Fluorescent peptide point and preparation method thereof

Abstract

The invention relates to the technical field of fluorescent nano materials, and discloses a fluorescent peptide point and a preparation method thereof, and the preparation method comprises the following steps: (1) a precursor solution preparation stage: dispersing amino acid and a regulator in a solvent to prepare a precursor solution; (2) a synthesis stage: heating and reacting the precursor solution, and cooling to room temperature to obtain a reaction solution; and (3) a post-treatment stage: purifying and drying the reaction solution to obtain fluorescent peptide point powder. Amino acid is selected as a precursor, amino acid molecules are subjected to dehydration condensation through heating to form amino acid polymer nanoparticles with conjugate luminescence characteristics, the fluorescent peptide point is synthesized through a one-step method, the process operation is simple, the heating temperature is low, the heating time is short, closed reaction is not needed, and the production difficulty of the fluorescent peptide point is effectively reduced; and a carbonization structure with potential safety hazards to a human body can be avoided, the device is suitable for food and medical detection, and the safety is further improved.

Description

Technical Field

[0001] The invention relates to the technical field of fluorescent nanomaterials, and in particular to a fluorescent peptide dot and a preparation method thereof. Background Art

[0002] Fluorescent nanomaterials mainly include rare earth nanomaterials, semiconductor quantum dots, graphene quantum dots and polymer carbon dots, which have very important applications in many fields such as optoelectronic devices, bioimaging, clinical diagnosis and food monitoring. However, the application of existing fluorescent nanomaterials in the field of food and medicine is mostly limited by their toxicity. The development of fluorescent materials with high safety and application in the field of food and medicine is one of the key issues to be solved in this field.

[0003] A method for synthesizing green fluorescent chiral carbon dots disclosed in the prior art CN103693633A comprises the following steps: the first step is to ultrasonically disperse a carbon precursor and an amino acid in deionized water to prepare a transparent aqueous solution or emulsion; the mass ratio of the carbon precursor to the amino acid is 200:1 to 5:1; the second step is to place the mixed solution obtained in the first step in a microwave heating device for microwave heating reaction to obtain a yellow or brownish yellow liquid. The third step is to dialyze the carbon quantum dot solution obtained in the second step with a dialysis bag with a molecular weight cutoff of 1,000 to 50,000 to remove the unreacted carbon precursor and amino acid, and obtain fluorescent carbon dots with a narrow particle size distribution without further purification. However, the fluorescent carbon quantum dots prepared by the prior art, even if they are basically non-toxic, are still potentially harmful to the human body due to the presence of a large number of "carbonized structures" such as aromatic compounds, polycyclic aromatic hydrocarbons (PAHs), and azo compounds.

[0004] In view of this, the development of a completely non-toxic, highly biocompatible fluorescent peptide dot and its preparation method can not only effectively make up for the shortcomings of existing technologies, but also further improve the safety of fluorescent materials. It is of great significance to produce safe and non-toxic fluorescent nanomaterials. Summary of the invention

[0005] The present invention aims to provide a fluorescent peptide dot and a preparation method thereof, so as to solve the technical problem that when fluorescent chiral carbon dots are prepared from carbon precursors in the prior art, there are many carbonized structures, which are potentially harmful to the human body and reduce the safety of their use.

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

[0007] The principles and advantages of this solution are:

[0008] 1. Compared with the fluorescent carbon quantum dots prepared by the prior art, which contain a large amount of "carbonized structures" and are not safe for consumption, this scheme can synthesize fluorescent peptide dots 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; and by limiting the reaction temperature and time, the peptide chain length of the synthesized fluorescent peptide dots is effectively limited, and the proportion of "carbonized structures" is effectively reduced, thereby improving the safety of the fluorescent peptide dots.

[0009] 2. This scheme can adjust the synthesis route and synthesis 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 and reducing the amount of fluorescent peptide points used in the experiment. While reducing costs, it also reduces possible environmental pollution and achieves the unity of economic and environmental benefits.

[0010] Preferably, as an improvement, the amino acids are one or more combinations of twenty natural amino acids.

[0011] Technical effect: This scheme adopts the above-mentioned setting, which is convenient for obtaining fluorescent peptide dots of different colors by using different amino acids or combinations as the reaction subjects, and adapting to different application requirements. The applicant has found through long-term experiments that, in theory, amino acids and their derivatives can be successfully used to prepare fluorescent peptide dots. Considering the safety of the prepared fluorescent peptide dots to the human body, reducing the carbon chain-modified natural amino acids can effectively reduce the carbonized structure content in the prepared fluorescent peptide dots, thereby further improving the safety of the use of fluorescent peptide dots.

[0012] Preferably, as an improvement, the regulator is one or more combinations of inorganic acids, bases, and metal ions. Preferably, the regulator is a combination of metal ions and inorganic acids or bases.

[0013] Technical effect: This scheme adopts the above-mentioned settings to facilitate providing a suitable environment for the synthesis of fluorescent peptide dots and accelerate the synthesis efficiency of fluorescent peptide dots. The applicant has found through long-term experiments that inorganic acids promote the synthesis of fluorescent peptide dots by promoting the protonation of amino acids and inhibiting side reactions; bases promote the synthesis of fluorescent peptide dots by promoting the formation of peptide bonds and preventing peptide chain degradation, and effectively stabilize fluorescent groups; metal ions catalyze peptide chain assembly through coordination, electron transfer and template effects, and stabilize fluorescent groups. In particular, when metal ions and inorganic acids / bases are combined, it can not only effectively accelerate the synthesis of fluorescent peptide dots, but also effectively stabilize fluorescent groups and increase fluorescence intensity. The applicant has found through long-term experiments that if an organic acid is used, it cannot act as a regulator, but will participate in the reaction as a substrate, change the structure of the peptide dot, and reduce the performance of the fluorescent peptide dot-producing material.

[0014] Preferably, as an improvement, the solvent is one or more combinations of water and an organic solvent.

[0015] Technical effect: This scheme adopts the above-mentioned settings to facilitate the rapid dissolution of amino acids, regulators and possible fluorescent groups synthesized by the reaction. Good solubility can ensure uniform mixing between reactants, avoid local excessive concentration or precipitation, and provide a suitable environment for peptide chain assembly, thereby improving the synthesis effect and yield of fluorescent peptide spots.

[0016] Preferably, as an improvement, the boiling point of the organic solvent is higher than 60°C.

[0017] Technical effect: This scheme adopts the above-mentioned setting, which is convenient for rapid removal after the reaction, while avoiding the situation that the boiling point is too low, which may cause rapid evaporation during the reaction and affect the controllability of the reaction.

[0018] Preferably, as an improvement, the mass concentration of amino acids in the precursor solution is 1.0-10 g / L.

[0019] Technical effect: This scheme adopts the above-mentioned setting, which facilitates the rapid protonation of amino acids and assembles them into peptide chains, thereby improving the synthesis efficiency of fluorescent peptide dots. The applicant has found through long-term experiments that if the amino acid concentration in the precursor solution is too low, the reaction efficiency will be reduced due to too few amino acids, thereby reducing the reaction efficiency; and if the amino acid concentration is too high, the reaction will be incomplete due to too many amino acids, resulting in a waste of raw materials.

[0020] Preferably, as an improvement, the mass concentration of the regulator contained in the precursor solution is 0 to 5.47 g / L.

[0021] Technical effect: This scheme adopts the above settings to effectively speed up the synthesis of fluorescent peptide dots and improve efficiency. The applicant has found through long-term experiments that if no regulator is added or the regulator content is too low, the fluorescence properties will remain unchanged due to insufficient functionalization; if the regulator content is too high, the fluorescence intensity will be too low due to changes in the fluorophore.

[0022] Preferably, as an improvement, the temperature and time of the heating reaction are 50-150° C. and 1-100 min, respectively.

[0023] Technical effect: This scheme adopts the above-mentioned setting to facilitate the full dehydration condensation reaction of amino acids into fluorescent peptide spots. The applicant has found through long-term experiments that if the reaction temperature is too low, the fluorescence intensity will decrease due to the low temperature; if the reaction temperature is too high, the reaction rate will be too fast and the reaction process will be difficult to control; if the reaction time is too short, the yield will be too low due to incomplete conversion of the precursor; and if the reaction time is too long, the dispersion and stability in the solution will be reduced due to agglomeration.

[0024] Preferably, as an improvement, the purification is performed by dialyzing for 24 to 48 hours using a 500 to 1000 Da dialysis bag, and the dialysate in the dialysis bag is taken to obtain a purified product solution.

[0025] Technical effect: This scheme adopts the above-mentioned setting, which is convenient for effectively removing the unreacted raw materials in the reaction solution, and the fluorescent peptide dot solution can be obtained, and then the high-purity fluorescent peptide dot can be obtained by drying. The applicant found through long-term experiments that the molecular weight of the fluorescent peptide dot obtained in this scheme is about 180-1000Da, and the fluorescent peptide dot can be completely intercepted by using a 500Da dialysis bag to prevent it from penetrating and reducing the product yield. If the molecular weight cutoff value of the dialysis bag is too small, the purity of the product will be reduced due to the interception of small molecule intermediates or amino acids, and the fluorescence intensity will be reduced; if the molecular weight cutoff value of the dialysis bag is too large, the product will flow out with the solvent because the molecular weight cutoff value of the dialysis bag is greater than the product molecular weight, reducing the product yield. If the dialysis time is too short, the purity of the carbon dots will be reduced due to the failure to fully remove small molecule impurities and unreacted precursor substances; if the dialysis time is too long, the carbon dots will be lost due to the escape of some carbon dots.

[0026] Preferably, as an improvement, the present solution also provides a fluorescent peptide dot, which is prepared according to the above method.

[0027] Preferably, as an improvement, the fluorescent peptide dots contain C64-72%, O19-26%, N2-9%, and the rest are trace elements.

[0028] Technical effect: This scheme adopts the above settings to facilitate pH sensing applications.

[0029] The technical advantages of this solution are summarized as follows:

[0030] 1. The present invention selects amino acids as precursors, and dehydrates and condenses amino acid molecules by heating to form amino acid polymer nanoparticles with conjugated luminescent properties, and synthesizes fluorescent peptide dots in one step. The process is simple to operate, the heating temperature is low, the time is short, and no closed reaction is required, which effectively reduces the difficulty of producing fluorescent peptide dots; and it can also avoid the formation of carbonized structures that pose a safety hazard to the human body, and is suitable for food and medical testing, further improving safety.

[0031] 2. The present 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 peptide dots with different emission wavelengths (colors), and the obtained fluorescent peptide dots have good dispersibility in aqueous solution and stable optical properties.

[0032] 3. This scheme uses inorganic acids and bases as regulators to adjust amino acids to form different amino acid ion forms, and uses metal ions as regulators to adjust amino acids to form different amino acid metal complexes, and finally adjusts the structure of the reaction product amino acid polymer, thereby adjusting the emission wavelength (color) of the fluorescent peptide dots. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the multi-wavelength excitation fluorescence emission spectrum of the fluorescent peptide dot (PD1, ie, S1 in the figure) obtained in Example 1 of the present invention.

[0034] Figure 2 This is the multi-wavelength excitation fluorescence emission spectrum of the fluorescent peptide dot (PD2, ie, S2 in the figure) obtained in Example 2 of the present invention.

[0035] Figure 3 This is the multi-wavelength excitation fluorescence emission spectrum of the fluorescent peptide dot (PD3, ie, S3 in the figure) obtained in Example 3 of the present invention.

[0036] Figure 4 This is the multi-wavelength excitation fluorescence emission spectrum of the fluorescent peptide dot (PD4, ie, S4 in the figure) obtained in Example 4 of the present invention.

[0037] Figure 5 Transmission electron microscope (TEM) images and particle size distributions of two types of fluorescent peptide dots (PD1 and PD2, corresponding to S1 and S2 in the figure, respectively) obtained in Examples 1 and 2 of the present invention.

[0038] Figure 6 Transmission electron microscope (TEM) images and particle size distribution of two kinds of fluorescent peptide dots (PD3 and PD4, corresponding to S3 and S4 in the figure, respectively) obtained in Examples 3 to 4 of the present invention.

[0039] Figure 7 These are the X-ray diffraction (XRD) patterns of the four fluorescent peptide dots obtained in Examples 1 to 4 of the present invention.

[0040] Figure 8 This is the full X-ray photoelectron spectrum (XPS) of the four fluorescent peptide dots obtained in Examples 1 to 4 of the present invention.

[0041] Fig. 9 This is the XPS high-resolution C1s spectra of the four fluorescent peptide dots obtained in Examples 1 to 4 of the present invention.

[0042] Fig.10 This is the XPS high-resolution O1s spectra of the four fluorescent peptide dots obtained in Examples 1 to 4 of the present invention.

[0043] Fig.11 This is the XPS high-resolution N1s spectra of the four fluorescent peptide dots obtained in Examples 1 to 4 of the present invention.

[0044] Fig.12 This is the XPS high-resolution Zn 1s spectra of the four fluorescent peptide dots obtained in Examples 1 to 4 of the present invention.

[0045] Fig.13 FTIR spectra of the four fluorescent peptide dots obtained in Examples 1 to 4 of the present invention.

[0046] Fig.14 This is the PL response curve of the fluorescent peptide dots with pH at an excitation wavelength of 320 nm in Experimental Example 1 of the present invention.

[0047] Fig.15 This is a graph showing the changing trend of the fluorescence intensity of the fluorescent peptide dots at pH 1 to 7 in Experimental Example 1 of the present invention.

[0048] Fig.16 This is the linear fitting curve of the fluorescent peptide dots in the pH range of 1 to 6 in Experimental Example 1 of the present invention.

[0049] Fig.17 This is a comparison chart of the fluorescence intensity of the fluorescent peptide dots prepared at different temperatures and times as the excitation wavelength changes.

[0050] Fig.18 The graph shows the change of fluorescence intensity of the fluorescent peptide dots prepared when only acid or base is used as a modulator versus excitation wavelength.

[0051] Fig.19 The fluorescence intensity change curve of the fluorescent peptide dots prepared at different wavelengths when only acid is used as a modulator.

[0052] Fig. 20 The fluorescence intensity change curve of the fluorescent peptide dots prepared at different wavelengths when only base is used as a regulator.

[0053] Fig.21 The graph shows the change of fluorescence intensity of the fluorescent peptide dots prepared without using a modulator or when the modulator is only a metal ion as a function of the excitation wavelength.

[0054] Fig. 22 The fluorescence intensity change curve of the fluorescent peptide dots prepared at different wavelengths when only metal ions are used as regulators.

[0055] Fig.23 This is the fluorescence intensity change curve of the fluorescent peptide dots prepared at different wavelengths when no modulator is used. DETAILED DESCRIPTION

[0056] The present invention is further described in detail below in conjunction with examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used can be obtained from commercial sources.

[0057] Program Overview

[0058] The present scheme provides a fluorescent peptide dot, which is obtained by a precursor solution containing an amino acid mass concentration of 1.0 to 10 g / L, a regulator mass concentration of 0 to 5.47 g / L, and a solvent as the remainder, and is subjected to a temperature reaction, cooling, purification, and drying.

[0059] Among them, the amino acid is 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, that is, 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°C. The temperature and time of the heating reaction are 50-150°C and 1-100 min, respectively. During purification, a 500-1000 dialysis bag is used for dialysis for 24-48 hours, and the dialysate in the dialysis bag is taken to obtain a purified fluorescent peptide dot solution.

[0060] This scheme also provides a method for preparing fluorescent peptide dots, comprising the following steps:

[0061] (i) a precursor solution preparation stage, wherein any one or more combinations of amino acids and a regulator are dispersed in a solvent to prepare a precursor solution;

[0062] (ii) in the synthesis stage, the precursor solution is heated to 50-150° C. for reaction for 1-100 min and then cooled to room temperature to obtain a reaction solution;

[0063] (iii) Post-processing stage: the reaction solution is purified and dried to obtain fluorescent peptide dot powder.

[0064] The purification is performed by dialyzing for 24 to 48 hours using a 500 to 1000 Da dialysis bag, and the dialysate in the dialysis bag is taken to obtain a purified product solution.

[0065] Drying was performed at 60°C in vacuum for 8 h.

[0066] Example 1

[0067] Preparation method of fluorescent peptide dots: weigh 25 mg of tyrosine solid and 25 mg of cysteine ​​solid in a beaker, add 10 mL of ethylene glycol and 300 μL of concentrated hydrochloric acid aqueous solution (37%) to dissolve, prepare a precursor solution (total amino acid concentration is 5 g / L, hydrochloric acid concentration is 5.47 g / L, and the solution pH is about 0.7), move it into a microwave oven and heat it at a temperature of 120° C. for 5 min with medium heat, stop heating, wait for the solution to cool to room temperature, dialyze it using a 3000Da dialysis bag for 1 day, take the dialysate in the dialysis bag to obtain a purified product solution, and finally freeze-dry to obtain a pure fluorescent peptide dot powder, named PD1, and calculate the yield (PD1) = (actual mass / theoretical mass) × 100% = (18 mg / 50 mg) × 100% = 36%; the peptide dot PD1 powder can be evenly and stably dispersed in water and ethanol, and its solution emits strong green fluorescence under irradiation of a 365nm ultraviolet lamp.

[0068] Fluorescence emission spectrum test results of multiple excitation wavelengths (320nm, 370nm, 420nm, 470nm and 520nm) Figure 1 ) showed that the obtained peptide dot PD1 exhibited the strongest fluorescence emission peak at 435nm under 320nm excitation, and a second emission peak appeared at about 525nm.

[0069] TEM test results ( Figure 5 ) showed that the obtained peptide dots PD1 were nearly spherical nanoparticles with uniform size, and the particle size D50 was about 6.42 nm. XRD test results ( Figure 7 ) showed that the obtained peptide dot PD1 did not show the crystal diffraction peak of graphite and did not form a carbonized structure.

[0070] XPS full spectrum test results ( Figure 8 ) showed that the obtained peptide point PD1 contained carbon, oxygen, nitrogen and sulfur elements; the XPS high-resolution element spectrum test results ( Figures 9 to 12 ) shows that the carbon structure is mainly CC single bond and a small amount of C=C, CO / C=O, and the oxygen and nitrogen structures are mainly OC and NC single bonds, respectively, and a small amount of O=C and N=C double bonds. FTIR spectrum test results ( Fig.13 ) showed that the peptide point PD1 had vibration peaks of OH, NH, CH, CC, C=C, CO, C=O and CN bonds in the amino acid and polypeptide structure, and a COC vibration peak also appeared, indicating that dehydration condensation occurred between carboxyl groups; in addition, the broader OH and NH peaks indicated the existence of hydrogen bonds.

[0071] Example 2

[0072] Preparation method of fluorescent peptide dots: 50 mg of tyrosine solid and 5.0 mg of zinc chloride solid were weighed in a beaker, 10 mL of deionized water and 200 μL of concentrated hydrochloric acid aqueous solution (37%) were added to dissolve, and a precursor solution (total amino acid concentration was 5.0 g / L, zinc ion concentration was 0.5 g / L, hydrochloric acid concentration was 3.65 g / L, and the solution pH was about 1.0) was prepared, and the precursor solution was transferred into an electric constant temperature blast drying oven and heated at 70°C for 60 min, and the heating was stopped and the solution was cooled to room temperature, and the reaction mixture was dried at a speed of 160 00 rpm, centrifuge at a centrifugal speed to remove the upper liquid, disperse with water, repeat centrifugation 3 times, and then vacuum dry the obtained solid at 60°C to finally obtain pure fluorescent peptide dot powder, named PD2, and calculate the yield (PD1) = (actual mass / theoretical mass) × 100% = (17 mg / 55 mg) × 100% = 34%; the peptide dot PD2 powder can be uniformly and stably dispersed in water and ethanol, and its solution emits strong blue fluorescence under 365nm ultraviolet light.

[0073] Fluorescence emission spectrum test results of multiple excitation wavelengths (320nm, 370nm, 420nm, 470nm and 520nm) Figure 2 ) showed that the obtained peptide dot PD2 exhibited the strongest fluorescence emission peak at 455nm under 370nm excitation.

[0074] TEM test results ( Figure 5 ) showed that the obtained peptide dots PD2 were nearly spherical nanoparticles with a relatively uniform size and a particle size of about 5.96 nm. XRD test results ( Figure 7 ) showed that the obtained peptide dot PD2 did not show the crystal diffraction peak of graphite and did not form a carbonized structure; however, an obvious zinc peak and a weaker diffraction peak that was more matched with the zinc ion complex appeared, indicating that there was a certain zinc ion coordination structure in the peptide dot.

[0075] XPS full spectrum test results ( Figure 8 ) showed that the obtained peptide dot PD2 contained carbon, oxygen, nitrogen and zinc elements; the XPS high-resolution element spectrum test results ( Figures 9 to 12 ) shows that the carbon structure is mainly CC single bond and a small amount of C=C, CO / C=O, and the oxygen and nitrogen structures are mainly OC and NC single bonds, respectively, and a small amount of O=C and N=C double bonds. FTIR spectrum test results ( Fig.13 ) showed that the peptide point PD1 had vibration peaks of OH, NH, CH, CC, C=C, CO, C=O and CN bonds in the amino acid and polypeptide structure, and a COC vibration peak also appeared, indicating that dehydration condensation occurred between carboxyl groups; in addition, the broader OH and NH peaks indicated the existence of hydrogen bonds.

[0076] Example 3

[0077] 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 in a beaker, add 10 mL of deionized water and 200 μL of sodium hydroxide aqueous solution (200 g / L) to dissolve, prepare a precursor solution (total amino acid concentration is 5.0 g / L, zinc ion concentration is 0.5 g / L, sodium hydroxide concentration is 4 g / L, and the pH value of the solution is about 13.5), move it into an electric constant temperature blast drying oven and heat it at 90°C for 30 minutes, stop heating, wait for the solution to cool to room temperature, and use The mixture was centrifuged at a speed of 16000 rpm, the upper liquid was removed, and the mixture was dispersed with water. The mixture was centrifuged three times, and the solid was vacuum dried at 60°C to finally obtain a pure fluorescent peptide dot powder, named PD3. The yield was calculated as (PD1) = (actual mass / theoretical mass) × 100% = (18.5 mg / 55 mg) × 100% = 37%. The peptide dot PD3 powder can be uniformly and stably dispersed in water and ethanol, and its solution emits a strong green fluorescence under 365nm ultraviolet light.

[0078] Fluorescence emission spectrum test results of multiple excitation wavelengths (320nm, 370nm, 420nm, 470nm and 520nm) Figure 3 ) showed that the obtained peptide dot PD3 exhibited the strongest fluorescence emission peak at 455nm under 370nm excitation.

[0079] TEM test results ( Figure 6 ) showed that the obtained peptide dots PD3 were nearly spherical nanoparticles with a relatively uniform size and a particle size of about 7.06 nm. XRD test results ( Figure 7 ) showed that the obtained peptide dot PD2 did not show the crystal diffraction peak of graphite and did not form a carbonized structure; however, an obvious zinc peak and a weaker diffraction peak that was more matched with the zinc ion complex appeared, indicating that there was a certain zinc ion coordination structure in the peptide dot.

[0080] XPS full spectrum test results ( Figure 8 ) showed that the obtained peptide dot PD2 contained carbon, oxygen, nitrogen and zinc elements; the XPS high-resolution element spectrum test results ( Figures 9 to 12 ) shows that the carbon structure is mainly CC single bond and a small amount of C=C, CO / C=O, and the oxygen and nitrogen structures are mainly OC and NC single bonds, respectively, and a small amount of O=C and N=C double bonds. FTIR spectrum test results ( Fig.13) showed that the peptide point PD1 had vibration peaks of OH, NH, CH, CC, C=C, CO, C=O and CN bonds in the amino acid and polypeptide structure, and a COC vibration peak also appeared, indicating that dehydration condensation occurred between carboxyl groups; in addition, the broader OH and NH peaks indicated the existence of hydrogen bonds.

[0081] Example 4

[0082] 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, prepare a precursor solution (total amino acid concentration is 5.0 g / L, hydrochloric acid concentration is 5.47 g / L, and the solution pH is about 0.7), transfer to a microwave oven and heat at high temperature at 150°C for 1 min, stop heating, wait for the solution to cool to room temperature, and use Dialysis was performed with a 5000Da dialysis bag for 2 days, and the dialysate in the dialysis bag was taken to obtain a purified product solution, which was finally freeze-dried to obtain a pure fluorescent peptide dot powder, named PD4, with a calculated yield (PD1) = (actual mass / theoretical mass) × 100% = (15.5 mg / 55 mg) × 100% = 31%; the peptide dot PD4 powder can be uniformly and stably dispersed in water and ethanol, and its solution emits a strong blue-green fluorescence under 365nm ultraviolet light.

[0083] Fluorescence emission spectrum test results of multiple excitation wavelengths (320nm, 370nm, 420nm, 470nm and 520nm) Figure 4 ) showed that the obtained peptide dot PD3 exhibited the strongest fluorescence emission peak at 425nm under 320nm excitation, and a second emission peak appeared at about 530nm.

[0084] TEM test results ( Figure 6 ) showed that the obtained peptide dots PD4 were nearly spherical nanoparticles with a relatively uniform size and a particle size of about 6.86 nm. XRD test results ( Figure 7 ) showed that the obtained peptide dot PD4 did not show the crystal diffraction peak of graphite and did not form a carbonized structure; but there was an obvious zinc peak and a weaker diffraction peak that was more matched with the zinc ion complex, indicating that there was a certain zinc ion site structure in the peptide dot.

[0085] XPS full spectrum test results ( Figure 8 ) showed that the obtained peptide dot PD4 contained carbon, oxygen, nitrogen and zinc elements; the XPS high-resolution element spectrum test results ( Figures 9 to 12 ) shows that the carbon structure is mainly CC single bond with a small amount of C=C and CO / C=O, and the oxygen and nitrogen structures are mainly OC and NC single bonds respectively, with a small amount of O=C and N=C double bonds.

[0086] FTIR spectrum test results ( Fig.13 ) showed that the peptide point PD4 showed vibration peaks of OH, NH, CH, CC, C=C, CO, C=O and CN bonds in the amino acid and polypeptide structure, and a COC vibration peak also appeared, indicating that dehydration condensation occurred between carboxyl groups; in addition, the broader OH and NH peaks indicated the existence of hydrogen bonds.

[0087] In addition, the above four peptide sites were characterized by X-ray photoelectrometry (XPS) analysis, and the results were as follows: Fig. 9 As shown. It can be seen from the figure:

[0088] 1) The overall spectrum results showed that all peptide spots contained carbon, oxygen and nitrogen elements. In addition, peptide spots PD1 and PD4 contained sulfur elements, and peptide spots PD2, PD3 and PD4 contained zinc elements. 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 integration of zinc into the peptide spots.

[0089] Table 1 Elements content of four peptide spots

[0090]

[0091] 2) The results of the fine spectrum of each element show that the carbon structure of peptide points PD1, PD2 and PD4 is mainly CC single bonds and a small amount of C=C, CO / C=O, while the C=C double bonds in peptide point PD3 account for more than half, and the nitrogen-containing carbon structure is not presented because the proportion is too small; the N1s spectrum has two peaks around 398ev and 400ev, which are pyridine N and graphite N respectively. Since there is no graphite N, it means that the synthesized fluorescent peptide point PD. The oxygen and nitrogen structures in all peptide points are mainly OC and NC single bonds, respectively, and there are a small amount or very little O=C and N=C double bonds; in addition, the zinc element in peptide points PD2 and PD3 exists as a divalent ion.

[0092] Experimental Example 1: pH Sensing Application of PD

[0093] This scheme provides an application of fluorescent peptide dots in pH sensing, including the following contents: 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. Take 2 mL of peptide dot solution with a concentration of 0.1 mg / mL, measure its fluorescence spectrum and record the fluorescence intensity as F0. Take 1.5 mL of carbon dot solution with the same concentration of 0.1 mg / mL and add 3 mL of buffer solutions with different pH values ​​to mix thoroughly. After the solution is stable for 6 minutes, the fluorescence emission spectrum is measured at the same excitation wavelength and the same slit width and the fluorescence intensity is recorded as F. The change value of fluorescence intensity is recorded as ΔF, and the relationship between the value of ΔF / F and pH value is found, and a linear fit is performed. The fluorescence intensity of a series of aqueous solutions was systematically detected with a pH range of 1 to 13. Acidic and alkaline environments have a significant effect on the fluorescence of PDs through the protonation and deprotonation of the surface part.

[0094] Fig.14 PL response curve with pH at an excitation wavelength of 320 nm. Fig.15 The fluorescence intensity changes in pH 1-7 showed a downward trend. On this basis, through linear fitting, it was found that the pH value was in the range of 1 to 6. Fig.16 As shown in Figure 2, the linear equation is y = -0.5034x + 2.9377, and the standard deviation R 2 is 0.9903. The results show that the prepared peptide dots have the potential to be effective pH sensors in acidic media. We found through linear fitting that the pH value is in the range of 1 to 6, as shown in Figure 2. Fig.16 As shown, the linear equation is y = -0.5034x + 2.9377, and the standard deviation R2 is 0.9903. The results indicate that the prepared peptide dots have the potential to serve as effective pH sensors in acidic media.

[0095] Experimental Example 2: Effect of heating temperature on the fluorescence intensity of fluorescent peptide dots

[0096] Referring to the preparation method of fluorescent peptide dots (PD1) in Example 1, the heating temperature and time of the precursor solution were adjusted to set control 1 (precursor solution heated at 40°C for 110 min) and control 2 (precursor solution heated at 120°C for 10 min).

[0097] Fig.17 The comparison chart of the fluorescence intensity of the fluorescent peptide dots prepared at different temperatures and times versus wavelength. The experimental results show that if the temperature is too low, even if the heating time is extended, the effect on the increase of fluorescence intensity is poor.

[0098] Experimental Example 3: Effect of using only acid / base as a modulator on the fluorescence intensity of fluorescent peptide dots

[0099] Referring to the preparation method of the fluorescent peptide dot (PD3) in Example 3, the regulator is adjusted to use only acid (specifically hydrochloric acid) or only base (specifically sodium hydroxide).

[0100] Fig.18 The graph shows the change of fluorescence intensity of the fluorescent peptide dots prepared when only acid or base is used as a modulator versus excitation wavelength. Fig.19 The fluorescence intensity change curve of the fluorescent peptide dots prepared at different wavelengths when only acid is used as a modulator. Fig. 20 The fluorescence intensity change curves of the fluorescent peptide dots prepared when only alkali is used as a modulator at different wavelengths are shown in Figure 2. The data show that the fluorescence intensity of the fluorescent peptide dots produced when only alkali is used as a modulator is higher than that of the fluorescent peptide dots produced when only alkali is used as a modulator.

[0101] Experimental Example 4: Effect of No Modulator or Metal Ions as Modulator on the Fluorescence Intensity of Fluorescent Peptide Dots

[0102] Referring to the preparation method of the fluorescent peptide dot (PD3) in Example 3, the modulator was adjusted to use only metal ions (specifically zinc chloride) or no modulator.

[0103] Fig.21 The graph shows the change of fluorescence intensity of the fluorescent peptide dots prepared without using a modulator or when the modulator is only a metal ion as a function of the excitation wavelength. Fig. 22 The fluorescence intensity change curve of the fluorescent peptide dots prepared at different wavelengths when only metal ions are used as regulators. Fig.23 The curves of fluorescence intensity changes of the prepared fluorescent peptide dots at different wavelengths without using a modifier are shown in Figure 2. The results show that fluorescent peptide dots with high fluorescence intensity can be produced whether or not a modifier is added. Among them, adding metal ions as a modifier can further improve the fluorescence intensity of the prepared fluorescent peptide dots.

[0104] The above is only an embodiment of the present invention, and the common knowledge such as the known specific technical solutions and / or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several modifications and improvements can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records 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: The method comprises: (i) a precursor solution preparation stage, in which amino acids and regulators are dispersed in a solvent to prepare a precursor solution; (ii) a synthesis stage, in which the precursor solution is heated for reaction and then cooled to room temperature to obtain a reaction solution; and (iii) a post-treatment stage, in which the reaction solution is purified and dried to obtain fluorescent peptide dot powder.

2. The method for preparing a fluorescent peptide dot according to claim 1, characterized in that: The amino acids are one or more combinations of twenty natural amino acids.

3. The method for preparing a fluorescent peptide dot according to claim 2, characterized in that: The regulator is one or more combinations of inorganic acids, bases, and metal ions.

4. The method for preparing a fluorescent peptide dot according to claim 1, characterized in that: The solvent is one or more combinations of water and organic solvents.

5. The method for preparing a fluorescent peptide dot according to claim 4, characterized in that: The boiling point of the organic solvent is higher than 60°C.

6. The method for preparing a fluorescent peptide dot according to claim 1, characterized in that: The mass concentration of amino acids in the precursor solution is 1.0-10 g / L.

7. The method for preparing a fluorescent peptide dot according to claim 1, characterized in that: The mass concentration of the regulator contained in the precursor solution is 0-5.47 g / L.

8. The method for preparing a fluorescent peptide dot according to claim 1, characterized in that: The temperature and time of the heating reaction are 50-150° C. and 1-100 min respectively.

9. The method for preparing a fluorescent peptide dot according to claim 8, characterized in that: The purification is performed by using a 500-1000 Da dialysis bag for dialysis for 24-48 hours, and the dialysate in the dialysis bag is taken to obtain a purified product solution.

10. A fluorescent peptide dot, characterized in that: The fluorescent peptide dots prepared according to the methods of claims 1 to 9 contain 64 to 72% C, 19 to 26% O, 2 to 9% N, and the rest are trace elements.

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