A polypeptide fluorescent probe, a preparation method and application thereof

Abstract

The application discloses a polypeptide fluorescent probe and a preparation method and application thereof, and belongs to the technical field of polypeptide fluorescent probes.The polypeptide fluorescent probe prepared by the application can realize accurate recognition and signal response of copper ions, sulfur ions and glutathione at the same time, and a high-efficiency sensing system for multi-index parallel detection is constructed.The polypeptide fluorescent probe can be flexibly adapted to detection requirements in key fields such as biological and medical diagnosis, environmental water pollutant monitoring and food safety screening.

Description

Technical Field

[0001] This invention relates to the field of polypeptide fluorescent probe technology, specifically to a polypeptide fluorescent probe, its preparation method, and its application. Background Technology

[0002] Copper ions are an indispensable trace element in living organisms, widely participating in key physiological processes such as enzyme catalysis, electron transport, and cell signal transduction. Imbalances in copper ions can induce neurodegenerative diseases such as Alzheimer's and Parkinson's. Simultaneously, excessive copper ions in environmental water and soil can cause serious ecological pollution, threatening the survival of plants and animals and human health. Sulfide ions are substances with both toxicity and physiological activity; they are typical pollutants in the environment and also participate in the regulation of the body's redox balance. Glutathione, as a core intracellular antioxidant, is closely related to abnormal levels in tumors, liver diseases, and cardiovascular diseases. Therefore, achieving highly sensitive, highly selective, and real-time simultaneous detection of copper ions, sulfur ions, and glutathione has significant research value and practical application significance in fields such as biomedical diagnostics, environmental pollutant monitoring, and food safety screening.

[0003] Currently, detection technologies for copper ions, sulfur ions, and glutathione are mainly divided into two categories: traditional instrumental analysis methods and novel sensor analysis methods. Traditional instrumental analysis methods include inductively coupled plasma mass spectrometry (ICP-MS), high-performance liquid chromatography (HPLC), atomic absorption spectrometry, and ion chromatography. Although these methods have high accuracy and good reproducibility, they suffer from drawbacks such as expensive equipment, large instrument size, complex operation procedures, and poor portability. They are difficult to achieve rapid on-site detection and real-time analysis in vivo, and cannot simultaneously perform trace detection of all three substances, thus limiting their application scenarios.

[0004] Fluorescence sensing technology has gradually become the mainstream technology for trace substance detection due to its advantages such as high sensitivity, fast response speed, real-time imaging, and adaptability to in vivo detection. Among them, peptide-based fluorescent probes have unique advantages such as excellent biocompatibility, easy modification of molecular structure, ability to simulate biological specific recognition, and good water solubility. Compared with fluorescent probes based on small organic molecules, polymers, and nanomaterials, they are more suitable for the detection of biological samples and complex aquatic environments, and have become a research hotspot in the field of fluorescence sensing.

[0005] While existing peptide-based fluorescent probes have achieved the detection of some metal ions or small biological molecules, they still suffer from several technical limitations: First, their detection functions are limited; most probes can only detect a single substance among copper ions, sulfur ions, and glutathione, lacking multi-response probes that can simultaneously identify all three. Second, their selectivity is insufficient; they are easily interfered with by common coexisting substances in the environment, such as metal ions, anions, and amino acids, resulting in poor reliability of detection results. Third, their sensitivity is low; the detection limits are mostly at the micromolar level, making it difficult to meet the detection requirements of trace substances in organisms and ultra-low concentration pollutants in the environment. Fourth, their biocompatibility is poor; some probes have high cytotoxicity and weak cell membrane penetration, making them unsuitable for in-situ imaging detection of substances within living cells. Fifth, their response modes are irreversible or have poor cyclicity, making it difficult to construct stable reversible fluorescent response systems, thus limiting the reusability and dynamic monitoring applications of the probes.

[0006] Based on this, the present invention designs a polypeptide fluorescent probe, its preparation method, and its application to solve the above problems. Summary of the Invention

[0007] To address the aforementioned shortcomings of existing technologies, this invention provides a method for preparing a polypeptide fluorescent probe, comprising the following steps:

[0008] S1. Weigh 0.2-0.25 g of Rink amide AM resin, place it in a peptide synthesis tube, add 4-6 mL of dichloromethane for swelling treatment for 20-40 min; then add 1-3 mL of piperidine for deprotection reaction for 10-20 min.

[0009] S2. Add 0.2-0.25 g of Fmoc-His(Trt)-OH and carry out the first amino acid condensation reaction for 30-60 min; add 1-3 mL of piperidine and carry out deprotection for 10-20 min; add 0.12-0.14 g of Fmoc-Pro-OH, 0.14-0.16 g of Fmoc-Thr(tBU)-OH, 0.2-0.25 g of Fmoc-His(Trt)-OH, and 0.13-0.15 g of Fmoc-Ahx-OH in sequence to carry out coupling reactions, with each coupling reaction taking 30-60 min;

[0010] S3. Add 0.03-0.05 g of fluorescein isothiocyanate and react at room temperature in the dark for 3-5 h to obtain a resin complex loaded with crude polypeptide fluorescent probe.

[0011] S4. Add 4-5 mL of lysis buffer to the resin complex and lyse at room temperature for 2-4 h to cleave and elute the crude peptide fluorescent probe from the resin; collect the lysis buffer, add 15-25 mL of cold diethyl ether for precipitation, then centrifuge, discard the supernatant, and obtain the crude peptide fluorescent probe precipitate.

[0012] S5. The crude precipitate of the polypeptide fluorescent probe was dissolved in an acetonitrile-water mixed solvent containing trifluoroacetic acid and purified by high performance liquid chromatography to obtain the polypeptide fluorescent probe.

[0013] Furthermore, the lysis solution is composed of trifluoroacetic acid, triisopropylsilane, and ultrapure water in a volume ratio of (3.5-4.0):(0.1-0.3):(0.1-0.3).

[0014] Furthermore, S5 specifically involves: dissolving the crude precipitate of the polypeptide fluorescent probe in an acetonitrile-water mixed solvent containing 0.05-0.15% trifluoroacetic acid (v / v), and purifying it using high-performance liquid chromatography (HPLC); using a C18 column, with the mobile phase being an acetonitrile solution containing 0.05-0.15% trifluoroacetic acid (v / v) and ultrapure water containing 0.05-0.15% trifluoroacetic acid (v / v), a gradient elution time of 15-25 min, an elution flow rate of 0.8-1.2 mL / min, and a detection wavelength of 210-220 nm; collecting the target elution fraction; concentrating the target fraction and then freeze-drying it to obtain the polypeptide fluorescent probe.

[0015] Furthermore, the volume fraction of acetonitrile in the acetonitrile-water mixed solvent is 5-70%, and the volume fraction of water is 30-95%.

[0016] A polypeptide fluorescent probe prepared according to the method described above.

[0017] An application of the described polypeptide fluorescent probe in the simultaneous detection of copper ions, sulfur ions and glutathione.

[0018] Compared with the prior art, the beneficial effects of this invention are as follows:

[0019] 1. The polypeptide fluorescent probe prepared in this invention can simultaneously achieve accurate identification and signal response of copper ions, sulfur ions, and glutathione, constructing a highly efficient sensing system for parallel detection of multiple indicators. It can be flexibly adapted to the detection needs of key fields such as biomedical diagnostics, environmental water pollutant monitoring, and food safety screening.

[0020] 2. This invention, through precise design of the polypeptide sequence structure and coupling with specific fluorescent groups, enables the probe to form a recognition environment highly matched to the target substance. The probe exhibits a specific fluorescence quenching response to copper ions, and the complex formed with copper ions can trigger specific fluorescence recovery signals for sulfide ions and glutathione, unaffected by interference from common metal ions, anions, amino acids, and various small biological molecules. This highly specific recognition mechanism ensures that the probe can obtain highly stable, reproducible, and reliable detection results even in complex matrices.

[0021] 3. The polypeptide fluorescent probe prepared in this invention achieves detection limits of 41 nM, 55 nM, and 101 nM for copper ions, sulfur ions, and glutathione, respectively, all reaching nanomolar-level detection levels. This sensitivity is sufficient to accurately capture trace amounts of physiologically active substances in organisms, and also enables quantitative analysis of ultra-low concentration pollutants in environmental systems, providing reliable technical support for the accurate detection of low-content target substances.

[0022] 4. The probe of this invention uses a polypeptide as its core backbone, possessing both good water solubility and extremely low biotoxicity, while also exhibiting excellent cell membrane penetration ability, enabling it to smoothly enter living cells. It can be directly used for in-situ visualization fluorescence imaging of copper ions, sulfur ions, and glutathione within living cells, clearly revealing the distribution and content changes of target substances within cells, providing a safe and efficient visualization detection method for the study of physiological processes at the living organism level.

[0023] 5. The polypeptide fluorescent probe of this invention can reversibly bind to copper ions. Upon addition of sulfide ions or glutathione, the probe fluorescence can be efficiently restored, forming a stable and reversible fluorescence response system. This probe can stably complete more than six "fluorescence quenching-fluorescence recovery" cycles, enabling both dynamic real-time monitoring of target substance concentration changes and probe reusability, effectively reducing detection costs and demonstrating significant advantages in long-term dynamic tracking detection scenarios. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0025] Figure 1 This is a flowchart of the preparation process of the polypeptide fluorescent probe (L);

[0026] Figure 2 High-performance liquid chromatography (HPLC) chromatogram of purified polypeptide fluorescent probe (L);

[0027] Figure 3 The image shows an electrospray ionization mass spectrometry (ESI-MS) image of a polypeptide fluorescent probe (L).

[0028] Figure 4 A magnified view of the protonated molecular ion peak of the L-peptide fluorescent probe in mass spectrometry.

[0029] Figure 5 The image shows the UV-Vis absorption spectrum of the L-type polypeptide fluorescent probe.

[0030] Figure 6 The UV-Vis absorption spectrum changes of the probe (L) under copper ion titration;

[0031] Figure 7 For copper ion gradient titration, the probe (L) 1 H NMR spectrum;

[0032] Figure 8 The bar chart shows the fluorescence selectivity response of the probe (L) to different metal ions.

[0033] Figure 9 The fluorescence spectrum changes of the probe (L) under copper ion gradient titration;

[0034] Figure 10 Job curve and fitting plot showing the binding ratio of probe (L) to copper ions;

[0035] Figure 11 For probe L-Cu 2+ Fluorescence response and cycling performance of the complex to sulfide ions, cysteine, and glutathione;

[0036] Figure 12 Standard curves showing the detection limits of the probe (L) for copper ions, sulfur ions, and glutathione.

[0037] Figure 13 Fluorescence imaging of the probe (L) in HeLa cells for detecting copper ions, sulfur ions, and glutathione. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0039] Example 1: This example provides a method for preparing a polypeptide fluorescent probe, including the following steps:

[0040] S1. Weigh 0.25 g of Rink amide AM resin, place it in a peptide synthesis tube, add 6 mL of dichloromethane for 40 min of swelling treatment; then add 3 mL of piperidine for 20 min of deprotection reaction.

[0041] S2. Add 0.25 g of Fmoc-His(Trt)-OH and carry out the first amino acid condensation reaction for 60 min; add 3 mL of piperidine and carry out deprotection for 20 min; add 0.14 g of Fmoc-Pro-OH, 0.16 g of Fmoc-Thr(tBU)-OH, 0.25 g of Fmoc-His(Trt)-OH, and 0.15 g of Fmoc-Ahx-OH in sequence to carry out coupling reactions, with each coupling reaction taking 30-60 min;

[0042] S3. Add 0.05 g of fluorescein isothiocyanate and react at room temperature in the dark for 5 h to stably couple fluorescein isothiocyanate to the end of the polypeptide chain, thus obtaining a resin complex loaded with crude polypeptide fluorescent probe.

[0043] S4. Add 5 mL of lysis buffer to the resin complex and lyse at room temperature for 4 h to elute the crude peptide fluorescent probe from the resin. Collect the lysis buffer, add 25 mL of cold ether for precipitation, and then centrifuge at 12000 rpm for 5 min. Discard the supernatant to obtain the crude peptide fluorescent probe precipitate.

[0044] The lysis buffer was prepared by mixing trifluoroacetic acid, triisopropylsilane, and ultrapure water in a volume ratio of 4.0:0.3:0.3.

[0045] S5. The crude precipitate of the polypeptide fluorescent probe was dissolved in an acetonitrile-water mixed solvent containing 0.15% trifluoroacetic acid (v / v), and purified by high-performance liquid chromatography (HPLC). A C18 column was used for purification. The mobile phase consisted of acetonitrile solution containing 0.15% trifluoroacetic acid and ultrapure water containing 0.15% trifluoroacetic acid (v / v). The gradient elution time was 25 min, the elution flow rate was 1.2 mL / min, and the detection wavelength was 220 nm. The target eluent was collected. The target fraction was concentrated and then freeze-dried at -40℃ to obtain a polypeptide fluorescent probe with a purity of not less than 95%.

[0046] The acetonitrile-water mixed solvent contains 70% acetonitrile by volume and 30% water by volume.

[0047] S6. Dissolve the purified polypeptide fluorescent probe in ultrapure water to prepare a 15mM polypeptide fluorescent probe stock solution. Store it in a sealed container at -15℃ in the dark for later use.

[0048] Example 2: This example provides a method for preparing a polypeptide fluorescent probe, including the following steps:

[0049] S1. Weigh 0.2 g of Rink amide AM resin and place it in a peptide synthesis tube. Add 4 mL of dichloromethane for 20 min of swelling treatment. Then add 1 mL of piperidine for 10 min of deprotection reaction.

[0050] S2. Add 0.2 g of Fmoc-His(Trt)-OH and carry out the first amino acid condensation reaction for 30 min; add 1 mL of piperidine and carry out deprotection for 10 min; add 0.12 g of Fmoc-Pro-OH, 0.14 g of Fmoc-Thr(tBU)-OH, 0.2 g of Fmoc-His(Trt)-OH, and 0.13 g of Fmoc-Ahx-OH in sequence to carry out coupling reactions, with each coupling reaction taking 30 min.

[0051] S3. Add 0.03 g of fluorescein isothiocyanate and react at room temperature in the dark for 3 h to stably couple fluorescein isothiocyanate to the end of the polypeptide chain, thus obtaining a resin complex loaded with crude polypeptide fluorescent probe.

[0052] S4. Add 4 mL of lysis buffer to the resin complex and lyse at room temperature for 2 h to elute the crude peptide fluorescent probe from the resin. Collect the lysis buffer, add 15 mL of cold diethyl ether for precipitation, then centrifuge at 8000 rpm for 2 min, discard the supernatant, and obtain the crude peptide fluorescent probe precipitate.

[0053] The lysis buffer is composed of trifluoroacetic acid, triisopropylsilane, and ultrapure water in a volume ratio of 3.5:0.1:0.1.

[0054] S5. The crude precipitate of the polypeptide fluorescent probe was dissolved in an acetonitrile-water mixed solvent containing 0.05% trifluoroacetic acid (v / v), and purified by high-performance liquid chromatography (HPLC). A C18 column was used for purification. The mobile phase consisted of acetonitrile solution containing 0.05% trifluoroacetic acid and ultrapure water containing 0.05% trifluoroacetic acid (v / v). The gradient elution time was 15 min, the elution flow rate was 0.8 mL / min, and the detection wavelength was 210 nm. The target elution fraction was collected. After concentration, the target fraction was freeze-dried at -40℃ to obtain a polypeptide fluorescent probe with a purity of not less than 95%.

[0055] The acetonitrile-water mixed solvent contains 5% acetonitrile by volume and 30% water by volume.

[0056] S6. Dissolve the purified polypeptide fluorescent probe in ultrapure water to prepare a 5mM polypeptide fluorescent probe stock solution. Store it in a sealed container at -25℃ in the dark for later use.

[0057] Example 3: This example provides a method for preparing a polypeptide fluorescent probe, including the following steps:

[0058] S1. Weigh 0.22 g of Rink amide AM resin and place it in a peptide synthesis tube. Add 5 mL of dichloromethane for 25 min of swelling treatment. Then add 1.8 mL of piperidine for 16 min of deprotection reaction.

[0059] S2. Add 0.24 g of Fmoc-His(Trt)-OH and carry out the first amino acid condensation reaction for 50 min; add 2.2 mL of piperidine and carry out deprotection for 14 min; add 0.13 g of Fmoc-Pro-OH, 0.14 g of Fmoc-Thr(tBU)-OH, 0.23 g of Fmoc-His(Trt)-OH, and 0.15 g of Fmoc-Ahx-OH in sequence to carry out coupling reactions, with each coupling reaction taking 40 min.

[0060] S3. Add 0.035 g of fluorescein isothiocyanate and react at room temperature in the dark for 3 h to stably couple fluorescein isothiocyanate to the end of the polypeptide chain, thus obtaining a resin complex loaded with crude polypeptide fluorescent probe.

[0061] S4. Add 4.5 mL of lysis buffer to the resin complex and lyse at room temperature for 3 h to cleave and elute the crude peptide fluorescent probe from the resin; collect the lysis buffer, add 21 mL of cold diethyl ether for precipitation, then centrifuge at 8000 rpm for 5 min, discard the supernatant to obtain the crude peptide fluorescent probe precipitate.

[0062] The lysis buffer was prepared by mixing trifluoroacetic acid, triisopropylsilane, and ultrapure water in a volume ratio of 3.8:0.2:0.1.

[0063] S5. The crude precipitate of the peptide fluorescent probe was dissolved in an acetonitrile-water mixed solvent containing 0.08% trifluoroacetic acid (v / v), and purified by high-performance liquid chromatography (HPLC). A C18 column was used for purification. The mobile phase consisted of acetonitrile solution containing 0.09% trifluoroacetic acid (v / v) and ultrapure water containing 0.11% trifluoroacetic acid (v / v). The gradient elution time was 20 min, the elution flow rate was 0.9 mL / min, and the detection wavelength was 220 nm. The target eluent was collected. The target fraction was concentrated and freeze-dried at -40°C to obtain a peptide fluorescent probe with a purity of not less than 95% (HPLC chromatogram shown in Figure 1). Figure 2 (as shown)

[0064] Figure 2 The data is integrated into Table 1;

[0065] Table 1:

[0066] Group time relative content Peak area 1 9.322 0.7382 42375 2 11.942 0.9722 55808 3 12.505 96.09 5515612 4 12.923 0.7667 44008 5 13.068 0.7332 42087 6 13.376 0.5487 31495 7 13.706 0.1567 8994

[0067] The acetonitrile-water mixed solvent has a volume fraction of 40% for acetonitrile and 60% for water.

[0068] S6. Dissolve the purified polypeptide fluorescent probe in ultrapure water to prepare a 10mM polypeptide fluorescent probe stock solution. Store it in a sealed container at -15℃ in the dark for later use.

[0069] Taking Example 3 as an example, the following experiment was conducted.

[0070] Experimental Example 1: Electrospray mass spectrometry (ESI-MS) characterization;

[0071] The purified probe L sample was analyzed using a Bruker Daltonics Esquire 6000 mass spectrometer; the molecular weight of probe L was determined to be 991.4. Figure 3 As shown, its protonated molecular ion peak is [M+H]. + .like Figure 4 As shown,

[0072] Experimental Example 2: Ultraviolet-Visible Absorption Spectroscopy Characterization;

[0073] Prepare a 10 μM probe L solution (10 mM HEPES buffer, pH 7.4) and measure using an Agilent Cary 8454 UV-Vis spectrophotometer; Figure 5 As shown, the excitation wavelength of probe L was determined to be 495 nm. Figure 6 As shown, after gradually adding 0-0.5 equivalent copper ions, the absorbance of probe L decreased at 245 nm and 495 nm, while new absorption peaks appeared at 225 nm and 235 nm with increased absorbance.

[0074] Experimental Example 3: Hydrogen Nuclear Magnetic Resonance (NMR) 1 Characterization by H NMR;

[0075] A 0.05 mmol / L deuterated dimethyl sulfoxide solution was prepared and measured using a Bruker Avance 400 MHz NMR spectrometer; 0, 0.1, 0.3, 0.5, and 1.0 equivalents of copper ions were added sequentially in the titration experiment; (The text abruptly ends here, so the translation stops as well.) Figure 7As shown, without the addition of copper ions, the chemical shift of the amino hydrogen of the L imidazole ring was δ=14.36ppm; after adding 0.2, 0.5, and 1.0 equivalents of copper ions, the proton peak of the imidazole ring hydrogen in the 7.15-8.94ppm range broadened, and the peak shape of the methylene-hydrogen in the 2.62-3.24ppm range gradually flattened.

[0076] Experiment Example 4: Fluorescence Selectivity Experiment (Metal Ions);

[0077] Prepare a 10 μM probe L solution (10 mM HEPES buffer, pH 7.4); add 16 metal ions of equal concentration (Na+, Na ... + K + Ag + Zn 2+ Pb 2+ Ca 2+ Mg 2+ Cd 2+ Co 2+ Cu 2+ Mn 2+ Ni 2+ Hg 2+ Fe 3+ Al 3+ Cr 3+ The results were obtained using an Agilent Cary Eclipse fluorescence spectrophotometer, with an excitation wavelength of 495 nm, an excitation slit of 1.5 nm, and an emission slit of 10 nm. Figure 8 As shown, only copper ions quenched the fluorescence of probe L to 1 / 9 of its original intensity, mercury ions only caused slight quenching, and the other metal ions had no interference; the addition of the above metal ions to the probe L-copper ion complex did not change the fluorescence quenching effect.

[0078] Experimental Example 5: Determination of the binding constant of probe L with copper ions;

[0079] Prepare a 10 μM probe L solution (10 mM HEPES buffer, pH 7.4); add 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 equivalents of copper ions sequentially, and measure the fluorescence intensity under 495 nm excitation. The results are as follows. Figure 9 As shown; the Job curve is fitted using the nonlinear least squares method (results are shown in the figure). Figure 10 As shown in the figure, the binding ratio was determined to be 2:1; the binding constant Ksp was calculated to be 1.81 × 10⁻⁶. 10 M -2 The correlation coefficient R = 0.9941.

[0080] Experiment Example 6: Sulfide ion / cysteine / glutathione response experiment;

[0081] Prepare a 10 μM probe L-copper ion complex solution (10 mM HEPES buffer, pH 7.4).

[0082] Sulfide ions: Sulfide ions were added sequentially from 0 to 5.0 equivalents. Fluorescence recovery saturation occurred at 4.0 equivalents. The remaining 21 anions showed no fluorescence recovery effect (e.g., ...). Figure 11 ab);

[0083] Cysteine: Cysteine ​​was added sequentially from 0 to 7.0 equivalents. Fluorescence recovery saturation occurred at 4.0 equivalents. The remaining 19 water-soluble amino acids showed no fluorescence recovery effect (e.g., ...). Figure 11 cd);

[0084] Glutathione: Add 0-4.5 equivalents of glutathione sequentially; fluorescence saturation is restored at 4.0 equivalents. Alternating addition of copper and sulfur ions allows for stable cycling up to 6 times (e.g., Figure 11 ef).

[0085] Experimental Example 7: Determination of detection limit;

[0086] The LOD was calculated using the formula LOD = 3σ / k (where σ is the blank standard deviation and k is the slope of the standard curve), and the result is as follows: Figure 12 As shown in ac;

[0087] Copper ions: good linearity in the concentration range of 0-0.7 μM, R=0.9976, detection limit 41 nM;

[0088] Sulfide ions: good linearity in the concentration range of 0-0.6 μM, R=0.9985, detection limit 55 nM;

[0089] Glutathione: good linearity in the concentration range of 0-2.0 μM, R=0.9926, and detection limit 101 nM.

[0090] Experiment Example 8: Live-cell fluorescence imaging experiment

[0091] HeLa cells were cultured in DMEM medium at 37°C, 5% CO2, and 95% humidity. After cell adhesion, 10 μM probe L, 10 μM copper ions, 30 μM sulfide ions, and 30 μM glutathione were added sequentially. After washing three times, the cells were incubated for 30 min and then imaged using a laser confocal microscope. Probe L could penetrate the cell membrane, and probe L showed fluorescence inside the cells. The fluorescence was quenched upon the addition of copper ions and restored upon the addition of sulfide ions / glutathione (results are shown in Figure 1). Figure 13 (As shown).

[0092] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions will not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing a polypeptide fluorescent probe, characterized in that, Includes the following steps: S1. Weigh 0.2-0.25 g of Rink amide AM resin, place it in a peptide synthesis tube, add 4-6 mL of dichloromethane for swelling treatment for 20-40 min; then add 1-3 mL of piperidine for deprotection reaction for 10-20 min. S2. Add 0.2-0.25 g of Fmoc-His(Trt)-OH and carry out the first amino acid condensation reaction for 30-60 min; add 1-3 mL of piperidine and carry out deprotection for 10-20 min; add 0.12-0.14 g of Fmoc-Pro-OH, 0.14-0.16 g of Fmoc-Thr(tBU)-OH, 0.2-0.25 g of Fmoc-His(Trt)-OH, and 0.13-0.15 g of Fmoc-Ahx-OH in sequence to carry out coupling reactions, with each coupling reaction taking 30-60 min; S3. Add 0.03-0.05 g of fluorescein isothiocyanate and react at room temperature in the dark for 3-5 h to obtain a resin complex loaded with crude polypeptide fluorescent probe. S4. Add 4-5 mL of lysis buffer to the resin complex and lyse at room temperature for 2-4 h to cleave and elute the crude peptide fluorescent probe from the resin; collect the lysis buffer, add 15-25 mL of cold diethyl ether for precipitation, then centrifuge, discard the supernatant, and obtain the crude peptide fluorescent probe precipitate. S5. The crude polypeptide fluorescent probe precipitate was dissolved in an acetonitrile-water mixed solvent containing trifluoroacetic acid and purified by high performance liquid chromatography to obtain the polypeptide fluorescent probe.

2. The method for preparing the polypeptide fluorescent probe according to claim 1, characterized in that, The lysis solution is composed of trifluoroacetic acid, triisopropylsilane, and ultrapure water in a volume ratio of (3.5-4.0):(0.1-0.3):(0.1-0.3).

3. The method for preparing the polypeptide fluorescent probe according to claim 1, characterized in that, S5 specifically involves: dissolving the crude precipitate of the polypeptide fluorescent probe in an acetonitrile-water mixed solvent containing 0.05-0.15% trifluoroacetic acid (v / v), and purifying it using high-performance liquid chromatography (HPLC). A C18 column was used for purification. The mobile phase consisted of an acetonitrile solution containing 0.05-0.15% trifluoroacetic acid (v / v) and ultrapure water containing 0.05-0.15% trifluoroacetic acid (v / v). The gradient elution time was 15-25 min, the elution flow rate was 0.8-1.2 mL / min, and the detection wavelength was 210-220 nm. The target eluted fraction was collected. After concentration, the target fraction was freeze-dried to obtain the polypeptide fluorescent probe.

4. The method for preparing the polypeptide fluorescent probe according to claim 1, characterized in that, The volume fraction of acetonitrile in the acetonitrile-water mixed solvent is 5-70%, and the volume fraction of water is 30-95%.

5. A polypeptide fluorescent probe prepared by the method according to any one of claims 1-4.

6. The application of the polypeptide fluorescent probe according to claim 5 in the simultaneous detection of copper ions, sulfur ions and glutathione.

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