CN113150770A - Molecular assembly fluorescent probe and preparation method and application thereof - Google Patents
Molecular assembly fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN113150770A CN113150770A CN202110206170.5A CN202110206170A CN113150770A CN 113150770 A CN113150770 A CN 113150770A CN 202110206170 A CN202110206170 A CN 202110206170A CN 113150770 A CN113150770 A CN 113150770A
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Abstract
The invention relates to a molecular assembly fluorescent probe and a preparation method and application thereof. The fluorescent probe is formed by molecular assembly of a hoechst fluorescent dye 33258 and supramolecular cucurbituril. The preparation method comprises the following steps: the polycondensation reaction of glycoluril and paraformaldehyde under acidic condition to prepare CB [ n ], and then separating and purifying to obtain the corresponding pure cucurbituril products by using their different solubilities in water and hydrochloric acids with different concentrations. Dissolving H33258 in water, adding CB [ n ] to make the molar ratio of H33258 to CB [ n ] be 1:1-2, stirring, rotary evaporating to concentrate solution, then freeze-drying so as to obtain the invented product. The fluorescent probe can rapidly and accurately identify Lys3 and P-Lys3, provides a brand-new detection means with high sensitivity and high specificity for methylated lysine and methylated peptide, and has great significance for deeply researching the regulation and control effect of protein methylation modification in the physiological process.
Description
Technical Field
The invention relates to the technical field of biochemistry, in particular to a molecular assembly fluorescent probe and a preparation method and application thereof.
Background
Lysine methylation is the transfer of one to three methyl groups from S-adenosylmethionine (SAM) to lysine epsilon-amine side chains catalyzed by lysine methyltransferases (KMTs) to form monomethylation (Me1), dimethylation (Me2), trimethylation (Me3), one of the most common post-translational modifications of proteins. Lysine methylation modification plays an important role in the regulation of many physiological processes. The research shows that: lysine methylation modifications in proteins can regulate intramolecular or intermolecular interactions of the target protein, affecting their affinity for RNA, and thus, affecting a variety of cellular processes, such as transcriptional regulation, cellular localization, ribosome assembly, RNA processing, maturation of heterogeneous riboriboriboribosomal proteins (hnRNPs), protein-protein interactions, accurate translation, nuclear trafficking, protein-nucleic acid trafficking and metabolism, intracellular signaling, and the like.
To date, proteomics research for methylation modification is a new field. The separation and enrichment of methylated peptide fragments is the key to the intensive research on methylated proteome. Affinity methods based on methylated lysine binding domains have also been introduced into the study of methylated proteins, primarily for the isolation of lysine methylated proteins. For example: the Gozani group isolated lysine-methylated proteins using proteins containing methylated lysine binding domains as enrichment tools. The triple malignant brain tumor domain (3 × MBT) of the L3MBTLI protein contains a binding pocket that recognizes methylated lysine, and its specificity is not affected by surrounding amino acid residues. However, the binding domains have weak acting force on methylated peptide fragments, so that the binding domains cannot be effectively applied to enrichment of methylated peptide fragments, and even if methylated proteins are separated, the complexity of samples after enzymolysis is high, and efficient identification of methylated sites is difficult to realize.
The future research focuses include the development of a methylated peptide fragment separation method with higher selectivity, high reliability identification of methylation modification sites in tissue samples and analysis of absolute occupancy of the methylation modification sites, and breakthrough of the research directions also provides important technical support for the scale analysis of methylation modification groups and the explanation of physiological processes in which methylation participates in regulation. At present, identification of methylation sites at the peptide fragment level is still difficult, mainly because introduction of methyl groups in a peptide chain does not significantly change net charge or isoelectric point of amino acid residues except for increasing steric hindrance of methylated amino acid residues and reducing hydrogen bonding force of the amino acid residues, which makes identification of methylated peptide fragments difficult and greatly affects analysis sensitivity. In conclusion, the development of a detection means with high sensitivity and high specificity aiming at methylated lysine and methylated peptide is significant for the deep research of the regulation and control effect of protein methylation modification in the physiological process.
Disclosure of Invention
The invention aims to provide a molecular assembly fluorescent probe and a preparation method and application thereof. The concentration of the molecular assembly fluorescent probe in the lower concentration range of 3.0-10.0 mu M can distinguish trimethyl substituted Fmoc protected lysine (Lys3) from other methylated or unmethylated lysine, and the fluorescent probe can be further used for identifying a peptide segment (P-Lys3) comprising trimethyl substituted Fmoc protected lysine.
The purpose of the invention is realized by adopting the following scheme:
a molecular assembly fluorescent probe has the following structural formula:
the molecular assembly fluorescent probe is assembled by H33258 and CB [ n ] through non-covalent bond interaction, wherein CB [ n ] is any one of three cucurbiturils, namely CB [6], CB [7] or CB [8 ].
The preparation method of the molecular assembly fluorescent probe comprises the following steps: dissolving H33258 in water, adding CB [ n ] to make the molar ratio of H33258 to CB [ n ] be 1:1-2, stirring for 1-4H, performing rotary evaporation to concentrate the solution, and then performing freeze drying to obtain the molecular assembly fluorescent probe.
The molecular assembly fluorescent probe is applied to identifying trimethyl substituted Fmoc protected lysine.
Preferably, the method comprises the following steps:
1) preparing three parts of fluorescent probe aqueous solution with the concentration of 3.0-10 mu M, measuring the fluorescence spectrum emitted by the three parts under the condition of the excitation wavelength of 361nm, and recording the maximum emitted fluorescence intensity I of each part of fluorescent probe aqueous solution at 400-650nm0A，I0B，I0C；
2) Adding Fmoc-protected lysine, dimethyl-substituted Fmoc-protected lysine and trimethyl-substituted Fmoc-protected lysine into the three solutions respectively and mixing uniformly, wherein the molar ratio of each type of lysine to the fluorescent probe is 0.3-0.5, measuring the fluorescence spectra emitted by the three solutions respectively under the condition of an excitation wavelength of 361nm, and recording the fluorescence intensity I at 400-650nm1A，I1B，I1C；
3) Repeating the step 2) n times to ensure that the amount of each type of lysine added dropwise is the same as that in the step 2), and respectively obtaining the Fmoc-protected lysine with the fluorescence intensity of I2ATo InA(ii) a Fluorescence intensity of dimethyl-substituted Fmoc-protected lysine is I2BTo InB(ii) a (ii) a Fluorescence intensity of trimethyl-substituted Fmoc-protected lysine is I2CTo InC；
4) Respectively with fluorescence intensity I0X,…InXThe ratio of the number of moles of the corresponding lysine species to the number of moles of the fluorescent probe is plotted on the abscissa as I0X,…InXPerforming nonlinear fitting for the ordinate to obtain fluorescence responsiveness curves of different types of lysine, wherein X is A, B or C; if InX/I0XWhen the content is less than or equal to 0.55, the solution contains trimethyl substituted Fmoc protected lysine.
Preferably, Tris-HCl buffer is added to the aqueous solution of the molecular assembly fluorescent probe in step 1) to adjust the pH to 2-7.
Preferably, the Fmoc-protected lysine is Lys, the dimethyl-substituted Fmoc-protected lysine is Lys2, and the trimethyl-substituted Fmoc-protected lysine is Lys3, which have the following structural formulas:
the molecular assembly fluorescent probe is applied to identifying peptides containing trimethyl substituted Fmoc protected lysine.
Preferably, the method comprises the following steps:
1) preparing four molecular assembly fluorescent probe aqueous solutions with the concentration of 3.3-10 mu M and the pH value of 2-7, measuring the fluorescence spectrum emitted by the molecular assembly fluorescent probe aqueous solutions under the condition of the excitation wavelength of 361nm, and recording the maximum emission fluorescence intensity I of each fluorescent probe aqueous solution at 400-650nm0a，I0b，I0c，I0d；
2) Providing four peptides P-Lys, P-Lys1, P-Lys2 and P-Lys3, adding the four peptides into the four solutions respectively and mixing uniformly, wherein the molar ratio of each type of peptide to the fluorescent probe is 0.3-0.5, measuring the fluorescence spectrum emitted by the four solutions respectively under the condition of an excitation wavelength of 361nm, and recording the fluorescence intensity I at 400-650nm1a，I1b，I1c，I1d；
3) Repeating the step 2) n times to ensure that the amount of each type of lysine added dropwise is the same as that in the step 2), ensuring that the final concentration of each type of peptide is between 45 and 75 mu M, and respectively obtaining the fluorescence intensity of P-Lys as I2aTo Ina(ii) a Fluorescence intensity of P-Lys1 is I2bTo Inb(ii) a (ii) a Fluorescence intensity of P-Lys2 is I2cTo Inc(ii) a Fluorescence intensity of P-Lys3 is I2dTo Ind；
4) If InY/I0YWhen the content is less than or equal to 0.55, the solution contains P-Lys3, wherein Y is a, b, c or d.
Preferably, the peptide sequences of the P-Lys, P-Lys1, P-Lys2 and P-Lys3 are as follows:
preferably, n is 15-25.
The invention has the beneficial effects that:
1. the fluorescent probe prepared by the invention has the advantages of high detection accuracy and high detection speed when detecting methylated and non-methylated lysine, and is very suitable for quickly identifying Lys3 in complex lysine methylation with different degrees;
2. when the fluorescent probe prepared by the invention is used, not only single Lys3 can be detected, but also the single Lys3 can be raised into a peptide fragment to identify trimethyl substituted Fmoc protected lysine (P-Lys3) in the peptide fragment.
3. The molecular assembly fluorescent probe prepared by the invention is designed from CB [ n ]]Good interaction with Lys3, via CB [ n ]]Hydrophobic cavity and carbonyl Port of (2) with the C-CH terminal Lys33The hydrophobic interaction and the ion-dipole interaction enter the cavity, so that the phenol part group of H33258 is extruded out of the cavity, the stoichiometric ratio of the molecular assembly type probe is changed, and the fluorescence property of the fluorescent probe is changed.
In conclusion, the invention avoids the problems that the introduction of methyl in a peptide chain increases the steric hindrance of methylated amino acid residues and reduces the hydrogen bonding force of the amino acid residues, and changes the stoichiometric ratio of a molecular assembly probe based on the good interaction between CB [ n ] and Lys3 so as to change the fluorescence property of a fluorescent probe and realize the accurate identification of Lys3 or P-Lys 3. The invention provides a brand-new detection means of methylated lysine and methylated peptide with high sensitivity and high specificity, and has great significance for deeply researching the regulation and control effect of protein methylation modification in the physiological process.
Drawings
FIG. 1 shows the chemical structure of Herster fluorochrome 33258 (H33258).
FIG. 2 is a chemical structural diagram of cucurbituril (CB [ n ]).
FIG. 3 is a chemical structural diagram of Lys, Lys2 and Lys 3.
FIG. 4 shows peptide sequences of P-Lys, P-Lys1, P-Lys2 and P-Lys 3.
FIG. 5 is a graph showing the fluorescence intensity of Fmoc-protected lysine (Lys3) added to a fluorescent probe at different molar ratios.
FIG. 6 is a graph showing the fluorescence intensity of Fmoc-protected lysine (Lys) added to a fluorescent probe at different molar ratios.
FIG. 7 is a graph showing the trend of the fluorescence intensity at 493nm of the fluorescence spectrum of a fluorescent probe added with different molar ratios of three types of lysine (including Lys, Lys2 and Lys3) along with the addition of the three types of lysine.
FIG. 8 is a graph showing the fluorescence intensity of P-Lys3 with different molar ratios added to the fluorescent probe.
FIG. 9 is a graph showing fluorescence intensity curves of fluorescent probes added with different molar ratios of P-Lys.
FIG. 10 is a schematic diagram of the "ON/OFF" state definition column.
Detailed Description
In order to make the contents, technical solutions and advantages of the present invention more apparent, the present invention is further described below with reference to specific examples and figures, which are only used for illustrating the present invention, and the present invention is not limited to the following examples.
Materials and apparatus used in the examples
H33258(1g, 98%), Tris reagent (100g, 97%) were purchased from Oakyka technologies, Beijing. Reagents such as urea (500g, 98%), glyoxal (500ml, 40%), paraformaldehyde (500g, 98%), concentrated sulfuric acid (500ml, 98%) and concentrated hydrochloric acid (500ml, 98%) required for synthesizing cucurbituril are purchased from national institute of medicine chemical agents, ltd. Lys, Lys2 and Lys3 are commercially available from Gill Biochemical (Shanghai) Co., Ltd., and have a purity of 98% or more. P-Lys, P-Lys1, P-Lys2 and P-Lys3 are purchased from Shanghai Qiaoqiao Biotechnology, and the purity is more than 98%. The water used in the present invention was prepared by a Milli-Q ultrapure water meter (resistivity: 18.2 M.OMEGA.. multidot.cm). Fluorescence spectra data were collected from a PerkinElmer LS55 fluorescence spectrophotometer.
Example 1
Preparation method of molecular assembly fluorescent probe
First, cucurbituril CB [ n ]:
80.0g of urea was weighed, dissolved in 250mL of deionized water with stirring, and the pH of the solution was adjusted to 1-2 with concentrated sulfuric acid. Under stirring, 62ml of glyoxal (40% aqueous solution) was slowly dropped into the solution with a dropping funnel at a dropping rate of about 3 seconds, the temperature of the oil bath was controlled to be lower than 70 ℃, after the dropping, the temperature was raised to about 75 ℃, and the reaction was completed for 6 hours. The reaction solution was filtered under reduced pressure, washed three times with water and acetone in this order, and vacuum-dried at 70 ℃ for 24 hours to obtain 55.8g of glycosidurea. 50.0g of the glycosidic urea was weighed out and dissolved in 150mL of concentrated hydrochloric acid. Stirring at normal temperature, placing in an ultrasonic dispersion machine, taking out after 10 minutes to ensure that the glycosidurea is completely dissolved, then adding 23.0g of paraformaldehyde, raising the temperature to 100 ℃ after the system becomes yellow gel-like substance, and refluxing by using a spherical condenser tube. After 5 minutes, the system turned from a gel-like state back to a fluid liquid. The reaction was carried out at this temperature for 10h to give a brownish yellow oily solution. The solution was concentrated to about 40mL by reduced pressure rotary evaporation, poured slowly into 200mL of acetone while hot, and the acetone was stirred vigorously during the pouring to prevent the precipitate from being poorly dispersed. Filtering under reduced pressure, washing out precipitate, washing with acetone three times, vacuum drying at 50 deg.C for 24 hr to obtain light yellow solid 47.8g, which is mixed cucurbituril homolog (CB [ n ]), including cucurbituril (CB [6]), cucurbituril (CB [7]), cucurbituril (CB [8]), and small amount of cucurbituril (5) urea (CB [5 ]). The cucurbituril homologues can be separated by utilizing the solubility difference of the cucurbituril homologues in water and hydrochloric acid with different concentrations. Then dissolving H33258 with ultrapure water, adding CB [ n ] to make the mole ratio of H33258 and CB [ n ] be 1:1-2, stirring for 1-4H, rotary evaporating to concentrate solution, then freeze-drying to obtain the fluorescent light probe. Dissolving H33258 in water, adding CB [ n ] to make the molar ratio of H33258 to CB [ n ] be 1:1-2, stirring for 1-4H, rotary evaporating to concentrate the solution, and freeze-drying to obtain the product.
Example 2
The application of the molecular assembly fluorescent probe in identifying trimethyl substituted Fmoc protected lysine comprises the following specific operation steps:
1) preparing 3 parts of fluorescent probe aqueous solution with the concentration of 3.0 mu M, and obtaining a fluorescent probe aqueous solution with the excitation wavelength of 361nmThe emitted fluorescence spectrum was measured under the test piece, and the maximum emitted fluorescence intensity I at 400-650nm was recorded for each sample0A，I0B，I0C；
2) Dropping Lys3 into any one solution and mixing uniformly to ensure that the molar ratio of the dropped Lys3 to the fluorescent probe is 0.3, measuring the fluorescence spectrum emitted by the solution under the condition of an excitation wavelength of 361nm, and recording the maximum emission fluorescence intensity I of the solution at 400-650nm1A；
3) Continuing to add Lys3 solution dropwise to the solution in the same amount as step 2), the maximum emitted fluorescence intensity I at 400-650nm was recorded2ARepeating for n times to obtain fluorescence intensity I2A,…InA(ii) a Superposing the fluorescence intensity curves obtained by each scanning to obtain a fluorescence spectrum titration curve, as shown in fig. 5, wherein n is 24;
4) with fluorescence intensity I0A,…InAThe ratio of the number of moles of Lys3 to the number of moles of fluorescent probe, respectively, is plotted on the abscissa as I0A,…InAAnd (3) performing nonlinear fitting to obtain a fluorescence responsiveness curve as a vertical coordinate, as shown in fig. 7.
Examples 3 to 4
The guest molecule Lys3 in the step 2) is respectively changed into Lys2 and Lys according to the embodiment 2, and the fluorescence spectra of the fluorescent probe added with Lys2 and Lys with different molar ratios are measured under the condition of not changing other conditions. The fluorescence spectrum of Lys is shown in FIG. 6. The resulting fluorescence response curve is shown in FIG. 7. It is clear that the Lys3 curve has the greatest tendency to change, and by calculation, InA/I0A0.44. Therefore, the trimethyl substituted Fmoc protected lysine can be accurately and effectively identified.
It will be appreciated that the guest molecules of examples 2, 3 or 4 were exchanged for lysine of unknown type and tested for In/I0If the value is 0.55 or less, Lys3 can be determined to be included in the solution.
Example 5
The fluorescent probe recognizes a peptide (P-Lys3) comprising an Fmoc-protected lysine substituted with a trimethyl group, and the specific steps are as follows:
1) four molecular assembly fluorescent probe aqueous solutions with the concentration of 10 mu M and the pH value of 7 are prepared, the numbers are 1, 2, 3 and 4, the fluorescence spectra emitted by the molecular assembly fluorescent probe aqueous solutions are respectively measured under the condition of the excitation wavelength of 361nm, and the maximum emission fluorescence intensity I of each fluorescent probe aqueous solution at the position of 400-650nm is recorded0a，I0b，I0c，I0d；
2) Gradually dripping P-Lys3 into the fluorescent probe solution with the number 1, wherein the dripping amount of each time ensures that the molar ratio of the P-Lys3 to the fluorescent probe is 0.3-0.5, the dripping is totally 15 times, the concentration of the P-Lys3 is finally 45-75 mu M, the mixture is uniformly mixed, measuring the fluorescence spectrum of the P-Lys3 solution of each time under the condition of the excitation wavelength of 361nm, and recording the maximum emission fluorescence intensity I at 400-650nm when the last dripping is carried outa(ii) a The fluorescence spectra obtained for each measurement were superimposed, as shown in FIG. 8, by calculating Ina/I0a＝0.45。
3) Repeating step 2), measuring other peptides P-Lys, P-Lys1 and P-Lys2 with fluorescent probe solutions numbered 2, 3 and 4, respectively, and recording their respective final maximum emitted fluorescence intensities Inb，IncAnd IndAnd calculate InY/I0YWherein Y is b, c or d, and is 0.67, 0.70 and 0.68, respectively. The fluorescence spectrum of P-Lys after the superposition is shown in FIG. 9.
4) Setting InY/I0YLess than P (0.55), is OFF, InY/I0YLess than P is in the "ON" state, P is the "ON"/"OFF" boundary, primarily to distinguish P-Lys3 from other methylated or unmethylated peptides;
5) with the type of methylated peptide as the abscissa and with the corresponding InY/I0YA two-dimensional histogram is built for the vertical axis, dividing the "ON"/"OFF" dividing line. As shown in fig. 10.
It will be appreciated that the guest molecule of example 5 was exchanged for an unknown type of peptide and its I was determinedn/I0When the value is 0.55 or less, it is judged that the solution contains a peptide including trimethyl-substituted Fmoc-protected lysine.
In conclusion, the invention avoids the problems that the introduction of methyl in a peptide chain increases the steric hindrance of methylated amino acid residues and reduces the hydrogen bonding force of the amino acid residues, and changes the stoichiometric ratio of a molecular assembly probe based on the good interaction between CB [ n ] and Lys3 so as to change the fluorescence property of a fluorescent probe and realize the accurate identification of Lys3 and P-Lys 3. Compared with the traditional detection method, the method has the advantages of high detection speed, low cost and strong anti-interference capability. Provides a brand-new detection means with high sensitivity and high specificity for methylated lysine and methylated peptide, and has great significance for deeply researching the regulation and control effect of protein methylation modification in the physiological process. Therefore, the method is expected to be applied to large-scale, high-throughput and high-precision methylated peptide detection and identification in a complex biological system.
Claims (10)
1. A molecular assembly fluorescent probe is characterized in that the structural formulas are respectively as follows:
the molecular assembly fluorescent probe is assembled by H33258 and CB [ n ] through non-covalent bond interaction, wherein CB [ n ] is any one of three cucurbiturils, namely CB [6], CB [7] or CB [8 ].
2. The method for preparing a molecular assembly fluorescent probe according to claim 1, comprising the steps of: dissolving H33258 in water, adding CB [ n ] to make the molar ratio of H33258 to CB [ n ] be 1:1-2, stirring for 1-4H, performing rotary evaporation to concentrate the solution, and then performing freeze drying to obtain the molecular assembly fluorescent probe.
3. Use of the molecular assembly fluorescent probe according to claim 1 for recognizing trimethyl substituted Fmoc protected lysine.
4. Use according to claim 3, characterized in that it comprises the following steps:
1) preparing three parts of fluorescent probe aqueous solution with the concentration of 3.0-10 mu M, measuring the fluorescence spectrum emitted by the three parts under the condition of the excitation wavelength of 361nm, and recording the maximum emitted fluorescence intensity I of each part of fluorescent probe aqueous solution at 400-650nm0A，I0B，I0C；
2) Adding Fmoc-protected lysine, dimethyl-substituted Fmoc-protected lysine and trimethyl-substituted Fmoc-protected lysine into the three solutions respectively and mixing uniformly, wherein the molar ratio of each type of lysine to the fluorescent probe is 0.3-0.5, measuring the fluorescence spectra emitted by the three solutions respectively under the condition of an excitation wavelength of 361nm, and recording the fluorescence intensity I at 400-650nm1A，I1B，I1C；
3) Repeating the step 2) n times to ensure that the amount of each type of lysine added dropwise is the same as that in the step 2), and respectively obtaining the Fmoc-protected lysine with the fluorescence intensity of I2ATo InA(ii) a Fluorescence intensity of dimethyl-substituted Fmoc-protected lysine is I2BTo InB(ii) a (ii) a Fluorescence intensity of trimethyl-substituted Fmoc-protected lysine is I2CTo InC；
4) Respectively with fluorescence intensity I0X,…InXThe ratio of the number of moles of the corresponding lysine species to the number of moles of the fluorescent probe is plotted on the abscissa as I0X,…InXPerforming nonlinear fitting for the ordinate to obtain fluorescence responsiveness curves of different types of lysine, wherein X is A, B or C; if InX/I0XWhen the content is less than or equal to 0.55, the solution contains trimethyl substituted Fmoc protected lysine.
5. The use of claim 4, wherein the pH of the molecular assembly fluorescent probe in step 1) is adjusted to 2-7 by adding Tris-HCl buffer solution.
6. The use of claim 4, wherein the Fmoc-protected lysine is Lys, the dimethyl-substituted Fmoc-protected lysine is Lys2, and the trimethyl-substituted Fmoc-protected lysine is Lys3, each having the formula:
7. use of the molecular assembly fluorescent probe according to claim 1 for identifying peptides comprising Fmoc-protected lysine substituted with trimethyl.
8. Use according to claim 7, characterized in that it comprises the following steps:
1) preparing four molecular assembly fluorescent probe aqueous solutions with the concentration of 3.3-10 mu M and the pH value of 2-7, measuring the fluorescence spectrum emitted by the molecular assembly fluorescent probe aqueous solutions under the condition of the excitation wavelength of 361nm, and recording the maximum emission fluorescence intensity I of each fluorescent probe aqueous solution at 400-650nm0a，I0b，I0c，I0d；
2) Providing four peptides P-Lys, P-Lys1, P-Lys2 and P-Lys3, adding the four peptides into the four solutions respectively and mixing uniformly, wherein the molar ratio of each type of peptide to the fluorescent probe is 0.3-0.5, measuring the fluorescence spectrum emitted by the four solutions respectively under the condition of an excitation wavelength of 361nm, and recording the fluorescence intensity I at 400-650nm1a，I1b，I1c，I1d；
3) Repeating the step 2) n times to ensure that the amount of each type of lysine added dropwise is the same as that in the step 2), ensuring that the final concentration of each type of peptide is between 45 and 75 mu M, and respectively obtaining the fluorescence intensity of P-Lys as I2aTo Ina(ii) a Fluorescence intensity of P-Lys1 is I2bTo Inb(ii) a (ii) a Fluorescence intensity of P-Lys2 is I2cTo Inc(ii) a Fluorescence intensity of P-Lys3 is I2dTo Ind；
4) If InY/I0YWhen the content is less than or equal to 0.55, the solution contains P-Lys3, wherein Y is a, b, c or d.
9. The use of claim 8, wherein the peptide sequences of P-Lys, P-Lys1, P-Lys2 and P-Lys3 are as follows:
10. the use according to claim 4 or 8, wherein n is 15 to 25.
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