Plug-and-play ultra-sensitive clustered peptidyl material and preparation method and application of visual ratio nano fluorescent probe of plug-and-play ultra-sensitive clustered peptidyl material

Abstract

The invention discloses a plug-and-play ultra-sensitive clustered peptidyl material and a preparation method and application of a visual ratio nano fluorescent probe of the plug-and-play ultra-sensitive clustered peptidyl material, and belongs to the technical field of nano composite material preparation. The invention solves the problems of complexity, low speed, low sensitivity and the like of the current bacillus anthracis marker and tetracycline detection method. A clustered peptidyl material PG-COOH is constructed, the PG-COOH and lanthanide ions are co-assembled to obtain a probe Eu / Tb-PG-COOH for detecting the DPA, meanwhile, a probe PNPG-Eu-LML for detecting the tetracycline in which the PG-COOH and luminol cooperatively bridge Eu < 3 + > is constructed, and the two probes both take clustered peptide-Eu < 3 + > as cores and have coordination stability; plug-and-play coupling of the identification unit and the fluorescence signal unit is realized, background interference is reduced in cooperation with a ratio fluorescence double-signal calibration mechanism, and visual output supports on-site rapid detection.

Description

Technical Field

[0001] This invention relates to the field of nanocomposite material preparation technology, and to a method for preparing and applying a plug-and-play ultrasensitive clustered peptide-based material and its visualized ratio fluorescent nanoprobe. Background Technology

[0002] In the field of public health security and environmental monitoring, anthrax pathogen early warning and antibiotic residue detection technologies face severe challenges. The core contradiction lies in the conflict between the high complexity of probe preparation and the environmental adaptability of the detection system. (Anthrax Bacillus) Bacillus anthracis Anthrax, as a highly hazardous pathogen, contains 2,6-pyridinedicarboxylic acid (DPA) unique to its spores, which is a key biomarker. Rapid detection of DPA is crucial for anthrax early warning and environmental safety monitoring. However, traditional detection methods suffer from systemic defects: smear microscopy requires complex pretreatment and lacks sufficient sensitivity; PCR technology, while highly specific, relies on laboratory equipment and specialized operation, with detection cycles lasting several hours; and enzyme-linked immunosorbent assay (ELISA) is limited by antibody stability and high-cost reagents. None of these methods can meet the needs of rapid on-site response. While immunofluorescence technology has the potential for rapid detection, the conventional organic fluorescent dyes used suffer from severe spectral crosstalk and background fluorescence interference in complex biological matrices due to their narrow Stokes shift and short fluorescence lifetime. Although rare-earth-doped nanomaterials can possess ultra-long fluorescence lifetimes and large Stokes shifts, improving detection sensitivity, their stability in complex matrices and their specific binding ability to the target analyte still need optimization. For example, ratiometric fluorescence detection technology based on lanthanide metal-organic frameworks (Ln-MOFs) can detect DPA by detecting terbium (Tb)... 3+ ) and europium (Eu) 3+ While dual-emission signals reduce interference, the coordination stability and target recognition efficiency of the framework structure in dynamic environments still have room for improvement. For example, ratiometric fluorescence detection technology based on lanthanide metal-organic frameworks (Ln-MOFs) can reduce interference by using terbium (Tb) as a precursor. 3+ ) and europium (Eu) 3+ The dual-transmission signal reduces interference, but there is still room for improvement in the coordination stability and target recognition efficiency of its framework structure in dynamic environments.

[0003] It is worth noting that such detection challenges are not limited to the field of pathogen biomarkers. In environmental monitoring and food safety, tetracycline antibiotics (TC), as widely used antibacterial drugs, pose a threat to human health and the ecological environment due to their residues, requiring technologies that can detect them rapidly. Fluorescence analysis has attracted much attention due to its advantages of portable devices and rapid response, but probe development faces more stringent challenges: although different tetracycline derivatives share the tetrabenzobenzene core, differences in their substituents lead to vastly different electron cloud density distributions. Traditional molecularly imprinted polymers (MIPs) or aptamer probes require re-optimization of synthetic parameters for each variant. The design and final functionalization of probes for different targets often take weeks, and synthetic routes need to be redesigned for different target molecules, severely restricting the field application of detection technologies.

[0004] Against this backdrop, the key to solving the problem lies in constructing a modular carrier platform with plug-and-play characteristics and using the carrier material to prepare probes. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a method for preparing and applying a plug-and-play, ultrasensitive clustering peptide-based material and its visualized ratiometric fluorescent nanoprobe: To achieve the above objectives, the technical solution of the present invention is as follows: One objective of this invention is to provide a clustered peptide-based material having the structure shown in Formula I: Formula I; In Equation I, n ranges from 1 to 200.

[0006] A second objective of this invention is to provide a method for preparing the above-mentioned clustered peptide-based material, the method comprising the following steps: S1, will N -Propyl-N-carboxylic anhydride monomer, organic solvent and initiator are mixed for monomer polymerization to obtain poly( N -propylated glycine). N -Prophylynyl-N-carboxylic anhydride monomers have the structure shown in Formula II, poly( N -propylated glycine) has the structure shown in Formula III: Formula II, Formula III; In Equation III, n ranges from 1 to 200; S2, gathering ( N The mixture of propyltriethynylglycine, benzoin dimethyl ether, mercaptopropionic acid, and organic solvents was subjected to photoclick chemistry under a protective atmosphere to obtain a clustered peptide-based probe material (PG-COOH).

[0007] Preferably,N The molar ratio of propargyl-N-carboxylic anhydride monomer to initiator is (10:1) to (100:1).

[0008] Preferably, poly( N The molar ratio of propargyl, benzoin dimethyl ether and mercaptopropionic acid in propargyl glycine is (10~40):1:(80~100).

[0009] Preferably, the photoclick chemistry is performed under a 270 nm wavelength ultraviolet lamp.

[0010] The third objective of this invention is to provide a method for preparing a visualized ratio nanofluorescent probe for anthrax spore markers using the aforementioned clustered peptide-based material. The method involves: dissolving the clustered peptide-based material in water to prepare a clustered peptide solution; and then... 3+ Salts are dissolved in water to prepare metal ion solution 1, and Tb-containing salts are added. 3+ Salts were dissolved in water to prepare metal ion solution 2. The cluster peptide solution, solution 1 and solution 2 were mixed and co-assembled to obtain a cluster peptide-based anthrax spore marker visualization ratio nanofluorescent probe (Eu / Tb@PG-COOH).

[0011] Preferably, Eu 3+ and Tb 3+ The sum of the molar numbers of the two metal ions and the molar ratio of the aggregated peptide are 1:1.

[0012] Preferably, Eu 3+ and Tb 3+ The ratio of the sum of the charges of the two metal ions to the charge of the carboxylic acid group in the clustered peptide is (1:10) to (10:1).

[0013] The fourth objective of this invention is to provide a visualization ratio nanofluorescent probe (Eu / Tb@PG-COOH) for clustered peptide-based Bacillus anthracis spore markers obtained by the above preparation method.

[0014] The fifth objective of this invention is to provide a method for preparing a clustered peptide-based tetracycline visual ratio fluorescent nanoprobe using the above-mentioned clustered peptide-based material. The method involves: dissolving the clustered peptide-based material in water to prepare a clustered peptide solution; and then... 3+ Salts were dissolved in water to prepare a metal ion solution, and luminol was dissolved in water to prepare a luminol solution. The cluster peptide solution, Tris-HCl buffer, and luminol solution were added to the reaction flask in sequence and stirred to obtain a mixed solution. The metal ion solution was added to the mixed solution and stirred for 2 h to obtain the cluster peptide tetracycline visualization ratio fluorescent nanoprobe (PNPG-Eu-LML).

[0015] Preferably, Eu 3+The ratio of the sum of the charges of luminol and the charge of the carboxylic acid group in the clustered peptide is (1:10) to (10:1).

[0016] The sixth objective of this invention is to provide a clustered peptide tetracycline visualization ratio fluorescent nanoprobe (PNPG-Eu-LML) prepared by the above method.

[0017] The seventh objective of this invention is to provide an application of the above-mentioned clustered peptide-based anthrax spore marker visualization ratio nanofluorescent probe in the detection of 2,6-pyridinedicarboxylic acid.

[0018] The eighth objective of this invention is to provide an application of the above-mentioned clustered peptide-based tetracycline visualization ratio nanofluorescent probe in the detection of tetracycline.

[0019] The beneficial effects of this invention are: This patent uses dicarboxylated polymer peptides and Eu 3+ / Tb 3+ During assembly, the bidentate coordination mode forms a more stable chelate structure, which has a significant advantage over the monodentate coordination of monocarboxylated peptides. The two-site coordination provided by the dicarboxyl groups enhances the thermodynamic stability of the complex, and the metal ions and carboxyl oxygen atoms in the structure form a more compact octahedral coordination environment, effectively suppressing metal ion dissociation and hydration. In terms of luminescence performance, the dicarboxylated coordination system significantly improves luminescence efficiency by reducing nonradiative energy loss caused by ligand vibration.

[0020] (1) The visualized ratiometric fluorescent nanoprobe of the present invention utilizes the unique modular assembly capability of clustered peptide-based materials to achieve efficient coupling between the recognition unit (target molecule) and the fluorescent signal unit, and improves the luminescence stability of rare earth ions by precisely controlling the coordination microenvironment. At the same time, the ratiometric fluorescence detection mode based on the clustered peptide-based carrier can further reduce background interference through dual signal calibration, and its visualized output characteristics meet the intuitive interpretation requirements of rapid on-site detection.

[0021] (2) This invention proposes to use carboxyl-modified clustered peptide materials as ligands to construct two probe systems: one is to use clustered peptide materials with Eu... 3+ / Tb 3+ Coordination to form an Eu / Tb@PG-COOH probe, by regulating the lanthanide ion (Eu 3+ / Tb 3+ The charge ratio between DPA and negatively charged PG-COOH achieves the purpose of assembly and binding, and modulates fluorescence colorimetry. 3+ / Tb 3+ The dual-emission fluorescence undergoes a visually perceptible transition from red to green when DPA specifically complexes with Tb. 3+ The intensity of the 545 nm green fluorescence is enhanced at that time, while Eu...3+ Using red light emission at 615 nm as an internal control, as the DPA concentration gradually increases, a visually perceptible red-to-green transition is simultaneously output under a 256 nm UV lamp, achieving highly sensitive detection of the anthrax Bacillus marker DPA. This solves the problems of background interference and low visualization in traditional single fluorescence detection, and the detection process is simple, rapid, and highly selective. Secondly, based on clustered peptide-based materials and Eu... 3+ Synergistic effect with luminol, utilizing the signal amplification effect of luminol and the β-diketone structure of TC to affect Eu 3+ By leveraging the specific coordination sensitization effect, a highly selective fluorescent probe PNPG-Eu-LML targeting tetracycline antibiotics was constructed. The addition of clustered peptides solves the problems of poor stability and significant matrix interference of traditional rare earth probes, enabling nanomolar-level highly sensitive detection of tetracycline in complex samples without the need for cumbersome sample pretreatment.

[0022] (3) This invention aims to enhance the stability of rare earth ions and reduce the interference of impurities on chromophores through a clustered peptide matrix. Both probe systems use PG-COOH as the core. Among the two visualized ratio nanofluorescent probes, the dicarboxylated clustered peptides form a more stable chelate structure when assembling with lanthanide metal ions due to their bidentate coordination mode, which has a significant advantage over the monodentate coordination of monocarboxylated clustered peptides. In addition, the dual-site coordination provided by the dicarboxylated groups in the dicarboxylated clustered peptides can improve the thermodynamic stability of the complexes formed with lanthanide metal ions. The lanthanide metal ions and carboxyl oxygen atoms form a more compact octahedral coordination environment, effectively inhibiting the dissociation and hydration of lanthanide metal ions. In terms of luminescence performance, the dicarboxylated coordination system significantly improves luminescence efficiency by reducing nonradiative energy loss caused by ligand vibration.

[0023] (3) It combines the advantages of coordination stability, visual ratio detection, and cost-effectiveness, and the combination of ratio fluorescence solves the problems of insufficient sensitivity and background interference of traditional fluorescent probes. In addition, the binding of PG-COOH with lanthanide metal ions or luminol chromophores effectively improves the stability and detection sensitivity of fluorescent probes, which helps to detect biomarkers quickly, accurately, and intuitively. This invention solves the problems of long preparation cycle, poor environmental adaptability, and difficulty in functional expansion of existing probes, and has broad application prospects in the fields of point-of-care diagnosis of anthrax bacteria and high-throughput monitoring of tetracycline antibiotic residues.

[0024] (4) This invention prepares a visual ratiometric nanofluorescent probe based on PG-COOH co-assembly via ring-opening polymerization (ROP) and post-modification, enabling rapid and visual detection of DPA and TC. The PG-COOH backbone structure is similar to a polypeptide with nitrogen-substituted side chains, which makes its backbone more flexible and has good biocompatibility and bioactivity. This gives PG-COOH a certain advantage when assembling with lanthanide metal ions, and makes the obtained probe have good stability and biocompatibility. Attached Figure Description

[0025] Figure 1 This is a TEM image of the visualization ratio fluorescent nanoprobe Eu / Tb@PG-COOH from Example 2 of the present invention; Figure 2 This is the three-dimensional fluorescence emission spectrum of the visualization ratiometric fluorescent nanoprobe Eu / Tb@PG-COOH in Example 2 of the present invention in response to different concentrations of DPA; Figure 3 This is a CIE coordinate plot of the visual ratiometric fluorescent nanoprobe Eu / Tb@PG-COOH responding to different concentrations of DPA in Example 2 of the present invention; Figure 4 This is a standard curve of the visualization ratio fluorescent nanoprobe Eu / Tb@PG-COOH as a function of different concentrations of DPA in Example 2 of the present invention. Figure 5 The fluorescence emission spectra of the visual ratiometric fluorescent nanoprobe PNPG-Eu-LML in Example 3 of this invention in response to different concentrations of TC are shown. Figure 6 This is a CIE coordinate plot of the visual ratiometric fluorescent nanoprobe Eu / Tb@PG-COOH responding to different concentrations of TC in Example 2 of the present invention; Figure 7 This is a standard curve of the visualization of the ratiometric fluorescent nanoprobe Eu / Tb@PG-COOH as a function of different concentrations of TC in Example 2 of the present invention; Figure 8 This is a visual detection effect diagram of DPA by the visualization ratio nanofluorescent probe Eu / Tb@PG-COOH in Example 2 of the present invention; Figure 9 This is a visual detection effect diagram of TC by the visualization ratio nanofluorescent probe PNPG-Eu-LML in Example 3 of the present invention. Detailed Implementation

[0026] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be described in detail below with reference to specific embodiments in the specification.

[0027] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0028] Unless otherwise specified, the experimental methods used in the following specific embodiments are conventional methods. Unless otherwise specified, the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0029] In the following specific embodiments and examples of the present invention, M represents mol / L, mM represents mmol / L, and μM represents μmol / L.

[0030] This invention provides a clustered peptide-based material, the probe material having the structure shown in Formula I: Formula I; In Equation I, n ranges from 1 to 200.

[0031] This invention provides a method for preparing the above-mentioned clustered peptide-based material, the method comprising the following steps: S1, will N -Propyl-N-carboxylic anhydride monomer, organic solvent and initiator are mixed for monomer polymerization to obtain poly( N -propylated glycine). N -Prophylynyl-N-carboxylic anhydride monomers have the structure shown in Formula II, poly( N -propylated glycine) has the structure shown in Formula III: Formula II, Formula III; In Equation III, n ranges from 1 to 200; S2, gathering ( N The mixture of propargyl glycine, benzoin dimethyl ether, mercaptopropionic acid, and organic solvents was subjected to photoclick chemistry under a protective atmosphere to obtain a clustered peptide-based probe material.

[0032] In this invention, the degree of polymerization of the polymeric peptide-based material is preferably 1 to 200.

[0033] In S1 of the present invention, the organic solvent is preferably an anhydrous solvent, and the initiator is preferably benzylamine.

[0034] In this invention, the monomer polymerization temperature is preferably 50~65 ℃, and the polymerization time is preferably 20~48 h.

[0035] In this invention, after monomer polymerization is completed, the monomer polymerization product is precipitated three times in excess ice-cold ether, and then successively centrifuged and vacuum dried to obtain polymer (…). N (-propargyl glycine), the centrifugation speed is preferably 8000 r / min, and the time is preferably 5 min. There is no special limitation on the specific method of vacuum drying, and any method known to those skilled in the art can be used.

[0036] This invention obtains poly( N After (-propargylglycine), the polymer ( N The mixture of propyltriethynylglycine, dimethyl ether, mercaptopropionic acid, and organic solvent was subjected to photoclick chemistry under a nitrogen protective atmosphere to obtain a polymeric peptide-based material (PG-COOH).

[0037] In this invention, the preferred temperature for photoclick chemistry is room temperature, and the preferred time is 3 hours.

[0038] After the photoclick chemistry in this invention is completed, the reaction system is placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in deionized water for three days, with the water changed multiple times during the period. Then, the solvent is removed by freeze drying to obtain the clustered peptide probe material (PG-COOH).

[0039] In this invention N The preparation method of propyl-N-carboxylic anhydride monomer includes the following steps: adding 150 g of glyoxylic acid aqueous solution (50% by mass) to a flask, adding 44.5 mL of propargylamine, adding 2 M HCl, and refluxing overnight to obtain... N -Prophylactic glycine hydrochloride, take 15 g N -Protylenol-substituted glycine hydrochloride was dissolved in 300 mL of water, then 70 g of di-tert-butyl dicarbonate and 46 g of triethylamine were added and reacted for 72 h. N -tert-Butyloxycarbonyl- N 15 g of propargyl-substituted glycine hydrochloride was dissolved in anhydrous dichloromethane, and phosphorus trichloride (1.5 eq) was added to obtain... N -propynyl- N -Carboxylic anhydride monomer.

[0040] This invention provides a method for preparing a visualized ratiometric nanofluorescent probe for anthrax spore markers using the aforementioned clustered peptide-based material. The method comprises: dissolving the clustered peptide-based material in water to prepare a clustered peptide solution; and then... 3+ Salts are dissolved in water to prepare metal ion solution 1, and Tb-containing salts are added. 3+ Salts were dissolved in water to prepare metal ion solution 2. The cluster peptide solution, solution 1 and solution 2 were mixed and stirred, and co-assembled to obtain a cluster peptide-based anthrax spore marker visualization ratio nanofluorescent probe (Eu / Tb@PG-COOH).

[0041] In this invention, the concentration of the polymeric peptide solution is preferably 0.05–10 mg / mL, more preferably 1 mg / mL.

[0042] In this invention, the metal ions are added under stirring conditions, and the stirring time after adding the metal ions is preferably 24 h.

[0043] In this invention, Eu 3+ and Tb 3+ The preferred molar ratio of the sum of the two metal ions to the aggregated peptide is 1:1.

[0044] In this invention, Eu 3+ and Tb 3+ The ratio of the sum of the charges of the two metal ions to the charge of the carboxylic acid group in the clustered peptide is preferably (1:10) to (10:1), more preferably 1:1.

[0045] This invention provides a method for preparing a clustered peptide-based tetracycline visual ratio fluorescent nanoprobe using the above-mentioned clustered peptide-based probe material. The method comprises: dissolving the clustered peptide-based material in water to prepare a clustered peptide solution; and then preparing a Eu-containing... 3+ Salts were dissolved in water to prepare a metal ion solution, and luminol was dissolved in water to prepare a luminol solution. The cluster peptide solution, Tris-HCl buffer, and luminol solution were added to the reaction flask in sequence and stirred to obtain a mixed solution. The metal ion solution was added to the mixed solution and stirred for 2 h to obtain the cluster peptide tetracycline visualization ratio fluorescent nanoprobe (PNPG-Eu-LML).

[0046] In this invention, clustered peptide-based materials, Eu 3+ The preferred molar ratio of luminol to luminol is (4.0~5.0):(0.5~2.0):(0.1~0.2), and more preferably 4.5:1:0.15.

[0047] In this invention, the concentration of the polymeric peptide solution is preferably 0.05–10 mg / mL, more preferably 1 mg / mL.

[0048] In this invention, the metal ions are added under stirring conditions, preferably for 24 hours.

[0049] To further illustrate the present invention, specific embodiments provided by the present invention are described in detail below with reference to examples, but these should not be construed as limiting the scope of protection of the present invention. The term "an embodiment" or "embodiment" as used below refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that excludes other embodiments.

[0050] Example 1 S1, Poly ( N Synthesis of -propargylglycine (PNPG): Add 150 g (50% by mass) of glyoxylic acid aqueous solution to a flask, add 44.5 mL of propargylamine, add 450 mL of 2 mol / L HCl and reflux overnight to obtain N-propargyl-substituted glycine hydrochloride. N 15 g of propargyl-substituted glycine hydrochloride was dissolved in 300 mL of water, and 70 g of di-tert-butyl dicarbonate and 46 g of triethylamine were added. The mixture was reacted at room temperature for 72 h to protect the amino group, yielding... N -tert-butyloxycarbonyl-N-propynyl-substituted glycine hydrochloride, 15 g N -tert-butyloxycarbonyl-N-propynyl-substituted glycine hydrochloride was dissolved in anhydrous dichloromethane, and then phosphorus trichloride (1.5 eq) was added. The reaction was carried out for 3 h to obtain... N -propynyl- N -Carboxylic anhydride monomer; Will N -propynyl- N 1) Carboxylic anhydride monomers are dissolved in anhydrous tetrahydrofuran, and benzylamine is used as an initiator to initiate the polymerization of the monomers. N -propynyl- N The molar ratio of α-carboxylic anhydride monomer to initiator was 40:1, and the reaction time was 24 h. After the reaction, the monomer polymerization product was precipitated three times in excess ice-cold diethyl ether, centrifuged at 8000 r / min for 5 min, and then vacuum dried to obtain a white polymer. N -propylated glycine); Synthesis of S2 and PG-COOH: PNPG was mixed with dimethyl benzoate (DMPA) and mercaptopropionic acid in a molar ratio of 10:1:100 and then added to 5 mL of anhydrous tetrahydrofuran. After removing oxygen from the system under vacuum, nitrogen gas was introduced for protection. The mixture was then irradiated at room temperature under a 270 nm UV lamp for 3 hours. After the reaction was completed, the reaction system was placed in a dialysis bag with a molecular weight cutoff of 1000 Da and dialyzed in pure water for three days, with the water changed multiple times. The solvent was then removed by freeze drying to obtain PG-COOH. In this example, the degree of polymerization n was 50.

[0051] Example 2 Preparation of a ratiometric fluorescent nanoprobe (Eu / Tb@PG-COOH) for visualizing clustered peptide-based Bacillus anthracis spore markers: A negatively charged PG-COOH solution was prepared by dissolving it in water to form a 1 mg / mL peptide-based polymer. A 1 mM / L Eu(NO3)3 solution was prepared by dissolving Eu(NO3)3·6H2O in water, and a 1 mM / L Tb(NO3)3 solution was prepared by dissolving Tb(NO3)3·5H2O in water. The Tb(NO3)3 and Eu(NO3)3 solutions were added to the peptide-based polymer solution under stirring, and stirring was continued for 24 h. The three solutions were then mixed and stirred for co-assembly to obtain Eu / Tb@PG-COOH. The carboxylic acid groups in the peptide-based polymer probe material interact with the Eu... 3+ and Tb 3+ The sum of the charges of the two metal ions is 1:1. The morphology and structure of Eu / Tb@PG-COOH are as follows: Figure 1 As shown, Eu / Tb@PG-COOH is uniformly sprayed onto a paper-based material, and after drying to remove the solvent, a portable DPA test paper is obtained. Example 3 Preparation of the clustered peptide-based tetracycline visualization ratio fluorescent nanoprobe (PNPG-Eu-LML): A 10 mg / L clustered peptide solution was prepared by dissolving the clustered peptide probe material in water. A 1 mM / L Eu(NO3)3 solution was prepared by dissolving Eu(NO3)3 in water. A 1 mM / L luminol solution was prepared by dissolving luminol in water. 0.15 mL of the clustered peptide solution, 0.8 mL of 50 mM Tris buffer (pH = 8.5), and 0.005 mL of luminol solution were added sequentially to a reaction flask and stirred for 2 h to obtain a homogeneous mixture. 0.05 mL of Eu(NO3)3 solution was then added to the mixture, and stirring continued for another 2 h to obtain PNPG-Eu-LML. PNPG-Eu-LML was uniformly sprayed onto a paper-based material, and after drying to remove the solvent, a portable TC test strip was obtained.

[0052] Application Example 1 The Eu / Tb@PG-COOH fluorescent probe from Example 2 was used to detect DPA. The method was as follows: DPA was dissolved in deionized water to prepare DPA solutions with a concentration gradient of 0–20 μM. The probe from Example 2 was dissolved in water to prepare a 1 mg / mL fluorescent probe stock solution. 100 μL of the fluorescent probe stock solution was taken and diluted to 2 mL with Tris-HCl (pH=8) buffer. Different concentrations of DPA solution (0–20 μM) were added to the diluted stock solution. The emission spectra and CIE coordinates of different concentrations (0–4.5 μM) of DPA were measured at an excitation wavelength of Ex=275 nm to investigate the sensitivity of the Eu / Tb@PG-COOH fluorescent probe in recognizing DPA. The results are as follows: Figure 2 and Figure 3As shown in the figure, it can be seen that after the addition of DPA, the fluorescence intensity of the nano-fluorescent probe at 545 nm significantly increased with increasing DPA concentration, while the red fluorescence at 615 nm showed no significant change; within the DPA solution range of 0–20 μM, a fluorescence change from red to green was achieved with increasing DPA concentration. Figure 3 As can be seen, the fluorescence changes from red to green as the arrow indicates the direction (which represents an increase in DPA concentration).

[0053] The relationship between different DPA concentrations and fluorescence intensity within the range of 0–20 μM was tested, and the results are as follows: Figure 4 As shown, the concentration of DPA exhibits a good linear relationship with fluorescence intensity, with a correlation coefficient R0. 2 =0.9927, and the relevant linear equation is I 545 / I 615 =0.01173C DPA +3.187, where C DPA The concentration of DPA (corresponding to) Figure 4 The detection limit (X) was as low as 5.73 nM, which was significantly lower than the infection limit of Bacillus anthracis spores (60 μM).

[0054] Different concentrations of DPA solution were added to the portable DPA test strip prepared in Example 2, and the color changes were observed under a 254 nm UV lamp. The results are as follows. Figure 8 As shown, the test strip undergoes a color change in the presence of DPA. As the concentration of DPA increases, the color changes from red to green, and the change is obvious and can be observed with the naked eye.

[0055] Application Example 2 TC was detected using PNPG-Eu-LML as described in Example 3. The method was as follows: TC was dissolved in deionized water to prepare TC solutions with a concentration gradient of 0–50 μM. The probe from Example 3 was dissolved in water to prepare a 1 mg / mL fluorescent probe stock solution. 100 μL of the fluorescent probe stock solution was taken and diluted to 2 mL with Tris-HCl (pH=8) buffer. Different concentrations of TC solution (0–50 μM) were added to the diluted stock solution. The emission spectra and CIE coordinates were measured at an excitation wavelength of Ex = 280 nm in the presence of different concentrations of DPA. The results are as follows: Figure 5 and Figure 6 As shown. From Figure 5 As can be seen, with the increase of TC concentration, the blue fluorescence of PNPG-Eu-LML at 430 nm gradually decreases, while the red fluorescence intensity at 614 nm significantly increases, and the emission color changes from blue to red. Figure 6As can be seen, the fluorescence changes from red to green as the arrow indicates the direction (representing an increase in DPA concentration). Within the TC solution range of 0–50 μM, such as Figure 7 As shown, the concentration of TC exhibits a good linear relationship with fluorescence intensity, with a correlation coefficient R0. 2 =0.9984, and the relevant linear equation is I 614 / I 430 =00.1194 C TC +0.02774, where C TC The concentration of TC (corresponding to) Figure 7 The detection limit for TC in milk is as low as 11.21 nM, which is far below the maximum residue limits for TC in milk set by the US and EU Food and Drug Administration (FDA) (0.676 μM and 0.225 μM, respectively).

[0056] Different concentrations of TC solution were added to the portable TC test strip prepared in Example 3, and the color change was observed under a 365 nm ultraviolet lamp. The results are as follows. Figure 9 As shown, it can be seen that the test paper undergoes a color change in the presence of TC. As the concentration of TC increases, the color change becomes more obvious and can be observed with the naked eye.

[0057] In summary, this invention provides a clustered peptide-based material and a clustered peptide-based Bacillus anthracis spore marker visualization ratio nanofluorescent probe and a clustered peptide-based tetracycline visualization ratio nanofluorescent probe based on this material. These two probes exhibit high sensitivity, a wide detection concentration range, and good selectivity, enabling rapid and visual detection of DPA and TC. Eu / Tb@PG-COOH, at an excitation wavelength of 275 nm, can specifically recognize DPA, and DPA and Tb... 3+ After coordination, the fluorescent probe emits characteristic green light at a wavelength of 545 nm through an antenna effect, changing the fluorescence from red to green, thus enabling rapid and visual detection of DPA. The detection limit for DPA by this probe is as low as 5.73 nM. PNPG-Eu-LML, at an excitation wavelength of 280 nm, can specifically recognize TC, achieving a fluorescence change from blue to red. TC and Eu... 3+ After coordination, the fluorescent probe emits characteristic red light at a wavelength of 616 nm through the antenna effect. The blue fluorescence emission intensity of luminol (430 nm) decreases with increasing tetracycline concentration. By recording the change in the ratio of red to blue emission, highly sensitive detection of tetracycline is achieved. The detection limit of this probe for TC is as low as 11.21 nM. These two peptide-based fluorescent probes have achieved accurate detection of DPA and TC, respectively, meeting the detection requirements of TC and DPA in food and environmental samples.

[0058] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A clustered peptide-based material, characterized in that, The material has the structure shown in Formula I: Formula I; In Equation I, n ranges from 1 to 200.

2. A method for preparing the clustered peptide-based material according to claim 1, characterized in that, The preparation method includes the following steps: S1, will N -Propyl-N-carboxylic anhydride monomer, organic solvent and initiator are mixed for monomer polymerization to obtain poly( N -propylated glycine). N -Prophylynyl-N-carboxylic anhydride monomers have the structure shown in Formula II, poly( N -propylated glycine) has the structure shown in Formula III: Formula II, Formula III; In Equation III, n ranges from 1 to 200; S2, gathering ( N A mixture of propargyl glycine, benzoin dimethyl ether, mercaptopropionic acid, and an organic solvent is subjected to photoclick chemistry under a protective atmosphere to obtain a clustered peptide-based material.

3. The preparation method according to claim 2, characterized in that, S1 N The molar ratio of propargyl-N-carboxylic anhydride monomer to initiator is (10:1) to (100:1); S2 medium-polymer ( N The molar ratio of propargyl, benzoin dimethyl ether and mercaptopropionic acid in propargyl glycine is (10~40):1:(80~100); photoclick chemistry is performed under a 270 nm wavelength ultraviolet lamp.

4. A method for preparing a clustered peptide-based anthrax spore marker visualization ratio nanofluorescent probe, characterized in that, The preparation method is as follows: dissolve the polymeric peptide-based material described in claim 1 in water to prepare a polymeric peptide solution, and then add Eu... 3+ Salts are dissolved in water to prepare metal ion solution 1, and Tb-containing salts are added. 3+ Salts were dissolved in water to prepare metal ion solution 2. The cluster peptide solution, solution 1 and solution 2 were mixed and co-assembled to obtain a cluster peptide-based anthrax spore marker visualization ratio nanofluorescent probe.

5. The preparation method according to claim 4, characterized in that, Eu 3+ and Tb 3+ The sum of the molar numbers of the two metal ions and the molar ratio of the aggregated peptide are 1:1; Eu 3+ and Tb 3+ The ratio of the sum of the charges of the two metal ions to the charge of the carboxylic acid groups in the clustered peptide material is (1:10) to (10:1).

6. A clustered peptide-based anthrax bacillus spore marker visualization ratio nanofluorescent probe, characterized in that, It is prepared by the method described in any one of claims 4 to 5.

7. A method for preparing a clustered peptide-based tetracycline-based visual ratio fluorescent nanoprobe, characterized in that, The preparation method is as follows: dissolve the polymeric peptide-based material described in claim 1 in water to prepare a polymeric peptide solution, and then add Eu... 3+ Salts were dissolved in water to prepare a metal ion solution, and luminol was dissolved in water to prepare a luminol solution. The cluster peptide solution, Tris-HCl buffer, and luminol solution were added to the reaction flask in sequence and stirred to obtain a mixed solution. The metal ion solution was added to the mixed solution and stirred for 2 h to obtain a cluster peptide-based tetracycline visual ratio fluorescent nanoprobe. The Eu 3+ The ratio of the sum of the charges of luminol and the charge of the carboxylic acid group in the aggregated peptide material is (1:10) to (10:1).

8. A clustered peptide-based tetracycline visual ratio fluorescent nanoprobe, characterized in that, It is prepared by the method described in claim 7.

9. The application of the clustered peptide-based anthrax bacillus spore marker visualization ratio nanofluorescent probe according to claim 6, characterized in that, This nanofluorescent probe is used to detect 2,6-pyridinedicarboxylic acid.

10. An application of the clustered peptide-based tetracycline visual ratio nanofluorescent probe of claim 8, characterized in that, This nanofluorescent probe is used to detect tetracycline.

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