has good biocompatibility, indicating that it has potential application value in the biological field.

[0007] The above technical purpose of the present invention is achieved through the following technical scheme: a FAM-GGH polypeptide fluorescent probe, which consists of a 5-FAM-labeled tripeptide FAM-Gly-Gly-His-NH2.

[0008] The present invention is further configured as follows: the probe and Cu 2+ Ion coordination forms FH-Cu with high affinity 2+ , the FH-Cu 2+ For S 2- It showed high sensitivity with a detection limit of 13.7 nM.

[0009] The present invention also provides a method for synthesizing a FAM-GGH polypeptide fluorescent probe, comprising the following steps:

[0010] S1, activating the resin and removing the original protecting groups of the resin to facilitate coupling with amino acids;

[0011] S2, adding amino acids with protecting groups in reverse order of the polypeptide sequence, washing the resin, removing the Fmoc protecting groups of the amino acids, washing the resin, and repeating this step until the complete polypeptide sequence is synthesized;

[0012] S3, modifying the polypeptide sequence with a fluorescent group and detecting the modification effect;

[0013] S4, adding lysis buffer to lyse the product to separate the fluorescent group-modified polypeptide from the resin;

[0014] S5, precipitating the polypeptide product and centrifuging to obtain a crude polypeptide;

[0015] S6. Purify the pure peptide using preparative HPLC and characterize it by mass spectrometry.

[0016] The present invention also provides an application of a FAM-GGH polypeptide fluorescent probe in the detection of copper ions in living cells of an organism.

[0017] The present invention also provides an application of a FAM-GGH polypeptide fluorescent probe in detecting sulfide ions in living cells of an organism.

[0018] The present invention also provides an application of a FAM-GGH polypeptide fluorescent probe in detecting biothiols in living cells of organisms.

[0019] In summary, the present invention has the following beneficial effects:

[0020] 1. The present invention constructs a new type of 5-FAM-based fluorescent sensor FAM-GGH, which can be used to detect Cu in HEPES solution system 2+ and S 2- The sensing mechanism was demonstrated by fluorescence, UV-visible, and theoretical simulation analysis. FAM-GGH is suitable for detecting Cu in actual samples. 2+ , the recovery rate was good, indicating that FAM-GGH has practical applicability. In addition, FAM-GGH also has good biocompatibility, indicating that it has potential application value in the biological field;

[0021] 2. The FAM-GGH polypeptide fluorescent probe in the present invention has excellent selectivity for copper ions and can specifically identify Cu among a variety of metal ions. 2+ , reducing the interference of other ions in the environment and biological samples, greatly improving the accuracy and reliability of detection;

[0022] 3. The FAM-GGH polypeptide fluorescent probe of the present invention can quickly respond to the presence of copper ions and hydrogen sulfide, is suitable for real-time and dynamic monitoring, and provides rapid feedback for environmental and health monitoring. Compared with traditional detection methods, the FAM-GGH polypeptide fluorescent probe has low cost, simple operation, and is easy to quickly deploy in the laboratory and on-site;

[0023] 4. The FAM-GGH polypeptide fluorescent probe of the present invention has good biocompatibility and is suitable for the detection of copper ions and hydrogen sulfide in biological systems, providing a new tool for biomedical research and clinical diagnosis. At the same time, the probe shows good stability and accuracy in both environmental and biological samples, broadening its application range in environmental monitoring and health monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a HPLC purification diagram of FAM-GGH in an embodiment of the present invention;

[0025] Figure 2 is a synthetic route diagram of FAM-GGH in an embodiment of the present invention;

[0026] Figure 3 is a mass spectrometry analysis diagram of FAM-GGH in an embodiment of the present invention;

[0027] Figure 4 is the ultraviolet absorbance spectrum of FAM-GGH in the embodiment of the present invention;

[0028] Figure 5 This is a schematic diagram of the change in fluorescence intensity of FAM-GGH after adding 17 metal ions in an embodiment of the present invention;

[0029] Figure 6 In the embodiment of the present invention, the cation pair FAM-GGH recognizes Cu 2+ The interference graph of

[0030] Figure 7 Cu in the embodiment of the present invention 2+ Fluorescence spectrum changes of titrated fluorescence sensor FAM-GGH and corresponding scatter plot;

[0031] Figure 8 In the embodiment of the present invention, FAM-GGH and Cu 2+ Job work curve chart;

[0032] Fig. 9 FAM-GGH titration of Cu in the embodiment of the present invention 2+ UV spectrum of

[0033] Fig.10 In the embodiment of the present invention, FAM-GGH and Cu 2+The structure diagram of the optimized complex;

[0034] Fig.11 It is FAM-GGH-Cu in the embodiment of the present invention. 2+ Schematic diagram of recognition of 18 anions;

[0035] Fig.12 It is FAM-GGH-Cu in the embodiment of the present invention. 2+ For S 2- , schematic diagram of fluorescence titration of Cys and GSH;

[0036] Fig.13 The FAM-GGH of the present invention is Cu 2+ , S 2- and schematic diagram of GSH detection limit;

[0037] Fig.14 Cu in the embodiment of the present invention 2+ and S 2- Fluorescence intensity profile of the cycles added to FAM-GGH;

[0038] Fig.15 In the embodiments of the present invention, FAM-GGH, FAM-GGH-Cu 2+ , FAM-GGH-Cu 2+ +S 2- Schematic diagram of fluorescence intensity. DETAILED DESCRIPTION

[0039] The following is combined with Figure 1-15 The present invention is described in further detail.

[0040] The drugs and reagents used in the embodiments of the present invention are shown in Table 1:

[0041] Table 1 List of drugs and reagents involved in the experiment

[0042] Drugs and reagents purity% Manufacturer RinkAmide Resins 98 Nanjing Taopu Biotechnology Co., Ltd. Fmoc-His(Trt)-OH 98 Nanjing Taopu Biotechnology Co., Ltd. Fmoc-Gly-OH 98 Nanjing Taopu Biotechnology Co., Ltd. (5-FAM) 5-Carboxyfluorescein 98 Nanjing Taopu Biotechnology Co., Ltd. (DMF)N,N-Dimethylformamide Analytical grade Chengdu Kelong Chemical Reagent Factory (DCM) Dichloromethane Analytical grade Zhengzhou Ruike Biochemical Technology Co., Ltd. (HEPES)4-Hydroxyethylpiperazineethanesulfonic acid 99 Anaiji (TFA) Trifluoroacetic acid 99 Chengdu Kelong Chemical Reagent Factory (TIS) Triisopropylsilane 98 Anaiji (EDT)1,2-Ethanedithiol Analytical grade Fuyu Fine Chemical Co., Ltd. (HOBT) 1-Hydroxybenzotriazole Analytical grade Fuyu Fine Chemical Co., Ltd. (DIC) N,N-Diisopropylcarbodiimide Analytical grade Nanjing Taopu Biotechnology Co., Ltd. Hexahydropyridine Analytical grade Fuyu Fine Chemical Co., Ltd. Anhydrous ethanol Analytical grade Fuyu Fine Chemical Co., Ltd. Thioanisole Analytical grade Fuyu Fine Chemical Co., Ltd. Anhydrous ether Analytical grade Fuyu Fine Chemical Co., Ltd. Sodium hydroxide Analytical grade Fuyu Fine Chemical Co., Ltd. hydrochloric acid Analytical grade Fuyu Fine Chemical Co., Ltd.

[0043] The experimental instruments used in the embodiments of the present invention are shown in Table 2:

[0044] Table 2 List of test instruments involved in the experiment

[0045] Experimental instruments Manufacturer or model 3D Shaker Haimen Qilin Clock TS-1000 Freeze Dryer Shanghai Tianfeng TF-FD-1 Medium Pressure High Performance Liquid Chromatograph Agilent Technologies ProStar High performance liquid chromatography Agilent Technologies 1260 Infinity SB-C18 Column Agilent Technologies Mass spectrometer Esquire6000 Ion Trap Mass Spectrometer Fluorescence spectrophotometer Japan Shimadzu RF-5301PC UV spectrophotometer Cary5000UV-Vis-NIR Transient and Steady-State Fluorescence Spectrometer EdinburghInstrumentFLS-920 pH Meter Leici PHS-2F

[0046] Example 1: Synthesis and characterization of FAM-GGH

[0047] 1) Activation of resin

[0048] Add 10 mL of DCM to the peptide synthesis tube, rinse and drain, weigh 1 g of Rink Amide resin, add it to a 25 mL peptide synthesis tube, add 10 mL of DCM to the tube, soak the resin for 25 minutes to make it fully swell, then drain the peptide synthesis tube, add 15 mL of the 20% hexahydropyridine solution prepared in advance, drain after 20 minutes, and add 10 mL to the tube each time. Wash the resin with DMF twice, then add 10mL of methanol to the tube each time to wash the resin, wash twice, continue to add 10mL of DMF to the tube each time, wash twice, take a few grains of the washed resin in a 5mL test tube, add 3 drops of ninhydrin solution to the test tube, add two drops of pyridine solution to react at 100℃ for three minutes, take out the test tube, if the resin in the test tube appears blue, it means that the Fmoc protecting group that protects the amino group on the original resin has been completely removed, if the resin in the test tube appears colorless, it is necessary to continue to add 15mL of 20% hexahydropyridine solution to react for 20 minutes to remove the Fmoc protecting group on the resin.

[0049] 2) Synthesis of peptides

[0050] Add 0.3g of Fmoc-His(Trt)-OH, 0.2g of HOBT, 1mL of DIC and 15mL of DMF solvent to the resin-activated peptide synthesis tube and react for 2h. When the time is up, drain the solution in the tube, add 10mL of DMF to the tube to wash the resin, drain it, then add 10mL of methanol to the tube to wash the resin, drain it, continue to add 10mL of DMF to the tube each time, wash the resin twice, do K test (same as the above washing and detection operations), if the resin does not change color, it means that the condensation reaction is complete, and you can proceed to the next step, add 2mL of pyridine and 2mL of acetic anhydride to the peptide synthesis tube, react for 40min, when the time is up, drain the solution in the tube, add 10mL of DMF to the tube to wash the resin, drain it, then add 10mL of methanol to the tube to wash the resin, drain it, continue to add 10mL to the tube each time DMF, wash the resin twice, do K's test (same as the above washing and detection operations). If the resin does not change color, it means that the condensation reaction is complete and you can proceed to the next step. Add 15 mL of the 20% hexahydropyridine solution prepared in advance. After 20 minutes, drain and add 10 mL of DMF to the tube each time to wash the resin. Wash twice, then add 10 mL of methanol to the tube each time to wash the resin. Wash twice, continue to add 10 mL of DMF to the tube each time, wash twice, do K's test (same as the above washing and detection operations). If the resin in the test tube appears blue, it means that the Fmoc protecting group that protects the amino group on the original resin has been completely removed, and you can proceed to the next step.

[0051] 3) Fluorescent group modification

[0052] Add 0.3 g of 5-FAM, 0.2 g of HOBT, 1 mL of DIC and 15 mL of DMF solvent to the peptide synthesis tube in the previous step, shield from light and react for 2 hours. When the time is up, drain the tube, add 10 mL of methanol to the tube to wash the resin, drain it, continue to add 10 mL of DMF to the tube each time, wash the resin twice, do K test (same as the above washing and detection operations), if the detection resin shows bright yellow, it means that the reaction is complete, add 20 mL of methanol to the tube, and drain it.

[0053] 4) Product cleavage

[0054] 30 mL of cleavage solution (prepared by adding 1.2 mL of EDT, 1.05 mL of thioanisole, 1.05 mL of TIS, and 0.6 mL of H2O to 26.1 mL of trifluoroacetic acid) was added to the peptide synthesis tube of the above reaction and reacted on an ice bath on a 3D shaker in the dark for 4 hours to cleave the product from the resin.

[0055] 5) Extraction of crude product

[0056] Filter the liquid after cleavage in the peptide synthesis tube with a sand core, discard the filter residue, transfer the filtrate to a 10mL special plastic tube per 7mL tube, fill the tube with ice ether, shake it well, let it stand for successful stratification, put it into the centrifuge, run it at 4000r for 3min, take out the tube, pour out the supernatant, continue to fill the precipitate with ice ether, shake it well, let it stand for stratification, and then centrifuge it for three minutes as above, and repeat this step once more. The crude product polypeptide obtained at the end is dried, put it in a freeze dryer and freeze-dried for two hours to obtain an orange block solid, which is stored in a -20℃ refrigerator.

[0057] 6) HPLC purification

[0058] The crude product was crushed, 5 mL of acetonitrile and 5 mL of ultrapure water were added to the tube, and the crude product was completely dissolved by ultrasound. The solution was filtered with a 0.45 μM filter membrane, and the filtrate was placed in a small liquid phase bottle. A 10 μM C18 reverse phase chromatography column was used for separation and purification on a high performance liquid phase, and a high performance liquid chromatography purification diagram ( Figure 1 ) and HPLC purification data sheet as shown below:

[0059] Table 3 HPLC purification data of FAM-GGH

[0060] Rank Time %Area Area 1 11.869 0.46 79505 2 13.000 1.27 218196 3 13.117 97.37 16669579 4 13.333 0.63 107364 5 13.676 0.27 45956

[0061] The HPLC purification data table shows that the purity of the product is as high as 97.37%, which has met the purity requirements of the subsequent experimental tests. The synthetic route of FAM-GGH is as follows: Figure 2 shown.

[0062] In the LC-MS and UV-visible spectroscopy characterization of FAM-GGH in this example, according to the structure of the peptide sensor FAM-GGH, the molecular weight is calculated to be 627.56 g / mol. Figure 3 ) we can see that the highest peak in the figure is 628.56 g / mol, which corresponds to [FAM-GGH+H] + It can be seen that the obtained product is the product designed in this embodiment. Because this embodiment uses 5-FAM as a fluorescent group, and the ultraviolet absorption peak of 5-FAM is about 494nm, the ultraviolet spectrum of the sensor FAM-GGH is measured ( Figure 4 )FAM-GGH has a strong ultraviolet absorption peak at 494nm, which is consistent with the excitation wavelength of 5-FAM. Based on this, this example determines that the synthesized compound is the target product FAM-GGH required for the experiment.

[0063] In this example, the reagents were prepared by diluting ultrapure water. + , K + , Ca 2+ , Fe 3+ , Fe 2+ , Sr 2+ 、Co 2+ 、Ni 2+ , Cu 2+ 、Zn 2+ , Mn 2+ Mg 2+ 、Cd 2+ , Hg 2+ 、Al 3+ , Ba 2+ and Pb 2+ ) Chloride and nitrate to evaluate different metal ions (10 -3 M) with FAM-GGH, anion stock solutions from the respective sodium or potassium salts were also prepared in ultrapure water (10 -3 M), this experiment measured the fluorescence emission spectrum of the fluorescence sensor in a 10.0 mm path quartz cuvette. The synthesized fluorescence sensor was configured as 10 -3 The mother solution of HEPES was stored in a refrigerator at -20°C away from light. In subsequent experiments, the polypeptide mother solution was diluted with 20 mM HEPES buffer solution (pH 7.4). The excitation and emission slit widths were both 1.5 nm. We measured the fluorescence emission spectrum of the fluorescent sensor at 496 nm. The absorption and fluorescence measurements were carried out in a 100% aqueous phase containing 20 mM HEPES buffer, pH 7.4, without any organic cosolvent. HEPES buffers with different pH values ​​(pH 2.0-12.0) were prepared with distilled water, and the pH was adjusted with sodium hydroxide solution and hydrochloric acid solution. All tests were carried out at room temperature.

[0064] Example 2: Metal ion selectivity experiment

[0065] Take a 50mL volumetric flask and add 20mM HEPES buffer solution (pH 7.4) and 500mL of 10 -3 10 μM FAM-GGH mother solution, continue to add buffer solution to the volumetric flask to the scale line, shake well and place in a brown wide-mouth bottle to prepare 50 mL of 10 μM FAM-GGH solution. Take 2 mL of 10 μM FAM-GGH solution in a cuvette each time, and then add 17 kinds of 20 μL of 10 -3 M metal ion solution to measure fluorescence spectra; Figure 5 As shown in the figure ((a) and (b) are the fluorescence spectra and histograms after adding 17 metal ions (20 μM) to FAM-GGH (10 μM), respectively, and (c) is a photo taken under natural light and 365 nm ultraviolet light after adding 17 metal ions (20 μM) to FAM-GGH (10 μM). The experimental results show that adding Cu 2+ The fluorescence intensity of FAM-30GGH was reduced, while no obvious change in fluorescence intensity was observed after adding other cations, which confirmed that FAM-GGH can selectively recognize Cu 2+ These observations suggest that Cu 2+ Potentially useful for detecting Cu in living organisms 2+ .

[0066] Example 3: Metal ion competition experiment

[0067] After the successful synthesis of the fluorescent sensor, this example firstly experiments on common metal ions under simulated physiological conditions (20 μM, pH 7.4, HEPES buffer solution), such as Figure 6 As shown, the free fluorescence sensor exhibited fluorescence at 522 nm (ex 496 nm). After treatment with the above metal ions, the fluorescence sensor only responded to copper ions. After the addition of copper ions, the fluorescence emission spectrum peak decreased, indicating that FAM-GGH can be used as a fluorescence sensor to detect copper ions.

[0068] Example 4: Cu 2+ Fluorescence titration experiment

[0069] To gain a deeper understanding of FAM-GGH as a probe for copper ions, fluorescence emission titration experiments were performed, such as Figure 7 As shown (where (a) and (b) are respectively in 20 μM pH 7.4 HEPES buffer solution with Cu 2+(0-3.0 equivalents) titration fluorescence sensor FAM-GGH fluorescence spectrum change and corresponding scatter plot), with the addition of copper ions, the intensity of the fluorescence emission spectrum of FAM-GGH at 522nm decreased, the results showed that Cu 2+ The reaction occurred between FAM-GGH and Cu in HEPES buffer solution at pH 7.4. 2+ The fluorescence spectrum of FAM-GGH was titrated (0-3.0 equivalents). Within a certain concentration range, the fluorescence intensity of FAM-GGH showed a good linear relationship with the copper ion concentration.

[0070] Example 5: Job working curve determination and UV titration experiment

[0071] In order to further study the FAM-GGH and Cu 2+ The stoichiometric ratio between Figure 8 ) determined the combined stoichiometric ratio of FAM-GGH and copper ions. Job curve analysis showed that at 0.5 mole fraction of Cu 2+ This indicates that FAM-GGH and Cu 2+ A 1:1 complex was formed between them.

[0072] In addition to Job's plot analysis, we also performed UV titration studies ( Fig. 9 ), Cu 2+ The solution was added dropwise to FAM-GGH, and the UV absorbance at 494 nm gradually decreased. 2+ When the titration was 1.0 eq, the UV absorbance remained basically unchanged, which indicated that the titration had basically reached saturation and the FAM-GGH and Cu 2+ The binding ratio of the formed complex is 1:1.

[0073] Example 6: Theoretical simulation calculation

[0074] In order to better prove the binding mechanism, theoretical calculation studies were performed to determine the 2+ The two complexes have the lowest energy and highest stability structure before and after combination. Fig.10 FAM-GGH and Cu 2+ The structure of the optimized complex (where (a) and (b) are FAM-GGH and Cu 2+ The structural diagram of the optimized complex is shown in Table 4. The energy contributions listed in Table 4 are as follows:

[0075] Table 4 Energy calculation table

[0076] / FAM-GGH FAM-GGH-Cu2+ Bond tensile energy 1.888 11.632 Corner Energy 39.018 75.160 Torsion Energy 14.552 72.065 Out-of-plane bending energy 0.083 17.085 1-4 Van der Waals energy 3.589 2.791 Van der Waals energy -10.859 -8.178 Fixed distance energy / 1.857 Fixed Angle Energy / 0.002

[0077] The energy contributions listed in Table 4 show that FAM-GGH-Cu 2+ The total energy is 181.031 kcals / mol, which is much higher than the total energy of FAM-GGH 42.826 kcals / mol, indicating that the coordination process is non-spontaneous. The theoretical simulation calculations are consistent with the fluorescence measurement results, providing a theoretical basis for the formation of the binding mechanism.

[0078] Example 7: Anion and amino acid interference study

[0079] like Fig.11 As shown (wherein in HEPES solution at pH 7.4, (10 μM) FAM-GGH-Cu 2+ For the recognition study of 18 anions (1 eq), the black bars indicate the addition of these ions (10 μM) to FAM-GGH-Cu 2+ The black bar indicates that S (20 μM) was subsequently added to the above solution. 2- ; (a) and (b) are FAM-GGH-Cu 2+ The fluorescence response of the sensor to 18 anions at an excitation wavelength of 496 nm in a pH 7.420 μM HEPES solution was shown in the bar graph of different anions and the photo under 365 nm UV light. The results showed that FAM-GGH has a strong fluorescence effect on S in these anions. 2- It exhibits excellent sensitivity and high selectivity, while having no response to various other anions.

[0080] Example 8: S 2- Fluorescence titration experiment of Cys and GSH

[0081] In this embodiment, S 2- The addition of Cys and GSH restored the 2+ The fluorescence emission peak is due to the thiol-containing S 2- , Cys, and GSH can be obtained from FAM-GGH-Cu 2+ Cu is taken away from the system 2+ And release FAM-GGH, S 2- 2.0 eq), 2.0 eq, ... 2+ The solution was placed in a cuvette and the fluorescence intensity was measured to make a titration curve ( Fig.12 ), when S 2- When the titration of Cys and GSH reached 3.0eq, 0.25eq and 1.0eq respectively, the titration reached saturation and the fluorescence intensity recovered (all recovered to the maximum level).

[0082] Example 9: Detection limit experiment

[0083] FAM-GGH for Cu 2+ , S 2- The detection limit of GSH was determined by comparing the fluorescence intensity at 522 nm with that of Cu 2+ , S 2- The good linear relationship between the concentration of Cu and GSH was calculated, and the detection limit (LOD) was calculated based on the fluorescence titration experimental data. The formula LOD = 3σ / k, where σ is the concentration in the absence of Cu 2+ , S 2- In the case of GSH or FAM-GGH, the standard deviation of blank measurement was obtained by measuring the fluorescence intensity of FAM-GGH 10 times, and k is the fluorescence intensity relative to Cu 2+ , S 2- Or the slope of GSH concentration, the detection limit of FAM-GGH was calculated using this formula, as shown in Fig.13 (a), (b), and (c) are the effects of FAM-GGH on Cu in HEPES solution with a pH of 7.4. 2+ ,S 2- , schematic diagram of the detection limits of GSH), which are 36nM, 73nM, and 43nM, respectively, which are lower than the maximum allowable level of H2S in drinking water recommended by the World Health Organization (WHO) (15μM).

[0084] Example 10: Cyclic assay experiment

[0085] In general, chemical sensors recognize single ions or molecules in a competing medium over a given concentration range in a reversible manner. For continuous monitoring, reversibility is an essential requirement. However, in the case of one-time measurements, such as analysis of blood and serum samples or blood glucose monitoring, such reversibility is not necessary. As the data show, S2- can be easily converted from FAM-GGH-Cu 2+ Substitution Cu 2+ , so in this embodiment, Cu 2+ and S 2- were added to the sensor system in sequence and the fluorescence intensity changes were measured ( Fig.14 ), the experimental results show that FAM-GGH has a 2+ and S 2- The detection can be well cycled multiple times, which means that FAM-GGH can be used as an "On-Off-On" type to achieve Cu 2+ and S 2- Detection.

[0086] Example 11: pH Response

[0087] A good fluorescent probe can be applicable to both actual water samples and biological systems. The key parameter usually used to compare the pH stability of fluorescent sensors is pKa. The fluorescence intensity of fluorescent sensors usually increases at higher pH values ​​and reaches a maximum at pH 8-9. Further increasing the pH value usually does not change the fluorescence. When the pH reaches 10-13, the protein will denature or the chromophore will degrade. Such pH-stable fluorescent sensors can be observed in acidic organelles such as lysosomes and Golgi apparatus and provide more reliable readings for quantitative detection. Therefore, the development of fluorescent sensors for biological H2S sensing is of great significance for understanding the biological function of hydrogen sulfide. Most endogenous H2S dissociates in physiological media at pH 7.4. Most reported H2S probes are designed based on two main methods, namely, H2S-induced reactions to change the fluorescence of conjugated fluorophores, and H2S-induced S 2- The second approach is often used in probe design because it results in a faster turn-on response than the first approach, except for the quenching metal ion.

[0088] Due to Cu 2+ The paramagnetism of Cu shows obvious emission quenching effect. 2+ It is the most commonly used paramagnetic metal for constructing H2S probes with fluorescent ligands, and S2- is beneficial for removing Cu 2+ Affinity for the sensor.

[0089] Example 12: Application experiment in actual water samples

[0090] In order to verify the practical application of the fluorescence sensor, we studied the analysis of Cu in real water samples using FAM-GGH. 2+ The practical application potential of Cu in ultrapure water was analyzed by spike recovery method. 2+ The Cu 2+ The spiked amount was 4 μmol·L-1, and the test result was 3.92±0.0086 μmol·L-1; Cu 2+ The spiked amount was 5 μmol·L-1, and the test result was 4.97±0.0040 μmol·L-1; Cu 2+ The spiked amount was 6 μmol·L-1, and the test result was 5.88±0.0071 μmol·L-1. FAM-GGH can accurately determine Cu in ultrapure water samples. 2+ Appropriate concentration gradient, Cu 2+ The recoveries were 98.1%-99.4%. These results indicate that FAM-GGH has good stability and accuracy for detecting Cu in real environmental samples. 2+ and S 2- Has application potential.

[0091] Summarize:

[0092] In summary, this example constructed a new 5-FAM-based fluorescent sensor FAM-GGH for detecting Cu in a HEPES (20 mM, pH = 7.4) solution system. 2+ and S 2- The sensing mechanism was demonstrated by fluorescence, UV-visible, and theoretical simulation analysis. FAM-GGH is suitable for detecting Cu in actual samples. 2+ , the recovery rate was good, indicating that FAM-GGH has practical applicability. In addition, FAM-GGH has good biocompatibility, indicating that it has potential application value in the biological field.

[0093] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A FAM-GGH polypeptide fluorescent probe, characterized in that: The probe consists of the tripeptide FAM-Gly-Gly-His-NH2 labeled with 5-FAM.

2. A FAM-GGH polypeptide fluorescent probe according to claim 1, characterized in that: The probe and Cu 2+ Ion coordination forms FH-Cu with high affinity 2+ , the FH-Cu 2+ For S 2- It showed high sensitivity with a detection limit of 13.7 nM.

3. The method for synthesizing a FAM-GGH polypeptide fluorescent probe according to claim 1, characterized in that: The following steps are involved: S1, activating the resin and removing the original protecting groups of the resin to facilitate coupling with amino acids; S2, adding amino acids with protecting groups in reverse order of the polypeptide sequence, washing the resin, removing the Fmoc protecting groups of the amino acids, washing the resin, and repeating this step until the complete polypeptide sequence is synthesized; S3, modifying the polypeptide sequence with a fluorescent group and detecting the modification effect; S4, adding lysis buffer to lyse the product to separate the fluorescent group-modified polypeptide from the resin; S5, precipitating the polypeptide product and centrifuging to obtain a crude polypeptide; S6. Purify the pure peptide using preparative HPLC and characterize it by mass spectrometry.

4. Use of a FAM-GGH polypeptide fluorescent probe according to claim 1 in the detection of copper ions in living cells of organisms.

5. Application of a FAM-GGH polypeptide fluorescent probe according to claim 1 in the detection of sulfide ions in living cells of organisms.

6. Use of a FAM-GGH polypeptide fluorescent probe according to claim 1 in the detection of biothiols in living cells of organisms.

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