Preparation of polyethyleneimine functionalized silver nanoparticles and application thereof in detection of monoamine oxidase b activity

Abstract

The application discloses a preparation method of polyethylene imine functionalized silver nanoparticles, which comprises the following steps: sequentially adding polyethylene imine solution, silver nitrate solution and sodium hydroxide solution into a centrifugal tube, stirring and reacting at 60-100 DEG C for 10-60 minutes, filtering, and adjusting pH to 7.0-9.0 for direct use in activity detection of B-type monoamine oxidase. The polyethylene imine functionalized silver nanoparticles prepared by the method are simple in synthesis and easy in raw material obtaining. The polyethylene imine functionalized silver nanoparticles prepared can be applied to activity detection of B-type monoamine oxidase. The polyethylene imine functionalized silver nanoparticles as the activity detection probe of B-type monoamine oxidase have the characteristics of stability and reliability. Compared with the previous activity detection method, the triple signal response of the polyethylene imine functionalized silver nanoparticles significantly improves the operability and universality of detection.

Description

Technical Field

[0001] This invention relates to a method for preparing polyethyleneimine-functionalized silver nanoparticles, which can be used as a detection probe for the detection of type B monoamine oxidase activity. Background Technology

[0002] Monoamine oxidase (B-type oxidase) is a highly active enzyme capable of oxidizing various amine biotransmitters, playing a crucial role in maintaining normal physiological function. Numerous studies have shown that B-type oxidase is a biomarker for neurodegenerative diseases (Alzheimer's disease, Parkinson's disease) and cirrhosis (hepatocellular carcinoma), and it also plays a significant role in the development and progression of many other diseases. Clinically, B-type oxidase activity is used as an indicator of tissue fibrosis. The aforementioned studies demonstrate that efficient and sensitive detection of this enzyme activity has significant application value in the diagnosis and treatment of related diseases.

[0003] Currently, most studies on the detection of type B monoamine oxidase activity rely on ultraviolet (UV) light, fluorescence, chromatography, and radiolabeling. Chromatography suffers from problems such as unintuitive results and complex operation; radiolabeling involves radioactivity, which can cause toxic side effects for operators; and using UV light or fluorescence alone has a high false-positive rate. Therefore, designing a simple, rapid, and intuitive method for detecting type B monoamine oxidase activity that responds to multiple signals is both necessary and urgent.

[0004] Silver nanoparticles have wide applications in antiviral, antifungal, and drug delivery systems, and their small size and bioinertness have led to their use in biomedical environmental detection. Recent research on silver nanoparticles has also confirmed that various molecules can serve as end-capping and protective agents in their synthesis. Changes in particle size, shape, end-capping agents, and the environment in which the end-capping agents are located significantly alter their optical properties. Changes in particle morphology and environmental state caused by the detection molecules produce corresponding signal responses, thus corresponding to the content or activity data of the detection molecules. In summary, the unique properties of silver nanoparticles make them highly promising for the construction of methods for detecting type B monoamine oxidase activity. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing polyethyleneimine-functionalized silver nanoparticles and to apply them to the detection of type B monoamine oxidase activity, which can be used to detect type B monoamine oxidase activity in serum.

[0006] This invention provides polyethyleneimine-functionalized silver nanoparticles, the preparation method of which is as follows: Add polyethyleneimine solution, silver nitrate solution, and sodium hydroxide solution sequentially to a centrifuge tube. Stir at 60–100°C (200–800 rpm) for 10–60 minutes, then adjust the pH to 7.0–9.0. Use 0.22… μ The polyethyleneimine-functionalized silver nanoparticle probe was obtained by filtration through an m-filter membrane.

[0007] Polyethyleneimine, with a molecular weight of 600-70000, silver nitrate solution concentration of 5-15 mM, polyethyleneimine solution concentration of 2-10%, sodium hydroxide solution concentration of 0.5-1.2 M, and the volume ratio of polyethyleneimine solution, silver nitrate solution, sodium hydroxide solution and deionized water is 5:5:2:8.

[0008] The aforementioned polyethyleneimine-functionalized silver nanoparticles can be used as probes for the detection of type B monoamine oxidase activity. The method for detecting type B monoamine oxidase activity is as follows: using polyethyleneimine-functionalized silver nanoparticles as probes, they bind to phenylacetaldehyde, the product obtained by the oxidation of phenylethylamine by type B monoamine oxidase. The polyethyleneimine-functionalized silver nanoparticles change from a pale yellow, clear, weakly fluorescent solution to a bright yellow, turbid, strongly fluorescent solution, producing changes in ultraviolet, fluorescence, and scattering signals. The intensity of the ultraviolet, fluorescence, and scattering signals is measured to obtain the activity intensity of type B monoamine oxidase.

[0009] The following section provides a more detailed explanation of the detection method for type B monoamine oxidase activity, the evaluation of selectivity and anti-interference ability, the evaluation of detection stability, and the experimental procedures for its application in serum testing.

[0010] 1. Method for detecting type B monoamine oxidase activity using polyethyleneimine-functionalized silver nanoparticle probes The method involves diluting the type B monoamine oxidase sample to be tested with a 4-hydroxyethylpiperazine ethanesulfonic acid buffer solution with a pH of 7.0-8.0, preheating at a constant temperature of 25-40°C, adding 5-15 mM phenylethylamine solution for reaction, and then adding polyethyleneimine-functionalized silver nanoparticles for further reaction. After the reaction is complete, the fluorescence, ultraviolet and scattering signal intensities are detected to obtain the activity of type B monoamine oxidase.

[0011] The concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid buffer was 10-40 mM. The enzyme reaction temperature after adding phenylethylamine was 25-40℃. The amount of polyethyleneimine-functionalized silver nanoparticles added was 10%-40% of the final solution volume, and the reaction temperature after adding the polyethyleneimine-functionalized silver nanoparticles was 30-70℃. Signal intensity was detected using a UV spectrophotometer and a fluorescence spectrophotometer. For the UV signal, the absorbance value at 405 nm was selected; for the fluorescence signal, the fluorescence intensity value at 386 nm under 308 nm excitation light was selected; and for the resonance Rayleigh scattering signal, the resonance Rayleigh scattering peak intensity value at 467 nm was selected.

[0012] 2. Evaluation of the selectivity and anti-interference ability of polyethyleneimine-functionalized silver nanoparticle probes in detecting type B monoamine oxidase. The detection method described above can selectively detect type B monoamine oxidase and obtain the activity of type B monoamine oxidase in the presence of interfering enzymes. The specific experimental steps are as follows: For the selectivity assessment experiment, different enzymes are reacted using the method described in "Method for Detecting Type B Monoamine Oxidase Activity with Polyethyleneimine Functionalized Silver Nanoparticle Probes"; for the anti-interference assessment experiment, 5%–15% of other enzymes are introduced into the reaction system during the type B monoamine oxidase enzymatic reaction, and the other steps are consistent with the method described in "Method for Detecting Type B Monoamine Oxidase Activity with Polyethyleneimine Functionalized Silver Nanoparticle Probes".

[0013] Figure 1 The selective ability of polyethyleneimine-functionalized silver nanoparticles to respond selectively to type B monoamine oxidase is evaluated under ultraviolet (A), fluorescence (B), and scattering (C) signals. Figure 2 The figure shows the anti-interference ability of polyethyleneimine-functionalized silver nanoparticles in response to type B monoamine oxidase under ultraviolet (A), fluorescence (B), and scattering (C) signals, in the presence of other enzymes. The enzymes in the figure, from left to right, are type B monoamine oxidase, type B monoamine oxidase, type C monoamine oxidase, type D monoamine oxidase, type E monoamine oxidase, type C monoamine oxidase, type D monoamine oxid α -glucosidase, β -Galactosidase, bovine serum albumin, β - Glucosidase, acid phosphatase.

[0014] 3. Stability of polyethyleneimine-functionalized silver nanoparticle probes for detecting type B monoamine oxidase activity Detection signal and time stability are important indicators of the performance of active detection probes. The stability of the polyethyleneimine functionalized silver nanoparticle probe was tested using the following methods: For detection signal stability, the absorbance value of the system after reaction at 405 nm was measured within 0-90 min; for time stability, the polyethyleneimine functionalized silver nanoparticle probe was stored in the dark at 10-25℃, and multiple detection experiments were conducted within 20 days. The change in absorbance value at 405 nm was used to represent the time stability of the detection performance of the polyethyleneimine functionalized silver nanoparticle probe.

[0015] Figure 3 The results showed that the detection signal (A) of the polyethyleneimine-functionalized silver nanoparticle probe remained stable within 90 min, indicating that the signal detection was not affected by time. The polyethyleneimine-functionalized silver nanoparticle probe maintained a good B-type monoamine oxidase signal response for 20 days, demonstrating good stability in the detection of B-type monoamine oxidase.

[0016] 4. Application of polyethyleneimine-functionalized silver nanoparticle probes in the detection of type B monoamine oxidase activity in serum. Serum contains various proteins and small molecules, the presence of which significantly interferes with the application of methods for detecting type B monoamine oxidase activity. In this invention, polyethyleneimine-functionalized silver nanoparticle probes have been successfully applied to the detection of type B monoamine oxidase activity in serum. The specific operating steps are as follows: Serum samples are diluted with 4-hydroxyethylpiperazine ethanesulfonic acid buffer, preheated at 25-40°C, and then reacted with 5-15 mM phenylethylamine solution. After the reaction, polyethyleneimine-functionalized silver nanoparticles are added and reacted again. After the reaction is complete, the fluorescence, ultraviolet, and scattering signal intensities are detected to obtain type B monoamine oxidase activity data.

[0017] The concentration of 4-hydroxyethylpiperazine ethanesulfonic acid buffer was 10–40 mM. The enzyme reaction temperature after adding phenylethylamine was 25–40 °C. The amount of polyethyleneimine-functionalized silver nanoparticles added was 10%–40% of the final solution volume. The reaction temperature after adding polyethyleneimine-functionalized silver nanoparticles was 30–70 °C. Signal intensity was detected using a UV spectrophotometer and a fluorescence spectrophotometer.

[0018] In summary, this invention develops a simple method for preparing polyethyleneimine-functionalized silver nanoparticles using readily available raw materials, and applies this method to the detection of monoamine oxidase (MAO) activity in standard MAO samples and serum. The results show that the MAO-functionalized silver nanoparticles prepared by this method exhibit excellent selectivity, anti-interference ability, and stability in MAO activity detection. Furthermore, the combined response of three optical signals significantly improves the false positive problem. Compared with existing methods, this method has the advantages of simple and rapid probe synthesis, and also offers advantages such as multi-mode response, strong operability, and good anti-interference ability in MAO detection. Attached Figure Description

[0019] Figure 1 To evaluate the selectivity of polyethyleneimine-functionalized silver nanoparticle probes for the detection of type B monoamine oxidase.

[0020] Figure 2 To evaluate the anti-interference ability of polyethyleneimine-functionalized silver nanoparticle probes for the detection of type B monoamine oxidase.

[0021] Figure 3 To evaluate the signal intensity and time stability of polyethyleneimine-functionalized silver nanoparticle probes for the detection of type B monoamine oxidase.

[0022] Figure 4 Infrared, ultraviolet and fluorescence properties of the polyethyleneimine-functionalized silver nanoparticle probe.

[0023] Figure 5 The UV, fluorescence, and resonance Rayleigh scattering response spectra and linear relationships of the polyethyleneimine-functionalized silver nanoparticle probe for detecting type B monoamine oxidase activity were obtained. Detailed Implementation

[0024] The following specific examples further illustrate the synthesis of the polyethyleneimine-functionalized silver nanoparticle detection probe, its application in the detection of standard type B monoamine oxidase activity, and its application in the detection of type B monoamine oxidase activity in serum.

[0025] Example 1: Preparation of a detection probe using polyethyleneimine-functionalized silver nanoparticles A mixture of 3.0 mL of 5% polyethyleneimine solution, 4.8 mL of deionized water, and 3.0 mL of silver nitrate solution (10 mM) was incubated at 100 °C for 10 min. Then, 2.4 mL of sodium hydroxide solution (1 M) was added, and the reaction was continued at 100 °C for 40 min. Stirring was maintained at 400 rpm throughout the reaction. The pH was adjusted to 8.0 with hydrochloric acid solution (1 M), and 0.22... μ Filter once with a water-based membrane and store at room temperature.

[0026] The volume ratio of the above-mentioned polyethyleneimine solution, silver nitrate solution, sodium hydroxide solution, and deionized water is 5:5:2:8.

[0027] Figure 4 The infrared (A), ultraviolet (B), and fluorescence (C) spectra of the obtained polyethyleneimine-functionalized silver nanoparticle probe are shown. In the infrared spectrum, 3389 cm⁻¹... -1 The nearby characteristic bands correspond to the stretching vibrations of various amino NH bonds in polyethyleneimine; 1612 cm⁻¹ -1 The peak at 1309 cm⁻¹ is related to the bending vibration of the NH bond; -1 The absorption peak is thought to be related to the stretching vibration of CN; 2957 cm⁻¹ -1 2841 cm -1 and 1466 cm -1 The absorption peaks are correlated with the stretching and bending vibrations of CH. These data successfully demonstrate that silver nanoparticles have been functionalized with polyethyleneimine.

[0028] Example 2: Assay of Standard Type B Monoamine Oxidase Activity Add 40 μ L of phenethylamine solution (10 mM) and monoamine oxidase of different activity levels were added, followed by 4-hydroxyethylpiperazine ethanesulfonic acid buffer to a final volume of 160. μ L. After reacting at 37°C and 400 rpm for 30 min with stirring, 40 L was added. μ L-type polyethyleneimine functionalized silver nanoparticle probes were reacted at 50 °C for 30 min, and then the intensity of ultraviolet, fluorescence, and resonance Rayleigh scattering signals was measured.

[0029] Figure 5 This paper presents the UV, fluorescence, and resonance Rayleigh scattering response spectra and linear relationships of polyethyleneimine-functionalized silver nanoparticles to type B monoamine oxidases with different activity levels. Using polyethyleneimine-functionalized silver nanoparticles as the detection molecule, the UV, fluorescence, and resonance Rayleigh scattering signal intensities of type B monoamine oxidase solutions with different activity levels were measured. Figure 5 As shown, with increasing activity of type B monoamine oxidase, the intensity of ultraviolet (UV), fluorescence, and resonance Rayleigh scattering signals significantly increased. Type B monoamine oxidase exhibited a good linear relationship with UV absorbance at 405 nm and resonance Rayleigh scattering intensity at 467 nm within the activity range of 0.05 to 0.5 U / mL, and also with UV absorbance at 386 nm within the activity range of 0.05 to 0.4 U / mL. The linear equations were: A = 4.55. c +0.14 (R 2 =0.9796), F=1050638c +140680(R 2 =0.9922) and RRS=188813 c +3901 (R 2 =0.9950), where A represents the absorbance value in the above formula; c The value represents the monoamine oxidase activity intensity; F represents the fluorescence intensity; and RRS represents the scattering intensity. Based on the signal-to-noise ratio (S:N=3), the calculated detection limits are 0.42 mU / mL (UV spectrophotometry), 3.49 mU / mL (fluorescence spectrophotometry), and 1.97 mU / mL (resonance Rayleigh scattering).

[0030] Example 3: Application of serum monoamine oxidase type B activity detection Place the blood sample in a centrifuge tube containing a coagulant, centrifuge at 1000 g for 5 min to obtain a serum sample (supernatant). Dilute the sample 10-fold and take 2... μ L added 40 μ Add 10 mM phenethylamine solution to L of 4-hydroxyethylpiperazine ethanesulfonic acid buffer to a final volume of 160 mL. μ L. After reacting at 37°C and 400 rpm for 30 min with stirring, 40 L was added. μ The L-polyethyleneimine functionalized silver nanoparticle probe was reacted at 50℃ for 30 min, and then the intensity of ultraviolet, fluorescence and resonance Rayleigh scattering signals was measured.

[0031] Table 1 shows the B-type monoamine oxidase activity obtained after applying the polyethyleneimine-functionalized silver nanoparticle probe to five serum samples.

Claims

1. The application of a polyethyleneimine-functionalized silver nanoparticle probe in the detection of type B monoamine oxidase activity, wherein the application is for non-diagnostic and non-therapeutic purposes, characterized in that, The preparation method of polyethyleneimine functionalized silver nanoparticle probe includes: adding polyethyleneimine solution, silver nitrate solution and sodium hydroxide solution to a centrifuge tube in sequence, stirring and reacting at 60~100℃ for 10~60 minutes, adjusting the pH to 7.0~9.0, and filtering with a 0.22 μm filter membrane to obtain polyethyleneimine functionalized silver nanoparticle probe. The type B monoamine oxidase sample to be tested was dissolved in 4-hydroxyethylpiperazine ethanesulfonic acid buffer to obtain the sample solution. After preheating at a constant temperature of 25-40℃, phenylethylamine solution was added and reacted at 25-40℃. After the reaction, polyethyleneimine-functionalized silver nanoparticles were added and reacted further at 30-70℃. After the reaction was complete, the fluorescence, ultraviolet and scattering signal intensities were detected to obtain the type B monoamine oxidase activity. The concentration of 4-hydroxyethylpiperazine ethanesulfonic acid buffer was 10-40 mM and the pH was 7.0-8.

0. For the ultraviolet signal, the absorbance value at 405 nm was selected; for the fluorescence signal, the fluorescence intensity value at 386 nm under 308 nm excitation light was selected; for the resonance Rayleigh scattering signal, the resonance Rayleigh scattering peak intensity value at 467 nm was selected.

2. The application of the polyethyleneimine-functionalized silver nanoparticle probe as described in claim 1 in the detection of type B monoamine oxidase activity, characterized in that: The concentration of silver nitrate solution is 5~15 mM, the concentration of polyethyleneimine solution is 2~10%, the concentration of sodium hydroxide solution is 0.5~1.2 M, and the volume ratio of polyethyleneimine solution, silver nitrate solution, sodium hydroxide solution and deionized water is 5:5:2:

8.

3. The application of the polyethyleneimine-functionalized silver nanoparticle probe as described in claim 1 in the detection of type B monoamine oxidase activity, characterized in that: The stirring speed is 200~800 rpm.

4. The application of the polyethyleneimine-functionalized silver nanoparticle probe as described in claim 3 in the detection of type B monoamine oxidase activity, characterized in that: Type B monoamine oxidase, α -glucosidase, β -Galactosidase, bovine serum albumin, β Glucosidase and acid phosphatase were dissolved separately in 4-hydroxyethylpiperazine ethanesulfonic acid buffer to obtain the enzyme sample solution to be tested. After preheating at a constant temperature of 25~40℃, phenylethylamine solution was added and reacted at 25~40℃. After the reaction, polyethyleneimine-functionalized silver nanoparticles were added and reacted further at 30~70℃. After the reaction was complete, the fluorescence, ultraviolet and scattering signal intensity were detected. Polyethyleneimine-functionalized silver nanoparticles can only make type B monoamine oxidase produce strong fluorescence, ultraviolet and scattering signal intensity, realizing the single selective detection of type B monoamine oxidase.

5. The application of the polyethyleneimine-functionalized silver nanoparticle probe as described in claim 1 in the detection of type B monoamine oxidase activity, characterized in that: The concentration of the phenethylamine solution was 5-15 mM; the concentration of the enzyme sample solution was 1 U / mL.

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