A Cu 2+ -PLL alkaline combination reagents and their applications

Abstract

This invention belongs to the field of polymer material analysis and detection technology, and provides a Cu 2+ -PLL alkaline combination reagents and their applications, using Cu provided by this invention 2+ -PLL alkaline combination reagents can accurately and efficiently test the polyethyleneimine content in mixtures of polyethyleneimine and polyamine compounds, solving the problems of complex operation, long analysis time, and poor accuracy of existing methods when determining the polyethyleneimine content in similar mixtures. It meets the needs of biomedicine, materials science and other fields for the analysis of such mixture components, and provides reliable technical support for the quality control and performance research of related products.

Description

Technical Field

[0001] This invention belongs to the field of polymer material analysis and detection technology, specifically relating to a Cu 2+ -PLL alkaline combination reagents and their applications. Background Technology

[0002] Polyethylene imine (PEI) is a water-soluble polymer with high cationicity and reactivity due to its rich amino group content. It exhibits unique application value in numerous fields. Peptides are compounds composed of amino acids linked by peptide bonds; they are polyamino compounds widely present in organisms and possess various important physiological functions. In practical applications, polyethyleneimine and peptides are often used in combination to achieve more functionalities. For example, in gene therapy, combining targeted peptides with polyethyleneimine can construct composite carriers with targeted gene delivery capabilities. This utilizes the efficient gene transfection ability of polyethyleneimine while leveraging the targeting properties of peptides to improve the precision of gene delivery, effectively enhancing the efficacy and safety of gene therapy. In drug sustained-release systems, the combined use of both can regulate the drug release rate, prolong the drug's duration of action, and improve bioavailability. In the field of in-situ hydrogels, polyethyleneimine is used to regulate the gelation rate of in-situ hydrogels, while the skeletal stability of peptides is used to regulate the degradation characteristics of the hydrogel, effectively expanding the application scope of in-situ hydrogels.

[0003] Products containing PEI have a wide range of applications and good efficacy; however, high concentrations of PEI are cytotoxic, meaning PEI poses certain biosafety risks. Therefore, quantitative analysis of the PEI content in these products and their degradation products is crucial. In vitro quantitative analysis of the PEI content range in these products and their degradation products facilitates rapid screening of product formulations and processes, quality control, and biosafety monitoring, ensuring the biosafety of products throughout their entire lifecycle and accelerating market approval and safe clinical application.

[0004] PEI is not a single compound, but is composed of polymers with different molecular weights, degrees of branching, and charge densities.

[0005] Currently, there are various methods for determining PEI content, such as high performance liquid chromatography (HPLC), nuclear magnetic resonance spectroscopy (NMR), and potentiometric titration.

[0006] High-performance liquid chromatography (HPLC) separates polyethyleneimine (PEI) and then uses a detector to quantify it. This method is generally suitable for samples containing only PEI. However, when used to determine mixed samples containing PEI and polyamine compounds, the presence of the polyamine compounds can interfere with the results. For example, peptides, as polyamine compounds, have similar chemical structures, molecular weights, and molecular weight distributions to PEI. Their retention behaviors on the chromatographic column are highly similar, making complete separation of their peaks difficult and resulting in low resolution, failing to meet the quantitative requirement of R ≥ 1.5, thus affecting the accuracy of PEI content determination. Furthermore, PEI readily forms electrostatic or hydrogen bonds with conventional silica-based HPLC columns, leading to decreased column efficiency or peak tailing, necessitating the use of specialized polymer or modified columns, which increases analytical costs. Since PEI molecules do not contain aromatic rings or other chromophores, conventional UV detectors cannot be used directly, or specialized equipment such as CAD / ELSD detectors must be employed as alternative detection methods.

[0007] Nuclear magnetic resonance (NMR) spectroscopy can determine the content of polyethyleneimine by analyzing the chemical shifts and peak areas of specific atoms in the molecule. However, this method requires high sample purity. In mixed systems or material degradation products, the composition is complex and may introduce many impurities. The atomic signals of impurity molecules can interfere with the signal of polyethyleneimine. Because polyamine compounds and polyethyleneimine have similar chemical structures, their signal distributions in NMR spectra are complex and difficult to distinguish and assign accurately, making quantitative analysis challenging. Furthermore, NMR spectrometers are expensive, resulting in high detection costs and long analysis times, which is not conducive to the rapid detection of large-scale samples.

[0008] Potentiometric titration is based on the basicity of polyethyleneimine (PEI) and determines its content by measuring the change in potential during titration. However, in mixtures, PEI exhibits inconsistent ionization behavior in different extraction solutions, making it difficult to quantify using conventional standard curve methods. Furthermore, the amino and carboxyl functional groups in polyamine compounds may participate in the titration reaction, altering the acid-base balance of the system and affecting the reaction equilibrium between PEI and the titrant. This makes it difficult to accurately determine the titration endpoint, introducing significant errors and reducing the precision of content determination.

[0009] In summary, existing methods for determining polyethyleneimine content generally suffer from inaccurate results, complex operation, and long analysis time when dealing with mixtures of polyethyleneimine and polyamine compounds. These methods cannot meet the needs of rapid and accurate determination of polyethyleneimine content in actual production and research in the biomedical field. Therefore, there is an urgent need to develop an analytical method that is highly resistant to interference and easy to operate. Summary of the Invention

[0010] The purpose of this invention is to provide an accurate and efficient analytical method for testing the polyethyleneimine content in a mixture of polyethyleneimine and polyamine compounds. This method addresses the problems of existing methods in determining the polyethyleneimine content in similar mixtures, such as complex operation, long analysis time, and poor accuracy. It meets the needs of biomedicine, materials science, and other fields for the analysis of the components of such mixtures and provides reliable technical support for the quality control and performance research of related products.

[0011] In a first aspect, the present invention provides a Cu 2+ -PLL alkaline reagent, the Cu 2+ - The PLL alkaline reagent consists of polylysine stock solution, copper ion donor stock solution, and alkaline buffer solution.

[0012] The polylysine stock solution is a 10 mg / g aqueous solution of polylysine dissolved in ultrapure water; the copper ion donor stock solution is a 31.3 mmol / L solution of a divalent copper ion donor compound dissolved in ultrapure water; the alkaline buffer solution is prepared by adjusting the pH of neutral PBS stock solution to 11-13 using NaOH; the PLL stock solution, copper ion donor stock solution, and alkaline buffer solution are mixed evenly at a volume ratio of 10:1:39 to form the Cu... 2+ -PLL alkaline combination reagents are used.

[0013] It should be noted that the polylysine mentioned above refers to ε-polylysine, whose chemical structural formula is shown below:

[0014]

[0015] ε-polylysine (ε-PLL) is a linear cationic polypeptide consisting of 23-33 L-lysine residues linked by amide bonds between ε-amino and α-carboxyl groups. ε-polylysine contains amino and carboxyl groups, and the peptide bond (–CO–NH–) is a saturated amide bond. Its electronic transition energy level is high, and it has absorption in the far ultraviolet region (< 190 nm), which is beyond the range of conventional laboratory ultraviolet detectors.

[0016] The divalent copper ion donor compound is selected from any one of CuSO4, CuCl2, Cu(NO3)2 or Cu(CH3COO)2; and preferably, the divalent copper ion donor compound is CuSO4;

[0017] Preferably, the neutral PBS stock solution contains 8 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, and 2.9 g of Na2HPO4•12H2O per liter, and is made up to 1 liter with ultrapure water.

[0018] Preferably, this application experimentally determined that the optimal pH of the alkaline buffer solution is 12.0 ± 0.5, at which point the amino deprotonation rate of PEI is high, and the reaction between PEI and Cu... 2+ Stronger complexing ability; at the same time, Cu 2+ The hydrolysis rate of PLL is ≤5%, ensuring the stability of the colorimetric reaction, and the absorbance fluctuates by ≤2% within 5~30 min.

[0019] Secondly, the present invention provides the above-mentioned Cu 2+ Application of PLL alkaline reagent in the quantitative determination of polyethyleneimine content in samples by ultraviolet-visible spectroscopy; the sample is a monolithic solution with polyethyleneimine as the sole solute, or a binary or ternary solution containing polyethyleneimine.

[0020] It should be noted that the molecular weight of the polyethyleneimine described in this invention ranges from 300 to 70,000 Da, and includes branched polyethyleneimine or linear polyethyleneimine. The structural formula of the branched polyethyleneimine is shown below:

[0021] ;

[0022] The structural formula of the linear polyethyleneimine is shown below:

[0023] ;

[0024] The PEI concentration in the unary system solution ranges from 100 μg / g to 500 μg / g;

[0025] The solute in the binary system solution is polyethyleneimine and a polyamine compound, for example, the binary system solution is a mixed aqueous solution of PEI and PLL, wherein the concentration of PEI is 100 μg / g to 500 μg / g and the concentration of PLL is 125 μg / g to 5000 μg / g.

[0026] The solute in the ternary system solution is polyethyleneimine, a polyamino compound, and a compound containing a benzene ring group. The compound containing a benzene ring group includes, for example, a benzaldehyde-modified polymer compound, benzaldehyde-terminated polyethylene glycol B-PEG. For example, the solute in the ternary system solution is PEI, PLL, and B-PEG, where the molecular weight of B-PEG is 2000~20000 Da, the concentration of PEI is 100μg / g~500μg / g, the concentration of PLL is 125μg / g~5000μg / g, and the concentration of B-PEG is ≤250μg / g.

[0027] It should be noted that the molecular weight of the above-mentioned benzaldehyde-terminated polyethylene glycol B-PEG is 5000~20000 Da, and its general chemical structure formula is shown below (x=4, 6 or 8):

[0028]

[0029] As a preferred implementation of the above application, the "blank subtraction-standard curve method" is adopted, that is, using the processed blank solution as a reference, the background absorption of the solvent and reaction solution itself is eliminated, so that the linear correlation coefficient R² ≥ 0.998 and the relative standard deviation (RSD) ≤ 2%. Specifically, the application steps include:

[0030] Step S1: Using ultrapure water as solvent, prepare PEI standard solutions with concentrations of 100 μg / g, 200 μg / g, 300 μg / g, 400 μg / g, and 500 μg / g by gradient dilution. After ultrasonic degassing, store at 4°C in the dark. Use ultrapure water as a blank solution.

[0031] Step S2, mix the above PEI standard solution and the Cu at a mass ratio of 1:4. 2+ - The PLL alkaline reagent mixture was thoroughly mixed and allowed to stand at room temperature to prepare the treated PEI standard solution; similarly, the blank solution and the Cu were mixed at a mass ratio of 1:4. 2+ - The PLL alkaline reagent mixture was thoroughly mixed and allowed to stand at room temperature as a reference solution; Cu 2+ -PLL alkaline combination reagents should be prepared and used immediately, that is, within 2 hours after preparation;

[0032] Step S3: Using a UV-Vis spectrophotometer, with the blank solution after step S2 (i.e., the reference solution) as a reference, measure the absorbance of the treated PEI standard solution at a single wavelength of 670 nm. Each sample is measured in parallel at least 3 times, and the average value is taken as the final absorbance value.

[0033] Step S4: Using the actual concentration of PEI (μg / g) in the treated sample as the x-axis and the corresponding average absorbance as the y-axis, linear regression is performed using the concentration method to obtain the linear equation.

[0034] Step S5: After processing the sample to be tested according to step S2, measure its absorbance at 670 nm according to step S3. Substitute it into the linear equation of step S4 to calculate the PEI concentration M in the processed sample to be tested. Then, according to the dilution factor, i.e., the volume ratio of sample to reaction liquid is 1:4, that is, the PEI concentration in the sample to be tested is 5 times the M value.

[0035] Thirdly, the present invention provides the above-mentioned Cu 2+ Application of PLL alkaline reagent in the quantitative detection of polyethyleneimine content in gene therapy composite vectors and their degradation products.

[0036] Polyethyleneimine (PEI) is a cationic polymer that binds to negatively charged DNA via electrostatic interactions, agglomerating into tightly packed nanoscale composite particles. PEI's coating and proton buffering capacity protect nucleic acids from enzymatic degradation. Subsequently, the PEI-DNA composite particles enter cells via endocytosis or extracellular microinjection, are released from the endosome into the cytoplasm, and finally absorb the DNA into the nucleus, thus achieving gene therapy. However, while PEI possesses these inherent advantages as a gene carrier, unmodified PEI exhibits cytotoxicity and aggregation under high ionic strength conditions, limiting its application in nucleic acid transport. To overcome these limitations, PEI typically requires modification, such as using polyamino compounds, especially peptides, to alter its physicochemical properties, while simultaneously providing ligand-mediated targeted delivery, improving biocompatibility, reducing cytotoxicity, and increasing transfection efficiency. Therefore, the gene therapy composite vector described in this invention refers to a non-viral transgenic vector constructed based on a modified polyethyleneimine complex, wherein the modified polyethyleneimine complex is polyethyleneimine modified with a polyamino compound; by introducing the therapeutic gene into the gene therapy composite vector described in this invention, the therapeutic gene can be introduced into target cells to achieve the purpose of treating tumors.

[0037] Fourthly, the present invention provides the above-mentioned Cu 2+ Application of PLL alkaline reagent in the quantitative determination of polyethyleneimine content in drug sustained-release complexes and their degradation products.

[0038] The drug sustained-release complex described herein refers to a composite drug carrier formed by PEI and polyamino compounds. The combined use of the two can regulate the drug release rate, prolong the drug action time, and improve the drug bioavailability. It can be used to load anionic drugs or nucleic acids.

[0039] Fifthly, the present invention provides the above-mentioned Cu 2+ - Application of PLL alkaline reagent in the quantitative detection of polyethyleneimine content in in-situ hydrogels and their degradation products.

[0040] It should be noted that the in-situ hydrogel described in the fifth aspect of this invention refers to an in-situ cross-linked hydrogel containing PEI and peptides. Polyethyleneimine is used to regulate the gelation rate of the in-situ hydrogel, and the degradation characteristics of the hydrogel are controlled by the skeletal stability of the peptides, effectively expanding the application fields of in-situ hydrogels. For example, a radiotherapy protective hydrogel utilizes a polyethylene glycol derivative solution and a polyamine cross-linking agent solution, which can be injected into the body via a dual-syringe to achieve in-situ curing. The curing rate and degradation rate can be controlled by the type of polyamine cross-linking agent. Similarly, a tumor embolization hydrogel utilizes a polyethylene glycol derivative solution and a polyamine cross-linking agent solution, which can be injected into the tumor-feeding artery in vivo via a coaxial double-lumen microcatheter, where it cures in situ to form an embolization gel. The curing rate and degradation rate can be controlled by the type of polyamine cross-linking agent.

[0041] Beneficial effects:

[0042] The Cu provided in the first aspect of the present invention 2+ -PLL alkaline reagent uses PLL as Cu 2+ The "slow-release ligand" of PLL, under alkaline conditions, first reacts with Cu. 2+ Forming stable complexes can improve Cu in alkaline environments. 2+ Regarding stability at high concentrations, specifically when used to detect samples containing PEI, existing data show that PEI has a pKb of 4.5, while PLL has a pKb of 6.0. PEI's amino group is more basic and can competitively bind Cu. 2+ Formation of more stable PEI-Cu 2+ Complex, with K=10¹ 5 The complex exhibits characteristic absorption at 670 nm and a molar absorptivity ε = 850 L·mol⁻¹ -1 ・cm -1 Other components in PEI-containing samples, such as PLL and B-PEG, do not absorb at this wavelength, thus fundamentally eliminating interference. Even under conditions where the concentration of PLL in the sample is as high as 5000 μg / g, for example, 10 times the concentration of PEI, and the concentration of B-PEG is 250 μg / g, the relative error of PEI determination is ≤±3%.

[0043] The second aspect of the present invention provides the above-described Cu 2+ The application of PLL alkaline reagent in the quantitative detection of polyethyleneimine content in samples by ultraviolet-visible spectroscopy: For the sample to be tested in a binary system solution, the total time from sample processing to determination is ≤15 min, while existing conventional detection methods such as NMR require at least 30 min. Moreover, the polyethyleneimine content detection method provided by this invention does not require complex pretreatment such as column separation and sample purification, and can realize batch sample detection.

[0044] The Cu provided by this invention 2+ -PLL alkaline reagent has a wide range of applications and can be used in products with PEI as the main raw material and their degradation products. Especially in the quantitative detection of gene therapy composite vectors or drug sustained-release complexes containing PEI, PEI is a key carrier material, and its content directly affects transfection efficiency and cytotoxicity. Accurate quantification helps to optimize the PEI / peptide / DNA ratio, improve the consistency and stability of the formulation, and has a significant cost advantage compared with existing detection reagents or detection methods. After processing the sample with the detection reagent, only a conventional UV-Vis spectrophotometer is needed to obtain quantitative detection results, resulting in low testing costs. Attached Figure Description

[0045] Figure 1 The spectral scan results are for the sample solution treated under acidic conditions in Experiment Example 1.

[0046] Figure 2 The spectral scan results are for the sample solution treated under neutral conditions in Experiment Example 1.

[0047] Figure 3 The spectral scan results are for the sample solution treated under alkaline conditions in Experiment Example 1.

[0048] Figure 4 The sample from Experiment 2, treated under acidic conditions, contained 10 μg / g Cu. 2+ The sample spectral scanning results;

[0049] Figure 5 The sample from Experiment 2, treated under acidic conditions, contained 20 μg / g Cu. 2+ The sample spectral scanning results;

[0050] Figure 6 The sample from Experiment 2, treated under acidic conditions, contained 100 μg / g Cu. 2+ The sample spectral scanning results;

[0051] Figure 7 The sample from Experiment 2, treated under acidic conditions, contained 250 μg / g Cu. 2+ The sample spectral scanning results;

[0052] Figure 8 The sample from Experiment 2, treated under acidic conditions, contained 400 μg / g Cu. 2+ The sample spectral scanning results;

[0053] Figure 9 The sample from Experiment 2, treated under alkaline conditions, contained 10 μg / g Cu. 2+ The sample spectral scanning results;

[0054] Figure 10 The sample treated under alkaline conditions in Example 2 contained 20 μg / g Cu. 2+ The sample spectral scanning results;

[0055] Figure 11 The sample treated under alkaline conditions in Example 2 contained 100 μg / g Cu. 2+ The sample spectral scanning results;

[0056] Figure 12 This is a line graph showing the absorbance at 670 nm corresponding to the PEI concentration of the sample solution after treatment in Experiment Example 3.

[0057] Figure 13 According to Figure 12 A linear relationship plot fitted to a line graph in the image;

[0058] Figure 14 For example 4, which contains a high concentration of Cu 2+ Spectral scanning results of samples in the alkaline PLL-Cu detection system;

[0059] Figure 15 The spectral scanning results are shown for the PEI sample in Experiment Example 5 after treatment with 100 PLL-10Cu.

[0060] Figure 16 The spectral scanning results of the PEI sample in Experiment Example 5 after treatment with 2000 PLL-100Cu;

[0061] Figure 17 The spectral scanning results of the PEI sample in Experiment Example 5 after treatment with 2000 PLL-200 Cu;

[0062] Figure 18 The spectral scanning results are those of the PEI sample in Experiment Example 5 after treatment with 4000 PLL-400 Cu.

[0063] Figure 19 The spectral scanning results of the PEI and B-PEG samples in Experiment Example 5 after treatment with 100PLL-10Cu;

[0064] Figure 20 The graph shows the linear relationship of the detection results based on PEI+2000PLL-100Cu in Experiment Example 6.

[0065] Figure 21 The linear relationship graph of the detection results based on PEI+4000PLL-400Cu in Experiment Example 7;

[0066] Figure 22This is the state of the blank sample solution after Cu treatment in Experiment Example 3, after standing at room temperature for 24 hours. Detailed Implementation

[0067] The present invention will be further described in detail below with reference to specific embodiments. The experimental examples given are only for illustrating the present invention and are not intended to limit the scope of the present invention.

[0068] In the following experimental examples, ε-polylysine (ε-PLL) is used as a representative substance of other polyamino compounds in the test sample. The PLL used in the detection system is also ε-polylysine. Benzaldehyde-terminated multi-arm polyethylene glycol (B-PEG) is used as a representative substance of benzene ring compounds in the test sample.

[0069] The main sources of raw materials in the experimental examples are as follows:

[0070] The PEI involved in this invention has the CAS number 9002-98-6. All PEI used in the following experimental examples is PEI 1800, purchased from Aladdin, Mw 1800, 98%.

[0071] The PLLs used in the following experimental examples are all ε-polylysine ε-PLLs, purchased from Aladdin, CAS No.: 28211-04-3, ≥95%, MV 2000-5000;

[0072] Preparation of B-PEG: According to the synthesis process of Thebenzaldehyde-terminated four-arm polyethylene glycol reported in Section 2.1 of the published literature Adv Healthc Mater. 2016 Nov;5(21):2813-2822.doi: 10.1002 / adhm.201600720. Epub 2016 Sep 26. except for the 4-arm-PEG-CHO, by replacing the 4-arm PEG in its preparation raw materials with 6-arm PEG and 8-arm PEG, respectively, benzaldehyde-terminated 8-arm polyethylene glycol 8-arm-PEG-CHO and benzaldehyde-terminated 6-arm polyethylene glycol 6-arm-PEG-CHO were prepared. The other preparation conditions were exactly the same as those in the above literature. The prepared 8-arm-PEG-CHO with a molecular weight of 20000 Da was used as the polyethylene glycol derivative raw material in the following experiments of this application.

[0073] Experimental Example 1: UV-Vis Spectroscopic Scanning Experiment of Mixed Sample PP + Water

[0074] (1) Sample preparation: ① PEI sample: Prepare a PEI aqueous solution of 500 ug / g using water as the solvent; ② PLL sample: Prepare a PLL aqueous solution of 500 ug / g using water as the solvent; ③ PP sample, i.e., a sample containing PEI and PLL: Prepare a PEI aqueous solution of 1000 ug / g and a PLL aqueous solution of 1000 ug / g, and mix them in a 1:1 mass ratio, which will contain 500 ug / g PEI and 500 ug / g PLL; ④ Blank sample: Water

[0075] (2) Sample preparation: PEI samples, PLL samples, PP samples, and blank samples were prepared as follows: 1 mL of sample was taken, 0.1 mL of water was added, and the samples were numbered according to Table 1 below. 3.9 mL of phosphate-buffered saline (PBS) at different pH values ​​was added to each sample. All samples were in solution state after preparation. The PEI and PLL concentrations mentioned in Table 1 are the concentrations of the corresponding components in the samples after the above preparation. The PBS at different pH values ​​were prepared as follows:

[0076] ①PBS7: 8g sodium chloride, 0.2g potassium chloride, 0.2g potassium dihydrogen phosphate, 2.9g disodium hydrogen phosphate dodecyl hydrate, added to 1L water, the specific pH of PBS7 after preparation is 7.36;

[0077] ②PBS2: 45g PBS7, add 0.5g 10% phosphoric acid aqueous solution, and the 10% phosphoric acid aqueous solution here is prepared by adding 9g water to 1g phosphoric acid. The specific pH of PBS2 after preparation is 2.32.

[0078] ③PBS12: 45g PBS7, add 0.5g 10mol / L sodium hydroxide aqueous solution, and here the 10mol / L sodium hydroxide aqueous solution is prepared by adding 5g sodium hydroxide to 12.5g water. The specific pH of PBS12 after preparation is 12.32.

[0079] (3) Ultraviolet-visible spectral scanning analysis: Using water as a reference, all samples shown in Table 1 were subjected to ultraviolet-visible spectral scanning, specifically within the wavelength range of 190~800nm. The results are as follows: Figures 1-3 As shown.

[0080] Table 1: Experiment Example 1

[0081]

[0082] PEI molecules contain nitrogen atoms, whose lone pair electrons can absorb ultraviolet light, exhibiting a characteristic absorption peak in the ultraviolet region. PEI has significant absorbance in the wavelength range of 190 nm to 250 nm. The presence of PLLs can interfere with PEI measurements. PLLs have absorbance around 190–250 nm. PLLs are compounds composed of amino acids linked by peptide bonds, and their structure contains amide bonds. The carbonyl group (C=O) in the amide bond and the lone pair electrons on the nitrogen atom can interact with ultraviolet light, resulting in absorption in the ultraviolet region. The absorption peaks of PLLs partially overlap with those of PEIs, making it impossible to accurately distinguish between the absorption signals of PEI and PLLs when using ultraviolet spectrophotometry to determine PEI content, thus affecting the accuracy of the measurement results.

[0083] like Figure 1 , Figure 2 and Figure 3 As shown, by comparing and analyzing the spectra of samples with different pH values ​​in Experiment 1, the following conclusions can be drawn: On the one hand, both PEI and PLL samples exhibit absorbance in the 190–250 nm wavelength range. With increasing wavelength, the absorbance of PLL is stronger than that of PEI; therefore, the presence of PLL interferes with the determination of PEI. On the other hand, as the pH of the samples increases, the full-spectrum curves of PEI and PP samples gradually overlap, and the absorption of UV-Vis light in the 190–800 nm range by PLL in PP is suppressed. Based on the results of Experiment 1, alkaline solution treatment is preferred and more conducive to testing the PEI content in PP mixtures.

[0084] Experiment Example 2: UV-Vis Spectroscopic Scanning Experiment of Mixed Sample PP+Cu Based on Copper Ions

[0085] (1) Sample preparation: ①PEI sample: Prepare a PEI aqueous solution of 500ug / g using water as the solvent; ②PLL sample: Prepare a PLL aqueous solution of 500ug / g using water as the solvent; ③PP sample: Sample containing PEI and PLL: Prepare a PEI aqueous solution of 1000ug / g and a PLL aqueous solution of 1000ug / g, and mix them in a 1:1 mass ratio, which will contain 500ug / g PEI and 500ug / g PLL; ④Blank sample: Water

[0086] (2) Sample preparation: The PEI, PLL, PP, and blank samples were prepared as follows: 1 mL of sample was taken, numbered according to Table 2 below, and 0.1 mL of CuSO4 aqueous solution of different concentrations and 3.9 mL of phosphate buffered saline (PBS) of different pH values ​​were added respectively. The state of the samples after treatment is shown in Table 2. In addition, the concentrations of PEI, PLL, and Cu in Table 2 are the concentrations of the corresponding components in the samples after the above treatment. The PBS of different pH values ​​were prepared in the same way as in Experiment 1. The CuSO4 aqueous solutions of different concentrations added in the above treatment steps were prepared with water as the solvent, and were 500, 1000, 5000, 12500, and 20000 ug / g CuSO4 solutions respectively.

[0087] (3) Ultraviolet-visible spectral scanning analysis: Using water as a reference, all samples shown in Table 2 were subjected to ultraviolet-visible spectral scanning, specifically a wavelength range of 190~800nm. The results are as follows: Figures 4-11 As shown.

[0088] Table 2

[0089]

[0090] Compared to Example 1, the introduction of copper ions into the sample, along with the presence of amino groups (-NH2) in both PEI and PLL, allows copper ions to coordinate with these amino groups, resulting in stronger absorbance in the 250–350 nm range. For example… Figure 4 The results shown, comparing samples 13, 16, 19, and 22, indicate that the specificity of PEI in PP cannot be determined under acidic conditions, and PLL interferes with the test; furthermore, according to... Figure 4 , Figure 5 , Figure 6 , Figure 7 and Figure 8 The comparison results show that increasing the copper ion concentration does not reduce the interference of PLL on the test results.

[0091] like Figure 9 As shown, comparing samples 15, 18, 21, and 24, under alkaline conditions, within the wavelength range of 250–800 nm, PEI showed a high degree of overlap with the mixed sample PP. Similarly, as... Figure 10 As shown, compared with samples 27, 30, 33, and 36, under alkaline conditions, at 250-800 nm, PEI showed a high degree of overlap with the mixed sample PP.

[0092] Compared to acidic samples, in alkaline environments, the amino groups in the samples are mostly deprotonated amino groups (–NH2, –NH–, –N<), which can contribute lone pair electrons to Cu. 2+ Coordinate bonds are formed; however, in acidic environments, the amino groups in the sample are mostly protonated amino groups (–NH3).+ –NH2 + –), which already carries a positive charge, is not conducive to interaction with Cu. 2+ Coordination.

[0093] like Figure 11 As shown, comparing samples 39, 42, 45, and 48, when excess Cu was introduced into the sample solution... 2+ At that time, due to the presence of precipitation in the sample, the coordination reaction had reached saturation and was not suitable for PEI analysis.

[0094] The experiment revealed that CuSO4 is extremely unstable in alkaline solutions. When a small amount of Cu is introduced into the blank samples, such as samples 15 and 27, the solution gradually precipitates within 5 hours. When a large amount of Cu is introduced into the blank samples, such as samples 39, 51, and 63, precipitation occurs within 30 minutes. The stability of the sample has a significant impact on the stability of the method; therefore, sample processing methods with more stable sample solutions should be preferred.

[0095] Experimental Example 3: PEI-Cu Full Spectrum Analysis and Quantitative Analysis

[0096] (1) Sample preparation: ① PEI sample: Prepare PEI solutions of 10, 50, 100, 200, 300, 400, and 500 ug / g using water as the solvent; ② Blank sample: Water.

[0097] (2) Sample preparation: The PEI sample and blank sample were prepared as follows: 1 mL of sample was taken and 0.1 mL of CuSO4 aqueous solution of different concentrations was added respectively, and 3.9 mL of PBS12 was added. The concentration of each component in the sample and the sample state after treatment are shown in Table 3. Table 3 shows the concentration of PEI and Cu in the sample after the above treatment. The PBS12 was prepared in the same way as in Experiment 1. The CuSO4 solutions of different concentrations added were prepared with water as solvent, and were 500, 1000, 2000, 3000, 4000 and 5000 ug / g CuSO4 solutions.

[0098] Table 3:

[0099]

[0100] Using the treated blank sample as the reference solution and the PEI sample from the treated group with a Cu ion concentration of 10 μg / g as the standard sample (i.e., the sample solutions labeled as standard samples 1-6 in Table 3), quantitative analysis was performed using a single wavelength. Table 4 shows the PEI concentration (μg / g) after treatment and the absorbance value obtained after irradiation at 670 nm for each standard sample. It should be noted that the PEI concentration (μg / g) after treatment for each standard sample in Table 4 is a theoretical concentration value calculated based on the concentration and amount of the prepared raw materials. For example, the calculation process for the PEI concentration after treatment of standard sample 1 is as follows:

[0101] 1 mL of PEI sample (1.0122 g) containing 50.0176 ug / g was added to 0.1 mL of CuSO4 solution (0.1016 g) containing 500 ug / g, and 3.9 mL of PBS12 (3.9002 g) was added. The theoretical PEI concentration can be calculated as 1.0122 * 50.0176 / (1.0122 + 0.1016 + 3.9002) = 10.0973.

[0102] Table 4

[0103]

[0104] Based on the conclusions of Experiments 1 and 2, it is evident that copper sulfate solution is unstable in alkaline environments and readily forms precipitates. When using a blank solution as a reference solution, it must be prepared fresh each time, which limits the testing time. If precipitation occurs in the reference solution, it will lead to significant errors in the test results.

[0105] like Figure 22 As shown, after the reference solution (i.e., the blank sample treated with Cu) was left to stand at room temperature for 24 hours, a precipitate appeared at the bottom of the sample. Therefore, the blank sample treated with Cu alone is very unstable as a reference solution and cannot be used directly. Furthermore, based on the results of Experiment 3, it can be seen that while increasing the Cu concentration can improve the sensitivity of the test, Cu is unstable in an alkaline environment, which will also cause precipitation in the PEI standard sample, forcing a higher lower limit of linearity and failing to meet the requirements for quantitative detection of low-concentration PEI.

[0106] Under the condition that standard samples 1-6 are prepared and used immediately, the results shown in Table 3 and the corresponding values ​​in Table 3 can be obtained. Figure 12 The line chart in the middle, Figure 13 It is based on Figure 12The linear relationship obtained from the line graph fitting shows that standard samples 1-6 have a good linear relationship in the range of 10-40 ug / g. However, after the Cu-treated PEI standard samples were left to stand at room temperature for 3-4 hours, the absorbance values ​​of samples 1-6 decreased to 0.0002-0.0007. This is due to the instability of Cu in an alkaline environment, which causes Cu in the standard samples to form particles (Cu(OH)2), which are not visible to the naked eye. After the standard samples were left to stand for 1 day, a precipitate appeared at the bottom of the solution.

[0107] This experimental example verifies that PEI complexation of Cu can be directly used. 2+ The detection range of PEI spectral quantitative analysis using this method is insufficient, and the testing time is limited.

[0108] Experiment Example 4: PLL-Cu Full Spectrum Analysis and Quantitative Analysis Experiment

[0109] (1) Sample preparation: Prepare PLL aqueous solutions of 100, 2000, and 4000 ug / g using water as the solvent;

[0110] (2) Sample processing: The PLL samples were grouped and processed as follows: Sample 73: Take 1 mL of 100 ug / g PLL aqueous solution, add 0.1 mL of 500 ug / g CuSO4, and add 3.9 mL of PBS12; Sample 74: Take 1 mL of 2000 ug / g PLL aqueous solution, add 0.1 mL of 10000 ug / g CuSO4, and add 3.9 mL of PBS12; Sample 75: Take 1 mL of 4000 ug / g PLL aqueous solution, add 0.1 mL of 20000 ug / g CuSO4, and add 3.9 mL of PBS12; The PBS12 used in this process was prepared in the same way as in Experiment 1; The CuSO4 solutions used in this process were all prepared with water as the solvent, and were 500, 10000, and 20000 ug / g CuSO4 solutions respectively. Table 5 shows the concentration and state of each component in the sample solution after processing.

[0111] Table 5

[0112]

[0113] Compared to Experiments 2 and 3, it can be verified that Cu in the alkaline PLL-Cu sample can be expressed as Cu in a high concentration range. 2+ It exists stably without forming sediment.

[0114] (3) Ultraviolet-visible spectral scanning analysis: Using water as a reference, the sample was subjected to ultraviolet-visible spectral scanning in the range of 190~800nm.

[0115] The results are as follows Figure 14 As shown, it can be seen that when high concentrations of PLL react with Cu 2+ After the complexation reaction occurs, the absorbance of the sample in the 200-400 nm range is particularly strong, reaching the limit of the equipment. At this point, the 200-400 nm range can no longer be used for PEI quantitative analysis. As the Cu concentration in the sample increases, in addition to the sharp increase in absorbance in the 200-400 nm range, the absorbance of the sample in the 400-800 nm range also gradually increases, and is still within the metrological range of the equipment, so it can be used as the range for PEI quantitative analysis. At the same time, selecting the 400-800 nm range can also eliminate the interference of compounds containing benzene rings.

[0116] Based on the experimental results of Examples 2-4, it can be verified that this application uses PLL as Cu 2+ The "slow-release ligand" can improve the Cu content in the detection system. 2+ By adjusting the concentration and selecting an appropriate detection wavelength, the detection range of PEI can be expanded and its sensitivity significantly improved.

[0117] Regarding Cu in the detection system 2+ To further investigate the effect of concentration on the accuracy of detection results, this application conducted the following experimental examples, namely Experimental Example 5 to Experimental Example 7.

[0118] Experimental Example 5: PEI+PLL-Cu Full Spectrum

[0119] (1) Sample preparation: ①PEI sample: Prepare PEI solutions of 50, 100, 200, 300, 400 and 500 ug / g using water as solvent; ②Blank sample: Water; ③B-PEG sample: Prepare B-PEG solution of 20000 ug / g using water as solvent.

[0120] (2) Sample preparation: The PEI sample, blank sample, and B-PEG sample were prepared as follows: Take 1 mL of sample and prepare according to Table 6 below, adding 4 mL of the corresponding PLL-Cu solution respectively; 100 PLL-10Cu solution was prepared by adding 0.1 mL of 500 ug / g CuSO4 to 1 mL of 100 ug / g PLL aqueous solution, followed by 3.9 mL of PBS12; 2000 PLL-100Cu solution was prepared by adding 0.1 mL of 5000 ug / g CuSO4 to 1 mL of 10000 ug / g PLL aqueous solution, followed by 3.9 mL of PBS12; 2000 PLL-200Cu solution was prepared by adding 0.1 mL of 10000 ug / g CuSO4 aqueous solution to 1 mL of 10000 ug / g PLL aqueous solution, followed by 3.9 mL of PBS12; 400 PLL-400Cu solution was prepared by adding 0.1 mL of 10000 ug / g CuSO4 aqueous solution to 1 mL of 20000 ug / g PLL aqueous solution, followed by 3.9 mL of PBS12; The solution was prepared by first adding 0.1 mL of 20000 ug / g CuSO4 aqueous solution to ug / g PLL aqueous solution, and then adding 3.9 mL of PBS12. The preparation method of PBS12 was the same as in Experiment 1.

[0121] Table 6:

[0122]

[0123] (3) Quantitative analysis of ultraviolet-visible light spectroscopy: Using the treated blank sample as a reference, the treated sample was subjected to ultraviolet-visible light (190~800nm) spectral scanning, and the results are as follows. Figures 15-19 As shown.

[0124] like Figure 15 As shown, when the PEI sample is treated with 100PLL-10Cu, the absorbance in the 190~800nm ​​band is too low. As the PEI concentration increases, the absorbance at 300nm no longer increases with the concentration. The linear trend of absorbance in the 600~800nm ​​band with the increase of PEI concentration is not significant. This indicates that the PEI concentration in the sample is too high and the Cu concentration is too low.

[0125] like Figure 16 As shown, after the PEI sample was treated with 2000 PLL-100 Cu, the absorbance at 300 nm and in the 190–800 nm band increased with increasing PEI concentration. In the 200–250 nm band, there was a characteristic peak that gradually increased negatively with increasing PEI concentration. This can be interpreted as PEI drawing Cu from the 2000 PLL-100 Cu solution to form PEI-Cu, indicating that the amount of Cu in the sample was appropriate.

[0126] like Figure 17 As shown, after the PEI sample was treated with 2000 PLL-200 Cu, the absorbance at 300 nm and in the 190–800 nm band increased with increasing PEI concentration. In the 200–250 nm band, no characteristic peak showing a gradual negative increase with increasing PEI concentration was observed. This can be interpreted as PEI drawing Cu from the 2000 PLL-200 Cu solution to form PEI-Cu, but the Cu in the sample remains in excess. The presence of excess Cu leads to a larger error in the quantitative determination of PEI in the mixed sample.

[0127] like Figure 18 As shown, after the PEI sample was treated with 4000 PLL-400 Cu, the absorbance at 300 nm and in the 190–800 nm band increased with increasing PEI concentration. In the 200–250 nm band, no characteristic peak showing a gradual negative increase with increasing PEI concentration was observed. This can be interpreted as PEI drawing Cu from the 4000 PLL-400 Cu solution to form PEI-Cu, but the Cu in the sample remains in excess. The presence of excess Cu leads to a larger error in the quantitative determination of PEI in the mixed sample.

[0128] like Figure 19 As shown, B-PEG samples treated in the same way exhibit strong UV absorption at 328 nm due to the presence of a benzene ring and the strong UV absorption characteristics of the benzene ring's conjugated π-electron system. When PEI and B-PEG are present in the sample, comparing the full spectra of PEI-Cu and B-PEG reveals that both exhibit strong absorption in the 200–400 nm range. Therefore, 200–400 nm cannot be selected as the characteristic wavelength for PEI quantification. B-PEG, on the other hand, shows no significant absorption in the 600–800 nm range and thus does not interfere with PEI-Cu.

[0129] Based on the results of Experiment 5, this application further verifies the accuracy of the detection effect of the 2000PLL-100Cu detection system, as shown in Experiment 6, and performs reverse verification by setting up an excess Cu detection system in Experiment 7.

[0130] Experimental Example 6: Quantitative Analysis of PEI + 2000 PLL - 100 Cu

[0131] (1) Sample preparation:

[0132] ①PEI samples: Prepare PEI aqueous solutions of 100, 200, 300, 400, and 500 ug / g using water as the solvent;

[0133] ②PP samples are those containing PEI and PLL:

[0134] 250 / 125PP sample: Prepare 500ug / g PEI aqueous solution and 250ug / g PLL aqueous solution, mix the two in a 1:1 mass ratio to obtain the 250 / 125PP sample, which actually contains 250ug / g PEI and 125ug / g PLL;

[0135] 250 / 250PP sample: Prepare 500ug / g PEI aqueous solution and 500ug / g PLL aqueous solution, mix the two in a 1:1 mass ratio to obtain the 250 / 250PP sample, which actually contains 250ug / g PEI and 250ug / g PLL.

[0136] 250 / 500PP sample: Prepare 500ug / g PEI aqueous solution and 1000ug / g PLL aqueous solution, mix the two in a 1:1 mass ratio to obtain 250 / 500PP, which actually contains 250ug / g PEI and 500ug / g PLL;

[0137] 250 / 5000PP sample: Prepare 500ug / g PEI aqueous solution and 10000ug / g PLL aqueous solution, mix the two in a 1:1 mass ratio to obtain the 250 / 5000PP sample, which actually contains 250ug / g PEI and 5000ug / g PLL.

[0138] ③BPP samples: Samples containing B-PEG, PEI, and PLL:

[0139] 500 / 250PP sample: Prepare 1000ug / g PEI aqueous solution and 500ug / g PLL aqueous solution, mix the two in a 1:1 mass ratio to obtain the 500 / 250PP sample, which actually contains 500ug / g PEI and 250ug / g PLL.

[0140] 500 / 500PP sample: Prepare 1000ug / g PEI aqueous solution and 1000ug / g PLL aqueous solution, mix them at a mass ratio of 1:1 to obtain the 500 / 500PP sample, which actually contains 500ug / g PEI and 500ug / g PLL.

[0141] 500 / 10000PP sample: Prepare 1000ug / g PEI aqueous solution and 20000ug / g PLL aqueous solution, mix the two in a 1:1 mass ratio to obtain the 500 / 10000PP sample, which actually contains 500ug / g PEI and 10000ug / g PLL.

[0142] 250 / 250 / 125BPP sample: Prepare 500 ug / g B-PEG aqueous solution and 500 / 250PP, mix them at a mass ratio of 1:1 to obtain the 250 / 250 / 125BPP sample, which actually contains 250 ug / g B-PEG, 250 ug / g PEI and 125 ug / g PLL.

[0143] 250 / 250 / 250BPP sample: Prepare 500 ug / g B-PEG aqueous solution and 500 / 500PP, mix the two in a 1:1 mass ratio to obtain the 250 / 250 / 250BPP sample, which actually contains 250 ug / g B-PEG, 250 ug / g PEI and 500 ug / g PLL.

[0144] 250 / 250 / 5000BPP sample: Prepare 500 ug / g B-PEG aqueous solution and 500 / 10000PP, mix them in a 1:1 mass ratio to obtain the 250 / 250 / 5000BPP sample, which contains 250 ug / g B-PEG, 250 ug / g PEI and 5000 ug / g PLL.

[0145] ④ Blank sample: water

[0146] (2) Sample preparation: The samples in ① to ④ above were prepared as follows: 1 mL of each sample was taken and 4 mL of 2000 PLL-100 Cu solution was added. 2000 PLL-100 Cu was prepared by adding 0.1 mL of 5000 ug / g CuSO4 aqueous solution to 1 mL of 10000 PLL aqueous solution and then adding 3.9 mL of PBS12. The preparation of PBS12 was the same as in Experiment 1.

[0147] (3) Quantitative analysis by ultraviolet-visible spectroscopy: Using the treated blank sample as a reference and the treated PEI sample as the standard sample, as shown in Table 7 (standard samples 1-5), a single-wavelength method was used for quantitative analysis to obtain the Abs-670nm value as shown in Table 7. It should be noted that the concentration of each standard sample in Table 7 is the PEI concentration after treatment (ug / g), which is a theoretical concentration value calculated based on the concentration and amount of the raw materials used. For example, the calculation process of the PEI concentration after treatment of standard sample 1 is as follows:

[0148] 1 mL of PEI sample (99.9271 ug / g, 1.0085 g) was added to 4 mL of 2000 PLL-100 Cu (3.9966 g). The theoretical PEI concentration can be calculated as: 1.0085 * 99.9271 / (1.0085 + 3.9966) = 20.1348.

[0149] Using the processed blank sample as a reference, and the processed PP and BPP samples as impurity samples, quantitative analysis was performed using a single wavelength.

[0150] Table 7: PEI+2000PLL-100Cu Standard Sample Abs

[0151]

[0152] The linear equation constructed based on Table 7 above is: Abs = 0.00043 * C + 0.00151, where R² ≥ 0.9997, C is the PEI concentration after treatment (μg / g), and Abs is the average absorbance of the sample at 670 nm. The corresponding line graph and the fitted linear relationship graph are shown below. Figure 20 As shown.

[0153] Table 8: Testing of PEI+2000PLL-100Cu Impurity Samples

[0154]

[0155] In Table 8, the relative error is calculated as (test value / theoretical value - 1) * 100%; and the results show that the relative error between the PEI test value and the theoretical value obtained using the 2000PLL-100Cu detection system is ≤ ±3%.

[0156] Experimental Example 7: Quantitative Analysis of PEI + 4000 PLL - 400 Cu

[0157] (1) Sample preparation: In this experiment, Example 7 prepared the same ①PEI sample, ②PP sample, ③BPP sample, and ④blank sample in the same way as in Example 6.

[0158] (2) Sample preparation: ①-④ were prepared. The only difference from Experiment 6 is that this experiment uses a Cu excess detection system, namely 4000PLL-400Cu solution. The specific sample preparation is as follows: Take 1 mL of sample and add 4 mL of 4000PLL-400Cu solution. 4000PLL-400Cu: Take 1 mL of 20000 ug / g PLL aqueous solution, add 0.1 mL of 20000 ug / g CuSO4 aqueous solution, and add 3.9 mL of PBS12. The preparation of PBS12 is the same as in Experiment 1.

[0159] (3) Quantitative analysis by ultraviolet-visible spectroscopy: Using the treated blank sample as a reference and the treated PEI sample as the standard sample, as shown in Table 9 below (standard samples 1-5), a single-wavelength method was used for quantitative analysis to obtain the Abs-670nm value as shown in Table 9. It should be noted that the concentration of each standard sample in Table 9 is the PEI concentration after treatment (ug / g), which is a theoretical concentration value calculated based on the concentration and amount of the raw materials used. For example, the calculation process of the PEI concentration after treatment of standard sample 1 is as follows:

[0160] 1 mL of PEI sample (99.9271 ug / g, 0.9853 g) was added to 4 mL of 2000 PLL-400 Cu (4.0169 g). The theoretical PEI concentration can be calculated as 0.9853 * 99.9271 / (0.9853 + 4.0169) = 19.682.

[0161] Table 9: PEI+4000PLL-400Cu Standard Samples Abs

[0162]

[0163] The line graph constructed based on Table 9 above and the fitted linear relationship graph are as follows: Figure 21 As shown;

[0164] Table 10: Testing of PEI+4000PLL-400Cu Impurity Samples

[0165]

[0166] Compared to the test results of PEI+2000PLL-100Cu in Experiment 6, the relative error of the impurity sample PEI+4000PLL-400Cu in Experiment 7 is larger, and the error is even larger as the amount of PLL in the mixture increases, because PLL can still react with too much Cu, resulting in a larger error in the final test results.

Claims

1. Cu 2+ The application of PLL alkaline reagent in the quantitative determination of polyethyleneimine content in samples by ultraviolet-visible spectroscopy is characterized by, The Cu 2+ - The PLL alkaline reagent consists of a polylysine stock solution, a copper ion donor stock solution, and an alkaline buffer solution. The polylysine stock solution is a 10 mg / g aqueous solution of polylysine dissolved in ultrapure water. The copper ion donor stock solution is a 31.3 mmol / L solution of a divalent copper ion donor compound dissolved in ultrapure water. The alkaline buffer solution is prepared by adjusting the pH of neutral PBS stock solution to 11-13 using NaOH. The polylysine stock solution, copper ion donor stock solution, and alkaline buffer solution are mixed thoroughly at a volume ratio of 10:1:39 to form the Cu... 2+ -PLL basic combination reagent is used; the polylysine is ε-polylysine; the divalent copper ion donor compound is selected from any one of CuSO4, CuCl2, Cu(NO3)2 or Cu(CH3COO)2; The application steps include: Step S1: Using ultrapure water as solvent, prepare PEI standard solutions with concentrations of 100 μg / g, 200 μg / g, 300 μg / g, 400 μg / g, and 500 μg / g by gradient dilution. After ultrasonic degassing, store at 4°C in the dark. Use ultrapure water as a blank solution. Step S2, mix the above PEI standard solution and the Cu at a mass ratio of 1:

4. 2+ - The PLL alkaline reagent mixture was thoroughly mixed and allowed to stand at room temperature to prepare the treated PEI standard solution; similarly, the blank solution and the Cu were mixed at a mass ratio of 1:

4. 2+ - The PLL alkaline reagent mixture was thoroughly mixed and allowed to stand at room temperature as a reference solution; Cu 2+ -PLL alkaline combination reagents should be prepared and used immediately, that is, within 2 hours after preparation; Step S3: Using a UV-Vis spectrophotometer, with the blank solution after step S2 (i.e., the reference solution) as a reference, measure the absorbance of the treated PEI standard solution at a single wavelength of 670 nm. Each sample is measured in parallel at least 3 times, and the average value is taken as the final absorbance value. Step S4: Using the actual concentration of PEI (μg / g) in the treated sample as the x-axis and the corresponding average absorbance as the y-axis, linear regression is performed using the concentration method to obtain the linear equation. Step S5: After processing the sample to be tested according to step S2, measure its absorbance at 670 nm according to step S3. Substitute it into the linear equation of step S4 to calculate the PEI concentration M in the processed sample to be tested. Then, according to the dilution factor, i.e., the volume ratio of sample to reaction liquid is 1:4, that is, the PEI concentration in the sample to be tested is 5 times the M value.

2. The application as described in claim 1, characterized in that, The neutral PBS stock solution contains 8 g of NaCl, 0.2 g of KCl, 0.2 g of KH2PO4, and 2.9 g of Na2HPO4·12H2O per liter, and is made up to 1 liter with ultrapure water.

3. The application as described in claim 1, characterized in that, The pH value of the alkaline buffer solution is 12.0 ± 0.

5.

4. The application as described in claim 1, characterized in that, The sample is a mono-component solution of polyethyleneimine, or a binary or ternary solution containing polyethyleneimine. The solute in the binary system solution is polyethyleneimine and a polyamine compound, and the solute in the ternary system solution is polyethyleneimine, a polyamine compound, and a compound containing a benzene ring group. The compound containing a benzene ring group includes benzaldehyde-modified polymers, and the benzaldehyde-modified polymers include benzaldehyde-terminated polyethylene glycol B-PEG.

5. The application as described in claim 1, characterized in that, The "blank subtraction-standard curve method" is used, which uses the processed blank solution as a reference to eliminate background absorption from the solvent and reaction solution itself, thus ensuring a linear correlation coefficient R. 2 ≥0.998, relative standard deviation ≤2%.

6. The application according to claim 1, characterized in that, The sample is a gene therapy composite vector and its degradation products. The gene therapy composite vector refers to a non-viral transgenic vector constructed based on a modified polyethyleneimine complex, and the modified polyethyleneimine complex is polyethyleneimine modified with a polyamine compound. By introducing therapeutic genes into the gene therapy composite vector, the therapeutic genes are delivered to target cells to achieve the purpose of treating tumors.

7. The application according to claim 1, characterized in that, The sample is a drug sustained-release complex and its degradation products. The drug sustained-release complex refers to a composite drug carrier formed by PEI and polyamino compounds.

8. The application according to claim 1, characterized in that, The sample is an in-situ hydrogel and its degradation products. The in-situ hydrogel refers to an in-situ cross-linked hydrogel containing PEI and polypeptides.

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