A fluorescent peptide dot and a preparation method thereof

Abstract

The present application relates to fluorescent nanomaterials technical field, disclose a kind of fluorescent peptide point and preparation method thereof, including (one) precursor solution configuration stage, amino acid is dispersed with regulator in solvent and is prepared into precursor solution;(two) synthesis stage, precursor solution is heated after reaction and is cooled to room temperature, obtain reaction solution;(three) post-processing stage, reaction solution is purified, dried, to obtain fluorescent peptide point powder.The present application selects amino acid as precursor, by heating to make amino acid intermolecular dehydration condensation, form amino acid polymer nanoparticle with conjugated luminescence characteristics, one-step synthesis fluorescent peptide point, process operation is simple, heating temperature is low, time is short, and need not closed reaction, effectively reduce the difficulty of fluorescent peptide point production;And also can avoid forming carbonization structure with security risk to human body, applicable to food and medical detection, further improve safety.

Description

Technical Field

[0001] This invention relates to the field of fluorescent nanomaterials technology, specifically to a fluorescent peptide dot and its preparation method. Background Technology

[0002] Fluorescent nanomaterials, mainly including rare-earth nanomaterials, semiconductor quantum dots, graphene quantum dots, and polymer carbon dots, have important applications in many fields such as optoelectronic devices, bioimaging, clinical diagnostics, and food monitoring. However, the application of existing fluorescent nanomaterials in the food and pharmaceutical fields is mostly limited by their toxicity. Developing highly safe fluorescent materials applicable to the food and pharmaceutical fields is one of the key issues that urgently needs to be addressed in this field.

[0003] The prior art CN103693633A discloses a green method for synthesizing fluorescent chiral carbon dots, comprising the following steps: First, a carbon precursor and amino acids are ultrasonically dispersed in deionized water to prepare a transparent aqueous solution or emulsion; the mass ratio of the carbon precursor to amino acids is 200:1 to 5:1. Second, the mixed solution obtained in the first step is placed in a microwave heating device for microwave heating reaction to obtain a yellow or brownish-yellow liquid. Third, the carbon quantum dot solution obtained in the second step is dialyzed using a dialysis bag with a molecular weight cutoff of 1,000 to 50,000 to remove unreacted carbon precursor and amino acids, yielding fluorescent carbon dots with a narrow particle size distribution without further purification. However, even though the fluorescent carbon quantum dots prepared by the prior art are essentially non-toxic, they still pose a potential hazard to human health due to the presence of a large number of aromatic compounds, polycyclic aromatic hydrocarbons (PAHs), azo compounds, and other "carbonized structures."

[0004] Therefore, developing a completely non-toxic and highly biocompatible fluorescent peptide and its preparation method is of great significance. It can not only effectively make up for the shortcomings of existing technologies, but also further improve the safety of fluorescent materials. Summary of the Invention

[0005] The present invention aims to provide a fluorescent peptide dot and its preparation method to solve the technical problem that the existing technology for preparing fluorescent chiral carbon dots using carbon precursors has many carbonized structures that pose potential hazards to the human body and reduce the safety of their use.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: a method for preparing fluorescent peptide dots, comprising (i) a precursor solution preparation stage, wherein amino acids and regulators are dispersed in a solvent to prepare a precursor solution; (ii) a synthesis stage, wherein the precursor solution is heated to react and then cooled to room temperature to obtain a reaction solution; and (iii) a post-processing stage, wherein the reaction solution is purified and dried to obtain fluorescent peptide dot powder.

[0007] The principles and advantages of this scheme are: 1. Compared to existing technologies that produce fluorescent carbon quantum dots containing a large number of "carbonized structures" which reduce food safety, this method synthesizes fluorescent peptides by dispersing amino acids and regulators in a solvent and then heating the reaction. During the process, the regulator effectively regulates the dehydration condensation reaction of the substrate amino acids to obtain amino acid polymer nanoparticles with conjugated luminescence properties. Furthermore, by limiting the reaction temperature and time, the peptide chain length of the synthesized fluorescent peptides is effectively limited, and the proportion of "carbonized structures" is effectively reduced, thereby improving the safety of the fluorescent peptides.

[0008] 2. This scheme can regulate the synthesis process and environment through regulators during the synthesis stage. On the one hand, it can effectively improve the efficiency of the synthesis reaction. On the other hand, it can also effectively increase the concentration of fluorescent groups, thereby increasing the fluorescence intensity, reducing the amount of fluorescent peptides used in the experiment, reducing costs, and reducing potential environmental pollution, thus achieving a balance between economic and environmental benefits.

[0009] Preferably, as an improvement, the amino acid is one or more combinations of twenty natural amino acids.

[0010] Technical Effects: This solution, employing the aforementioned setup, facilitates the acquisition of fluorescent peptides of different colors using different amino acids or combinations as the main reaction agents, adapting to diverse application needs. Through long-term experiments, the applicant has discovered that, theoretically, amino acids and their derivatives can be successfully used to prepare fluorescent peptides. Considering the safety of the prepared fluorescent peptides for human use, reducing the amount of carbon-chain modified natural amino acids can effectively lower the carbonized structure content in the prepared fluorescent peptides, thereby further improving the safety of their use.

[0011] Preferably, as an improvement, the regulator is one or more combinations of inorganic acids, bases, and metal ions; more preferably, the regulator is a combination of metal ions and inorganic acids or bases.

[0012] Technical Effects: This scheme, employing the aforementioned settings, facilitates a suitable environment for fluorescent peptide synthesis, accelerating its efficiency. Through long-term experiments, the applicant has discovered that inorganic acids promote fluorescent peptide synthesis by facilitating amino acid protonation and inhibiting side reactions; bases promote synthesis by promoting peptide bond formation and preventing peptide chain degradation, effectively stabilizing the fluorescent group; and metal ions catalyze peptide chain assembly and stabilize the fluorescent group through coordination, electron transfer, and template effects. In particular, combining metal ions with inorganic acids / bases not only effectively accelerates fluorescent peptide synthesis but also effectively stabilizes the fluorescent group and enhances fluorescence intensity. Long-term experiments have also revealed that organic acids, instead of acting as regulators, participate in the reaction as substrates, altering the peptide structure and consequently reducing the performance of the fluorescent peptides produced.

[0013] Preferably, as an improvement, the solvent is one or more combinations of water and organic solvents.

[0014] Technical benefits: The above-mentioned setup facilitates the rapid dissolution of amino acids, regulators, and potential fluorescent groups for reaction synthesis. Good solubility ensures uniform mixing of reactants, avoids excessively high local concentrations or precipitation, and provides a suitable environment for peptide chain assembly, thereby improving the synthesis effect and yield of fluorescent peptides.

[0015] Preferably, as an improvement, the organic solvent has a boiling point higher than 60 °C.

[0016] Technical effect: The above settings facilitate rapid removal after the reaction while avoiding excessively low boiling points that could lead to rapid evaporation during the reaction and affect the controllability of the reaction.

[0017] Preferably, as an improvement, the mass concentration of amino acids in the precursor solution is 1.0~10 g / L.

[0018] Technical Effects: This scheme, employing the aforementioned setup, facilitates the rapid protonation and assembly of amino acids into peptide chains, thereby improving the efficiency of fluorescent peptide synthesis. Through long-term experiments, the applicant discovered that if the amino acid concentration in the precursor solution is too low, the reaction efficiency will decrease due to insufficient amino acids; conversely, if the amino acid concentration is too high, the reaction will be incomplete due to excessive amino acids, leading to waste of raw materials.

[0019] Preferably, as an improvement, the precursor solution contains a regulator with a mass concentration of 0~5.47 g / L.

[0020] Technical Effects: This solution, employing the above-mentioned settings, effectively accelerates the synthesis of fluorescent peptides and improves efficiency. Through long-term experiments, the applicant discovered that if no regulator is added or the regulator content is too low, insufficient functionalization will result in unchanged fluorescence properties; conversely, if the regulator content is too high, alterations to the fluorophore will lead to excessively low fluorescence intensity.

[0021] Preferably, as an improvement, the temperature and time of the heating reaction are 50~150 °C and 1~100 min, respectively.

[0022] Technical Effects: The above-mentioned setup facilitates the complete dehydration and condensation reaction of amino acids into fluorescent peptides. Through long-term experiments, the applicant discovered that if the reaction temperature is too low, the fluorescence intensity will decrease; if the reaction temperature is too high, the reaction rate will be too fast, making the reaction process difficult to control; if the reaction time is too short, the yield will be too low due to incomplete precursor conversion; and if the reaction time is too long, aggregation will occur, reducing its dispersibility and stability in solution.

[0023] Preferably, as an improvement, the purification is performed by dialysis using a 500-1000 Da dialysis bag for 24-48 hours, and the dialysis solution in the dialysis bag is taken to obtain the purified product solution.

[0024] Technical Effects: This scheme, employing the above-described setup, facilitates the effective removal of unreacted raw materials from the reaction solution, yielding a fluorescent peptide solution. Further drying yields high-purity fluorescent peptides. Through long-term experiments, the applicant has found that the molecular weight of the fluorescent peptides obtained using this scheme is approximately 180-1000 Da. A 500 Da dialysis bag is sufficient to completely intercept the fluorescent peptides, preventing them from permeating and reducing product yield. If the molecular weight cutoff of the dialysis bag is too low, the product purity will decrease due to the retention of small molecule intermediates or amino acids, thus reducing fluorescence intensity. If the molecular weight cutoff is too high, the product will flow out with the solvent, reducing product yield. If the dialysis time is too short, the purity of the carbon dots will decrease due to insufficient removal of small molecule impurities and unreacted precursors. If the dialysis time is too long, some carbon dots will escape, resulting in carbon dot loss.

[0025] Preferably, as an improvement, this solution also provides a fluorescent peptide spot, which is prepared according to the above method.

[0026] Preferably, as an improvement, the fluorescent peptide has 64-72% C, 19-26% O, 2-9% N, and the remainder is trace elements.

[0027] Technical benefits: The above settings facilitate pH sensing applications.

[0028] The technical advantages of this solution are summarized as follows: 1. This invention selects amino acids as precursors and uses heating to cause dehydration and condensation between amino acid molecules to form amino acid polymer nanoparticles with conjugated luminescence properties. Fluorescent peptide dots are synthesized in one step. The process is simple to operate, with low heating temperature and short time, and does not require a closed reaction, effectively reducing the difficulty of fluorescent peptide dot production. It can also avoid the formation of carbonized structures that pose safety hazards to the human body, making it suitable for food and medical testing and further improving safety.

[0029] 2. This invention selects different amino acids, including one amino acid or a combination of multiple amino acids, to regulate the synthesis of amino acid polymers with different structures, thereby obtaining fluorescent peptides with different emission wavelengths (colors). The obtained fluorescent peptides have good dispersibility in aqueous solution and stable optical properties.

[0030] 3. This scheme uses inorganic acids and bases as regulators to adjust the amino acid to form different amino acid ion forms, and uses metal ions as regulators to adjust the amino acid to form different amino acid metal complexes, ultimately adjusting the structure of the amino acid polymer of the reaction product, and thereby adjusting the emission wavelength (color) of the fluorescent peptide. Attached Figure Description

[0031] Figure 1 The image shows the multi-wavelength excitation fluorescence emission spectrum of the fluorescent peptide spot (PD1, i.e., S1 in the figure) obtained in Example 1 of this invention.

[0032] Figure 2 The image shows the multi-wavelength excitation fluorescence emission spectrum of the fluorescent peptide spot (PD2, i.e., S2 in the figure) obtained in Example 2 of this invention.

[0033] Figure 3 The image shows the multi-wavelength excitation fluorescence emission spectrum of the fluorescent peptide spot (PD3, i.e., S3 in the figure) obtained in Example 3 of this invention.

[0034] Figure 4 The image shows the multi-wavelength excitation fluorescence emission spectrum of the fluorescent peptide spot (PD4, i.e., S4 in the figure) obtained in Example 4 of this invention.

[0035] Figure 5 The images show transmission electron microscopy (TEM) images and particle size distributions of the two fluorescent peptide spots (PD1 and PD2, corresponding to S1 and S2 in the figure, respectively) obtained in Examples 1 and 2 of this invention.

[0036] Figure 6 The images show transmission electron microscopy (TEM) images and particle size distributions of the two fluorescent peptide spots (PD3 and PD4, corresponding to S3 and S4 in the figure, respectively) obtained in Examples 3-4 of this invention.

[0037] Figure 7 The X-ray diffraction (XRD) patterns of the four fluorescent peptides obtained in Examples 1-4 of this invention are shown.

[0038] Figure 8 The X-ray photoelectron spectroscopy (XPS) spectra of the four fluorescent peptides obtained in Examples 1-4 of this invention are shown.

[0039] Figure 9 These are the XPS high-resolution C1s spectra of the four fluorescent peptides obtained in Examples 1-4 of this invention.

[0040] Figure 10 These are the XPS high-resolution O 1s spectra of the four fluorescent peptides obtained in Examples 1-4 of this invention.

[0041] Figure 11 These are the XPS high-resolution N 1s spectra of the four fluorescent peptides obtained in Examples 1-4 of this invention.

[0042] Figure 12 These are the XPS high-resolution Zn 1s spectra of the four fluorescent peptides obtained in Examples 1-4 of this invention.

[0043] Figure 13 The Fourier transform infrared (FTIR) spectra of the four fluorescent peptides obtained in Examples 1-4 of this invention are shown.

[0044] Figure 14 This is the PL response curve of the fluorescent peptide in Experiment Example 1 of the present invention with pH at an excitation wavelength of 320 nm.

[0045] Figure 15 This is a graph showing the trend of fluorescence intensity of the fluorescent peptide spot at pH 1 to 7 in Experimental Example 1 of this invention.

[0046] Figure 16 This is a linear fitting curve of the fluorescent peptide spot in Experimental Example 1 of the present invention within the pH range of 1 to 6.

[0047] Figure 17 A comparison of the fluorescence intensity of fluorescent peptides prepared at different temperatures and times as a function of excitation wavelength.

[0048] Figure 18 The graph shows the change in fluorescence intensity of fluorescent peptides prepared using only acid or base as regulators as a function of excitation wavelength.

[0049] Figure 19 The fluorescence intensity curves of the fluorescent peptides prepared using only acid as a regulator are shown at different wavelengths.

[0050] Figure 20 The fluorescence intensity curves of the fluorescent peptides prepared using only alkali as a regulator are shown at different wavelengths.

[0051] Figure 21 The graph shows the fluorescence intensity of fluorescent peptides prepared without a regulator or with only metal ions as the regulator, as a function of excitation wavelength.

[0052] Figure 22 The fluorescence intensity curves of the fluorescent peptides prepared using only metal ions as regulators at different wavelengths are shown.

[0053] Figure 23 The fluorescence intensity curves of the fluorescent peptides prepared without a modulator are shown at different wavelengths. Detailed Implementation

[0054] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the following embodiments and experimental examples are conventional means well known to those skilled in the art, and the materials and reagents used can all be obtained commercially.

[0055] Overview of the Plan This method provides a fluorescent peptide, which is obtained by purifying and drying a precursor solution containing 1.0~10 g / L amino acids, 0~5.47 g / L regulator, and the balance being solvent, after heating, cooling and rehydration.

[0056] The amino acids are one or more combinations of 20 natural amino acids; the regulator is one or more of an inorganic acid, a base, and a metal ion (preferably a combination of a metal ion and one of an inorganic acid or a base, i.e., a combination of an inorganic acid and a metal ion, or a combination of a base and a metal ion). The solvent is one or more combinations of water and an organic solvent. The boiling point of the organic solvent is higher than 60℃. The heating temperature and time are 50~150℃ and 1~100min, respectively. For purification, dialysis is performed using a 500~1000 dialysis bag for 24~48h, and the dialysate in the dialysis bag is used to obtain the purified fluorescent peptide solution.

[0057] This solution also provides a method for preparing fluorescent peptide dots, including the following steps: (a) Precursor solution preparation stage: any one or more combinations of amino acids and regulators are dispersed in a solvent to prepare a precursor solution; (ii) Synthesis stage: The precursor solution is heated to 50~150 ℃ and reacted for 1~100 min, then cooled to room temperature to obtain the reaction solution; (III) Post-processing stage: The reaction solution is purified and dried to obtain fluorescent peptide powder.

[0058] The purification process involves dialysis with a 500-1000 Da dialysis bag for 24-48 hours, and then collecting the dialysis solution from the dialysis bag to obtain the purified product solution.

[0059] Drying was performed under vacuum at 60°C for 8 hours.

[0060] Example 1 Preparation method of fluorescent peptide dots: Weigh 25 mg of solid tyrosine and 25 mg of solid cysteine ​​in a beaker, add 10 mL of ethylene glycol and 300 μL of concentrated hydrochloric acid aqueous solution (37%) to dissolve them, and prepare a precursor solution (total amino acid concentration of 5 g / L, hydrochloric acid concentration of 5.47 g / L, solution pH of approximately 0.7). Transfer the solution to a microwave oven and heat at 120 ℃ for 5 min on medium heat. Stop heating and let the solution cool to room temperature. Dialyze the solution using a 3000 Da dialysis bag for 1 day. Take the dialysate from the dialysis bag to obtain a purified product solution. Finally, freeze-dry the solution to obtain pure fluorescent peptide powder, named PD1. Calculate the yield = (actual mass obtained / theoretical mass) × 100% = (18 mg / 50 mg) × 100% = 36%. Peptide dot PD1 powder can be uniformly and stably dispersed in water and ethanol. Its solution emits strong green fluorescence under 365 nm ultraviolet light irradiation.

[0061] Fluorescence emission spectra at multiple excitation wavelengths (320 nm, 370 nm, 420 nm, 470 nm, and 520 nm) Figure 1 The results showed that the obtained peptide PD1 exhibited the strongest fluorescence emission peak at 435 nm under 320 nm excitation, and a second emission peak at about 525 nm.

[0062] TEM test results ( Figure 5 The results showed that the obtained peptide PD1 were nearly spherical nanoparticles with relatively uniform size and a particle size D50 of approximately 6.42 nm. XRD test results ( Figure 7 This indicates that the obtained peptide PD1 did not show graphite crystal diffraction peaks and did not form a carbonized structure.

[0063] XPS full spectrum test results ( Figure 8 The results indicate that the obtained peptide PD1 contains carbon, oxygen, nitrogen, and sulfur elements; XPS high-resolution elemental spectroscopy results ( Figures 9-12 The results show that carbon exists primarily as C=C single bonds with minor amounts of C=C and CO / C=O, while oxygen and nitrogen exist primarily as O=C and NC single bonds, respectively, with minor amounts of O=C and N=C double bonds. FTIR spectral results ( Figure 13 The results show that the peptide site PD1 exhibits vibrational peaks for the OH, NH, CH, CC, C=C, CO, C=O, and CN bonds in the amino acid and polypeptide structures, and also shows a COC vibrational peak, indicating that dehydration condensation between carboxyl groups has occurred; in addition, the relatively broad OH and NH peaks indicate the presence of hydrogen bonds.

[0064] Example 2 Preparation method of fluorescent peptide dots: Weigh 50 mg of tyrosine solid and 5.0 mg of zinc chloride solid in a beaker, add 10 mL of deionized water and 200 μL of concentrated hydrochloric acid aqueous solution (37%) to dissolve them, and prepare a precursor solution (total amino acid concentration of 5.0 g / L, zinc ion concentration of 0.5 g / L, hydrochloric acid concentration of 3.65 g / L, and solution pH of approximately 1.0). Transfer the solution to an electric thermostatic drying oven and heat it at 70 ℃ for 60 min. Stop heating and allow the solution to cool to room temperature. Centrifuge at 16000 rpm to remove the supernatant, then disperse it in water and repeat the centrifugation three times. Then, vacuum dry the obtained solid at 60 ℃ to finally obtain pure fluorescent peptide powder, named PD2. Calculate the yield = (actual mass obtained / theoretical mass) × 100% = (17 mg / 55 mg) ×100%=34%; Peptide PD2 powder can be uniformly and stably dispersed in water and ethanol, and its solution emits strong blue fluorescence under 365nm ultraviolet light.

[0065] Fluorescence emission spectra at multiple excitation wavelengths (320 nm, 370 nm, 420 nm, 470 nm, and 520 nm) Figure 2 The results showed that the obtained peptide PD2 exhibited the strongest fluorescence emission peak at 455 nm under 370 nm excitation.

[0066] TEM test results ( Figure 5 The results showed that the obtained peptide PD2 were nearly spherical nanoparticles with relatively uniform size and a particle size of approximately 5.96 nm. XRD test results ( Figure 7 The results showed that the obtained peptide PD2 did not exhibit graphite crystal diffraction peaks and did not form a carbonized structure; however, it showed obvious zinc peaks and relatively weak diffraction peaks that matched the zinc ion complex well, indicating that there was a certain zinc ion coordination structure in the peptide.

[0067] XPS full spectrum test results ( Figure 8 The results indicate that the obtained peptide PD2 contains carbon, oxygen, nitrogen, and zinc elements; XPS high-resolution elemental spectroscopy results ( Figures 9-12 The results show that carbon exists primarily as C=C single bonds with minor amounts of C=C and CO / C=O, while oxygen and nitrogen exist primarily as O=C and NC single bonds, respectively, with minor amounts of O=C and N=C double bonds. FTIR spectral results ( Figure 13 The results show that the peptide PD2 exhibits vibrational peaks for the OH, NH, CH, CC, C=C, CO, C=O, and CN bonds in the amino acid and polypeptide structures, as well as a COC vibrational peak, indicating that dehydration condensation between carboxyl groups has occurred; in addition, the relatively broad OH and NH peaks indicate the presence of hydrogen bonds.

[0068] Example 3 Preparation method of fluorescent peptide dots: Weigh 25 mg of tyrosine solid, 25 mg of alanine solid, and 5.0 mg of zinc chloride solid into a beaker, add 10 mL of deionized water and 200 μL of sodium hydroxide aqueous solution (200 g / L) to dissolve, and prepare a precursor solution (total amino acid concentration of 5.0 g / L, zinc ion concentration of 0.5 g / L, sodium hydroxide concentration of 4 g / L, and pH of the solution of approximately 13.5). Transfer the solution to an electric thermostatic drying oven and heat at 90 ℃ for 30 min. Stop heating and allow the solution to cool to room temperature. Centrifuge at 16000 rpm to remove the supernatant, then disperse in water and repeat centrifugation 3 times. Finally, vacuum dry the obtained solid at 60 ℃ to obtain pure fluorescent peptide powder, named PD3. Calculate the yield = (actual mass obtained / theoretical mass) × 100% = (18.5 mg / 55 mg). ×100%=37%; Peptide PD3 powder can be uniformly and stably dispersed in water and ethanol, and its solution emits strong green fluorescence under 365nm ultraviolet light.

[0069] Fluorescence emission spectra at multiple excitation wavelengths (320 nm, 370 nm, 420 nm, 470 nm, and 520 nm) Figure 3 The results showed that the obtained peptide PD3 exhibited the strongest fluorescence emission peak at 455 nm under 370 nm excitation.

[0070] TEM test results ( Figure 6 The results showed that the obtained peptide PD3 were nearly spherical nanoparticles with relatively uniform size and a particle size of approximately 7.06 nm. XRD test results ( Figure 7 The results showed that the obtained peptide PD3 did not exhibit graphite crystal diffraction peaks and did not form a carbonized structure; however, it showed obvious zinc peaks and relatively weak diffraction peaks that matched the zinc ion complex well, indicating that there was a certain zinc ion coordination structure in the peptide.

[0071] XPS full spectrum test results ( Figure 8 The results indicate that the obtained peptide PD3 contains carbon, oxygen, nitrogen, and zinc elements; XPS high-resolution elemental spectroscopy results ( Figures 9-12 The results show that carbon exists primarily as C=C single bonds with minor amounts of C=C and CO / C=O, while oxygen and nitrogen exist primarily as O=C and NC single bonds, respectively, with minor amounts of O=C and N=C double bonds. FTIR spectral results ( Figure 13The results show that the peptide PD3 exhibits vibrational peaks for the OH, NH, CH, CC, C=C, CO, C=O, and CN bonds in the amino acid and polypeptide structures, as well as a COC vibrational peak, indicating that dehydration condensation between carboxyl groups has occurred; in addition, the relatively broad OH and NH peaks indicate the presence of hydrogen bonds.

[0072] Example 4 Preparation method of fluorescent peptide dots: Weigh 25 mg of tyrosine solid, 25 mg of cysteine ​​solid and 5.0 mg of zinc chloride solid in a beaker, add 10 mL of ethylene glycol and 300 μL of concentrated hydrochloric acid aqueous solution (37%) to dissolve, and prepare a precursor solution (total amino acid concentration of 5.0 g / L, hydrochloric acid concentration of 5.47 g / L, solution pH of approximately 0.7). Transfer to a microwave oven and heat at 150 ℃ for 1 min on high power. Stop heating and let the solution cool to room temperature. Dialyze using a 5000 Da dialysis bag for 2 days. Take the dialysate from the dialysis bag to obtain a purified product solution. Finally, freeze-dry to obtain pure fluorescent peptide dot powder, named PD4. Calculate the yield = (actual mass obtained / theoretical mass) × 100% = (15.5 mg / 55 mg) × 100% = 31%. Peptide dot PD4 powder can be uniformly and stably dispersed in water and ethanol. Its solution emits strong blue-green fluorescence under 365 nm ultraviolet light irradiation.

[0073] Fluorescence emission spectra at multiple excitation wavelengths (320 nm, 370 nm, 420 nm, 470 nm, and 520 nm) Figure 4 The results showed that the obtained peptide PD4 exhibited the strongest fluorescence emission peak at 425 nm under 320 nm excitation, and a second emission peak at about 530 nm.

[0074] TEM test results ( Figure 6 The results showed that the obtained peptide PD4 were nearly spherical nanoparticles with relatively uniform size and a particle size of approximately 6.86 nm. XRD test results ( Figure 7 The results showed that the obtained peptide PD4 did not exhibit graphite crystal diffraction peaks and did not form a carbonized structure; however, it showed obvious zinc peaks and relatively weak diffraction peaks that matched the zinc ion complex well, indicating that there is a certain zinc ion site structure in this peptide.

[0075] XPS full spectrum test results ( Figure 8 The results showed that the obtained peptide PD4 contained carbon, oxygen, nitrogen, and zinc; XPS high-resolution elemental spectroscopy results ( Figures 9-12 This indicates that the carbon structure is mainly composed of C=C single bonds and a small amount of C=C and CO / C=O, while the oxygen and nitrogen structures are mainly composed of OC and NC single bonds, respectively, and a small amount of O=C and N=C double bonds.

[0076] FTIR spectral test results ( Figure 13 The results show that the peptide site PD4 exhibits vibrational peaks for the OH, NH, CH, CC, C=C, CO, C=O, and CN bonds in the amino acid and polypeptide structures, and also shows a COC vibrational peak, indicating that dehydration condensation between carboxyl groups has occurred; in addition, the relatively broad OH and NH peaks indicate the presence of hydrogen bonds.

[0077] Furthermore, X-ray photoelectric spectroscopy (XPS) characterization analysis was performed on the above four peptides, and the results are as follows: Figure 9 As shown in the figure. 1) The overall spectrum results showed that all peptide spots contained carbon, oxygen and nitrogen; in addition, peptide spots PD1 and PD4 contained sulfur, and peptide spots PD2, PD3 and PD4 contained zinc; the element contents of the four peptide spots are listed in Table 1. It is worth noting that the zinc content in peptide spot PD4 is extremely low, indicating that acidic conditions are not conducive to the incorporation of zinc into peptide spots.

[0078] Table 14 shows the elemental content of 14 peptides.

[0079] 2) Fine elemental spectroscopy results showed that the carbon structures of peptides PD1, PD2, and PD4 were mainly C=C single bonds with small amounts of C=C and CO / C=O, while C=C double bonds accounted for more than half of peptide PD3. Nitrogen-containing carbon structures were not observed due to their small proportion. The N1s spectrum showed two peaks around 398 eV and 400 eV, representing pyridine N and graphitic N, respectively. The absence of graphitic N indicates that the synthesized peptide PD is a fluorescent peptide. In all peptides, oxygen and nitrogen were mainly O=C and NC single bonds, respectively, with small or very few O=C and N=C double bonds. Additionally, zinc in peptides PD2 and PD3 existed as divalent ions.

[0080] Experimental Example 1: pH Sensing Application of PD This method provides an application of fluorescent peptide dots in pH sensing, including the following steps: First, PD1, hydrochloric acid, and sodium hydroxide are prepared into buffer solutions with pH values ​​of 1, 3, 5, 7, 9, 11, and 13, respectively. 2 mL of a 0.1 mg / mL peptide dot solution is taken, and its fluorescence spectrum is measured, with the fluorescence intensity recorded as F0. 1.5 mL of a 0.1 mg / mL carbon dot solution is taken and added to 3 mL of buffer solutions with different pH values, respectively, and mixed thoroughly. After the solutions stabilize for 6 minutes, the fluorescence emission spectra are measured again at the same excitation wavelength and the same slit width, and the fluorescence intensity is recorded as F. The change in fluorescence intensity is recorded as ΔF. The relationship between ΔF / F and pH value is investigated, and linear fitting is performed. A series of aqueous solutions with pH values ​​from 1 to 13 are systematically tested. Acidic and alkaline environments significantly affect the fluorescence of PDs through protonation and deprotonation of the surface portion.

[0081] Figure 14 The PL response curve with pH is shown at an excitation wavelength of 320 nm. Figure 15 The fluorescence intensity showed a decreasing trend from pH 1 to 7. Based on this, linear fitting revealed that within the pH range of 1 to 6, the fluorescence intensity... Figure 16 As shown, the linear equation is y = -0.5034x + 2.9377, and the standard deviation R0 is... 2 The value was 0.9903. The results indicate that the prepared peptides have the potential to serve as effective pH sensors in acidic media.

[0082] Experimental Example 2: Effect of Heating Temperature on Fluorescent Peptide Dot Fluorescence Intensity Referring to the preparation method of fluorescent peptide (PD1) in Example 1, the heating temperature and time of the precursor solution were adjusted, and Control 1 (precursor solution heated at 40°C for 110 min) and Control 2 (precursor solution heated at 120°C for 10 min) were set up.

[0083] Figure 17 A comparison of the fluorescence intensity of fluorescent peptides prepared at different temperatures and times as a function of wavelength. The experimental results show that if the temperature is too low, even with prolonged heating time, the effect on increasing fluorescence intensity is poor.

[0084] Experiment Example 3: Effect of using only acid / base as a modulator on the fluorescence intensity of fluorescent peptides Referring to the preparation method of fluorescent peptide (PD3) in Example 3, the regulator was adjusted to use only acid (specifically hydrochloric acid) or only base (specifically sodium hydroxide).

[0085] Figure 18The graph shows the change in fluorescence intensity of fluorescent peptides prepared using only acid or base as regulators as a function of excitation wavelength. Figure 19 The fluorescence intensity curves of the fluorescent peptides prepared using only acid as a regulator are shown at different wavelengths. Figure 20 The figures show the fluorescence intensity curves of fluorescent peptides prepared using only alkali as a modulator at different wavelengths. The data indicate that the fluorescence intensity of the fluorescent peptides prepared using only acid as a modulator is higher than that prepared using only alkali as a modulator.

[0086] Experiment Example 4: Effect of no modulator or metal ion as modulator on fluorescence intensity of fluorescent peptide dots Referring to the preparation method of fluorescent peptide (PD3) in Example 3, the regulator was adjusted to use only metal ions (specifically zinc chloride) or no regulator at all.

[0087] Figure 21 The graph shows the fluorescence intensity of fluorescent peptides prepared without a regulator or with only metal ions as the regulator, as a function of excitation wavelength. Figure 22 The fluorescence intensity curves of the fluorescent peptides prepared using only metal ions as regulators at different wavelengths are shown. Figure 23 The figures show the fluorescence intensity changes of the fluorescent peptides prepared without a regulator at different wavelengths. The results indicate that fluorescent peptides with high fluorescence intensity can be obtained regardless of whether a regulator is added. Furthermore, adding metal ions as a regulator can further enhance the fluorescence intensity of the prepared fluorescent peptides.

[0088] The above descriptions are merely embodiments of the present invention, and common knowledge such as specific technical solutions and / or characteristics are not described in detail here. It should be noted that those skilled in the art can make various modifications and improvements without departing from the technical solutions of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent. The scope of protection claimed in this application should be determined by the content of its claims, and the specific embodiments described in the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing fluorescent peptide dots, characterized in that: Includes the following steps: (i) Precursor solution preparation stage: Amino acids and regulators are dispersed in a solvent to prepare a precursor solution; the amino acids are tyrosine, a combination of tyrosine and cysteine, or a combination of tyrosine and alanine; the regulator is concentrated hydrochloric acid; the solvent is one or more combinations of water and organic solvents; the mass concentration of amino acids in the precursor solution is 1.0~10 g / L, and the mass concentration of regulators in the precursor solution is 3.65~5.47 g / L; (ii) Synthesis stage: The precursor solution is heated to react and then cooled to room temperature to obtain the reaction solution; the temperature and time of the heating reaction are 50~150 ℃ and 1~100 min, respectively; (III) Post-processing stage: The reaction solution is purified and dried to obtain fluorescent peptide powder.

2. The method for preparing fluorescent peptide dots according to claim 1, characterized in that: The regulator also includes metal ions.

3. The method for preparing fluorescent peptide dots according to claim 1, characterized in that: The organic solvent has a boiling point above 60 °C.

4. The method for preparing fluorescent peptide dots according to claim 1, characterized in that: Drying was performed under vacuum at 60°C for 8 hours.

5. The method for preparing fluorescent peptide dots according to claim 1, characterized in that: The purification process involves dialysis using a 500-1000 Da dialysis bag for 24-48 hours, followed by collection of the dialysis solution from the dialysis bag to obtain the purified product solution.

6. A fluorescent peptide dot, characterized in that: The fluorescent peptide dots prepared by the method according to any one of claims 1 to 5 contain 64-72% C, 19-26% O, 2-9% N, and the remainder are trace elements.

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