A method for qualitative and quantitative detection of MAO-B concentration and its application

Abstract

The present invention discloses a method and application for qualitatively and quantitatively detecting the concentration of MAO-B, wherein MAO-B is mixed with a substrate benzylamine solution, AuNS nanoparticles and Tollen reagent are added after water bath incubation, and a UV-visible absorption spectrum and a dark field spectrometer are used for quantitative analysis and detection respectively after continuing the water bath reaction, and the concentration of MAO-B is obtained according to the LSPR peak shift value, solution color change, peak blue shift value detected by a dark field spectrometer, and scattered light color change obtained when the UV-visible absorption spectrum is used for detection. The present invention can detect targets as low as nanograms, significantly improves the sensitivity of detection, and does not require the assistance of other biological enzymes, nor does it involve any complex modification and labeling process, effectively reducing the detection cost. The present invention is simple in principle, not only simplifies the detection process, does not require large instruments, has low cost, and the detection results are more sensitive and intuitive, and different concentrations of MAO-B can be distinguished by the naked eye.

Description

Technical Field

[0001] The present invention relates to a method for establishing a colorimetric and dark field dual-mode qualitative and quantitative detection of MAO-B concentration based on AuNS nanoparticles and its application, belonging to the field of biosensing technology. Background Art

[0002] Monoamine oxidases (MAOs) are flavoenzymes located in the outer mitochondrial membranes of neurons, glial cells, and others, mainly involved in the regulation of monoamine neurotransmission and oxidative stress in the brain. MAOs play an important role in maintaining the balance of biogenic amines, but abnormal changes in MAOs will disrupt the homeostasis of biogenic amine metabolism. If MAOs are overactivated in the brain, the increase in ROS levels will lead to oxidative stress and ultimately accelerate the development of neurodegenerative diseases such as Parkinson's disease (PD), Alzheimer's disease (AD), and amyotrophic lateral sclerosis (ALS). It is reported that the activity and expression of monoamine oxidase B (MAO-B) gradually increase with age in glial cells, up to about 4-fold, which will lead to more dopamine degradation, while generating high levels of hydrogen peroxide, forming oxidative stress, and finally resulting in apoptosis of dopaminergic neurons in the substantia nigra, which is considered to be one of the important events in the pathogenesis of Parkinson's disease. In addition, MAO-B can also metabolize 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to generate a neurotoxin 1-methyl-4-phenyl-pyridinium ion (MPP + ), further exacerbating the pathological process. Therefore, the development of a specific detection method for MAO-B activity is helpful for studying the biological functions of MAO-B in complex biological systems.

[0003] In recent years, optical analysis methods based on the local surface plasmon resonance (LSPR) characteristics of noble metal nanomaterials have developed rapidly. Due to the tunability of LSPR properties, they show great potential in biosensing and biomedical applications. Gold-based nanoparticles have broad prospects in many fields such as environmental monitoring, bioimaging, disease monitoring, catalysis, and optoelectronics due to their good biocompatibility, stability, visible light scattering band, and environmentally friendly synthesis methods, especially having high application value in the field of biosensing. Among them, gold nanorods (AuNS) are anisotropic plasmonic gold nanostructures with a high electromagnetic field enhancement effect, being more sensitive to changes in the microenvironment of the nanoparticle surface interface, and having stronger attraction for LSPR biosensing applications.

[0004] Currently, most of the developed methods for determining MAO-B activity are based on the quantitative analysis of hydrogen peroxide generated during the enzymatic catalysis process. For example, the established high-performance liquid chromatography-diode array (HPLC-DAD) detection method utilizes the generated hydrogen peroxide to combine with horseradish peroxidase (HRP) and then oxidizes the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) or the fluorescent substrate Amplex Red. There is also an electrochemical method that detects the current signal generated by the redox reaction of hydrogen peroxide catalyzed by peroxidase or materials with enzyme-like activity. However, the methods using the product hydrogen peroxide for detection have the following limitations: they require the assistance of external biological enzymes, so the detection cost is relatively high; the amount of hydrogen peroxide generated by enzymatic catalysis is very small, and quantitative analysis requires micromolar or even higher concentrations of hydrogen peroxide to be achieved, which indicates that the sensitivity of the method is poor. In addition, the established spectrophotometric method is based on the product aldehyde or imine generated by the enzymatic catalysis of the substrate amine combining with other organic derivatization reagents to form a chromogenic substance. However, the extinction coefficient of the organic chromogenic agent is relatively low. This traditional colorimetric strategy relies on the change in the optical density of the colored product, and the human eye is not sensitive to the depth of color, making it difficult to distinguish the target substance between samples with the naked eye.

[0005] Since MAO-B is closely related to a variety of neurodegenerative diseases, establishing a detection method for MAO-B has far-reaching significance in medical research. How to find a more suitable detection method for MAO-B and overcome the defects of existing detection methods has become one of the focuses widely concerned by researchers. Summary of the Invention

[0006] Object of the Invention: To solve the above technical problems in the background art, the first object of the present invention is to provide a method for qualitatively and quantitatively detecting the concentration of monoamine oxidase B (MAO-B) based on AuNS nanoparticles by establishing a colorimetric and dark-field dual mode; the second object of the present invention is to provide the application of the method for qualitatively and quantitatively detecting the concentration of MAO-B in the field of biosensing.

[0007] Technical Solution: The method for qualitatively and quantitatively detecting the concentration of MAO-B described in the present invention includes the following steps:

[0008] (1) Mix solutions of different concentrations of MAO-B with a benzylamine solution substrate respectively, and incubate in a water bath to obtain a reaction solution;

[0009] (2) Add AuNS nanoparticles and Tollen's reagent to the above reaction solution, and continue the reaction in a water bath to obtain a post-reaction solution;

[0010] (3) Establish the corresponding relationship between the color change of the post-reaction solution and the concentration of MAO-B in the post-reaction solution;

[0011] (4) Quantitatively analyze and detect the solution after the reaction at room temperature using an ultraviolet-visible absorption spectrometer and a dark-field spectrometer respectively. Establish a corresponding standard curve for the relationship between the LSPR peak shift value Δλ obtained by ultraviolet-visible absorption spectroscopy detection and the concentration of MAO-B, and establish a corresponding standard curve for the relationship between the blue shift value Δλ of the scattering peak and the concentration of MAO-B when detected by a dark-field spectrometer;

[0012] (5) Replace the MAO-B solutions with different concentrations in step (1) with the sample to be tested, and repeat the operations in steps (1)-(2);

[0013] (6) Qualitatively analyze MAO-B in the sample to be tested and preliminarily quantify the concentration of MAO-B according to the corresponding relationship in step (3) based on the color of the solution after the reaction of the sample to be tested.

[0014] (7) Quantitatively analyze and detect the solution after the reaction of the sample to be tested using an ultraviolet-visible absorption spectrometer and a dark-field spectrometer respectively. According to the obtained LSPR peak shift value Δλ and the blue shift value Δλ of the scattering peak, look up the concentration of MAO-B in the sample to be tested on the standard curves in step (4) respectively.

[0015] Further, in step (1), the volume ratio of MAO-B to the benzylamine solution is 10:300 to 3, the water bath temperature is 36.5 to 37.5 °C, and the incubation time is 1.5 to 2.5 h.

[0016] Further, in step (2), the particle size of the AuNS nanoparticles is 46.6 nm and the Zeta potential is -38.2 mV.

[0017] Further, in step (2), the volume ratio of MAO-B, the AuNS nanoparticle solution, and the Tollen reagent is 2:3:1 to 1.

[0018] Further, in step (2), the temperature for the continued water bath reaction is 36.5 to 37.5 °C, and the reaction time is 1.5 to 2 h.

[0019] Further, when analyzed and detected using an ultraviolet-visible absorption spectrometer, the concentration of MAO-B is 0.01 to 1 μg / mL, and when analyzed and detected using a dark-field spectrometer, the concentration of MAO-B is 0.5 to 20 ng / mL.

[0020] Further, in step (3), when detected using ultraviolet-visible absorption spectroscopy, the LSPR peak shift value changes from 0 nm to 80 nm, and the color of the solution changes from blue to yellow.

[0021] Further, in step (3), when detected using a dark-field spectrometer, the color of the scattered light changes from red to green.

[0022] Application of the method for detecting MAO-B in the field of biosensing.

[0023] Furthermore, the biological sample in the application of the method for detecting MAO-B in the field of biosensing is serum.

[0024] Mechanism of action of the present invention: As Figure 1 shown, when MAO-B is present, the substrate benzylamine is oxidized and deaminated to produce benzaldehyde. Benzaldehyde can undergo a silver mirror reaction with Tollen's reagent, and silver is deposited in-situ on the surface of AuNS nanoparticles, thereby triggering crystal growth and finally forming silver-coated AuNS nanoparticles (AuNS@Ag). This in-situ reduction of silver ions on AuNS nanoparticles induced by MAO-B can lead to a blue shift of the LSPR peak, a change in the solution color, and a change in the color of the scattered light. When MAO-B is absent, the substrate amine will not be oxidized, and the subsequent silver mirror reaction will not occur. Therefore, based on the spectral shift value and color change, multi-mode sensitive detection of MAO-B can be achieved and successfully applied to the analysis of serum samples.

[0025] The present invention does not use the enzyme-catalyzed product hydrogen peroxide, and neither requires external biological enzyme assistance nor uses organic chromogenic reagents with low extinction coefficients in the detection. It utilizes the benzaldehyde generated during the MAO-B catalysis process and combines with the classical silver mirror reaction to regulate the LSPR properties of AuNS nanoparticles, achieving a multi-color response according to the change in the target concentration. The detection limit of MAO-B can be lower, significantly improving the sensitivity, and it also has good selectivity.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0027] The present invention uses the benzaldehyde generated during the enzyme catalysis process for detection, and then uses the classical silver mirror reaction to generate silver nanoparticles to regulate the LSPR properties of AuNS nanoparticles, achieving a multi-color response according to the change in the target concentration. Compared with the traditional colorimetric method, the rich color changes of the multi-color sensing platform overcome the difficulty of distinguishing the single color of light and dark. Compared with the method based on hydrogen peroxide, the present invention can detect the target as low as nanograms, significantly improving the detection sensitivity. In addition, the present invention does not require the assistance of other biological enzymes and does not involve any complex modification and labeling processes, effectively reducing the detection cost. The principle of the present invention is simple, which not only simplifies the detection process, does not require large-scale instruments, has low cost, but also has more sensitive and intuitive detection results, and different concentrations of MAO-B can be distinguished by the naked eye. Description of the Drawings

[0028] Figure 1 It is the schematic diagram of the method for detecting MAO-B of the present invention;

[0029] Figure 2 UV-Vis absorption spectral peak shift and solution color change diagram for the feasibility of the colorimetric method based on AuNS nanoparticles in detecting MAO-B;

[0030] Figure 3 TEM images of AuNS nanoparticles and AuNS@Ag nanoparticles;

[0031] Figure 4 LSPR absorption peak shift change diagram of AuNS nanoparticles in the system under different conditions when the concentration of MAO-B is 0.5 μg / mL;

[0032] Figure 5 Absorption spectrum and corresponding solution color change diagram of the colorimetric method based on AuNS nanoparticles for detecting the target MAO-B in the range of 0.01 - 1 μg / mL;

[0033] Figure 6 Function relationship diagram of the degree of LSPR absorption peak blue shift (Δλ) with the change of MAO-B concentration;

[0034] Figure 7 Linear relationship diagram of the degree of LSPR absorption peak blue shift (Δλ) with the change of MAO-B;

[0035] Figure 8 Selectivity diagram of the colorimetric method based on AuNS nanoparticles for detecting the target;

[0036] Figure 9 Scattering spectra of AuNS nanoparticles and AuNS@Ag nanoparticles in dark field mode;

[0037] Figure 10 Scattering light color diagram of AuNS nanoparticles and AuNS@Ag nanoparticles in dark field;

[0038] Figure 11 Scattering spectrum and corresponding scattering light color change diagram of the dark field method based on AuNS nanoparticles for detecting the target MAO-B in the range of 0.5 ng / mL - 20 ng / mL;

[0039] Figure 12 Sensing performance diagram of the dark field analysis method based on AuNS nanoparticles;

[0040] Figure 13 Scattering light color change diagram corresponding to AuNS@Ag nanoparticles formed by different MAO-B concentrations;

[0041] Figure 14 Selectivity diagram of the dark field method based on AuNS nanoparticles for target detection. Specific implementation method

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0043] Description of Reagents and Instruments

[0044] Chloroauric acid (HAuCl 4 ·3H 2 O), ammonia water (NH 3 ·H 2 O) and potassium hydroxide (KOH) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Silver nitrate (AgNO 3 ) and ascorbic acid (AA) were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Sodium dodecyl sulfate (SDS) and benzylamine were purchased from Macklin Reagent (Shanghai, China). Monoamine oxidase B was purchased from Sigma-aldrich. All solutions used in the experiments were prepared with ultrapure water (resistance ≥ 18.2 MΩ / cm).

[0045] The ultraviolet-visible absorption spectrum was collected by an ultraviolet-visible spectrophotometer (Cary 100, Agilent, Singapore). The dark-field spectrum was measured using an inverted optical microscope (Eclipse Ti2-E, Nikon, Japan) equipped with a dark-field condenser (0.8 < NA < 0.95) and a 60× objective lens (NA = 0.7). The dark-field color image was taken by a true-color digital camera (Nikon DS-Fi3, Japan), and the scattering spectrum was recorded by a spectrometer (FERGIE, Princeton Instruments, USA).

[0046] Example 1

[0047] 1. Preparation of gold nanorods (AuNS) particles: The AuNS nanoparticles in the examples of the present invention were synthesized by the seed-mediated growth method. First, a gold seed solution was prepared. Under vigorous stirring, 15 mL of 1% citrate solution was added to 100 mL of boiling 1 mM HAuCl 4 solution. After boiling for 15 minutes, while keeping the solution volume stable, the solution was cooled and filtered through a 0.22 μm nitrocellulose membrane, and then stored at 4°C. 500 μL of the above gold seed solution (13 nm) was added to a round-bottom flask containing 50 ml of 0.25 mM HAuCl 4 solution (containing 50 μL of 1 M HCl). After stirring evenly, 1 mL of 0.6 mM AgNO 3And 250 μL of 100 mM ascorbic acid (AA) was added, and the solution was stirred for 30 seconds. It was observed that the color rapidly changed from light red to blue. Then, 500 μL of 10 mM sodium dodecyl sulfate (SDS) solution was continuously added, and the reaction continued at 25 °C for 5 min. Subsequently, centrifugal washing was carried out at a centrifugal force of 5000 g in a centrifuge tube for 15 min to stop nucleation. The solution was redispersed in 50 mL of deionized water and then stored at 4 °C for later use to obtain AuNS nanoparticles.

[0048] 2. Preparation of Tollen's reagent: The Tollen's reagent in the examples of the present invention was prepared according to the following steps: 100 mM AgNO 3 After the solution was mixed with 0.8 M KOH solution, it was observed that brown precipitate was produced. Subsequently, 0.6 M ammonia water was added dropwise until the precipitate dissolved. It was prepared immediately before use and placed in the dark.

[0049] 3. Preparation of amino-functionalized glass slides: The glass slides used in the examples of the present invention were amino-functionalized, and the modification steps were as follows: The glass slides were rinsed with clean water and then immersed in the alkaline water prepared from ethanol and sodium hydroxide for 2 h. Then, the glass slides were rinsed with deionized water and ethanol respectively. The cleaned glass slides were immersed in the ethanol solution containing 1% (3-aminopropyl)-triethoxysilane (APTES) for 2 h, and then rinsed with ethanol and dried with nitrogen. Finally, amino-functionalized glass slides were obtained.

[0050] 4. Preparation of polydimethylsiloxane (PDMS) film: The polydimethylsiloxane (PDMS) film in the examples of the present invention was self-made according to the following method: First, a mixed solution was prepared. The two required solutions were measured with a measuring cylinder and added to a beaker, and stirred evenly with a stirrer for 5 min, and then poured into a petri dish; after mixing evenly, matrix curing was continued. The petri dish was placed in a vacuum chamber for air extraction and defoaming for 20 min, and then cured at room temperature and placed in the vacuum chamber for 48 h to obtain the PDMS film.

[0051] 5. Preparation of AuNS@Ag nanoparticles: 3 μL of 100 mM benzylamine solution and 1 μL of 100 μg / mL MAO-B solution were taken, and deionized water was added to ensure that the total volume was 100 μL. After mixing evenly, it was incubated in a water bath at 37 °C for 2 h. Then, 150 μL of AuNS solution and 21 μL of 50 mM Tollen's reagent were added, and deionized water was added to control the volume at 300 μL. The reaction continued at 37 °C for 1 h. After the reaction was completed, AuNS@Ag nanoparticles were prepared.

[0052] 6. Transmission electron microscope scanning was performed on the prepared AuNS nanoparticles and AuNS@Ag nanoparticles, and the results were as Figure 3 shown.

[0053] Figure 3 TEM images of AuNS nanoparticles and AuNS@Ag nanoparticles. Among them, A is the TEM image of AuNS nanoparticles, and B is the TEM image of AuNS@Ag nanoparticles. It can be seen from Figures A and B that the successful deposition of silver on the surface of AuNS nanoparticles was confirmed by comparing the morphologies of AuNS nanoparticles and AuNS@Ag nanoparticles.

[0054] Feasibility study on the establishment of a colorimetric method for detecting MAO-B based on AuNS nanoparticles

[0055] Absorption spectra of reactants such as AuNS nanoparticles, Tollen's reagent, benzylamine, and the target analyte MAO-B were collected under different combinations. The following solutions were prepared separately and deionized water was added to make the total reaction volume 300 μL: (a) Take 150 μL of AuNS nanoparticles; (b) Take 3 μL of 100 mM benzylamine solution and 0.5 μL of 100 μg / mL MAO-B reaction solution, incubate at 37 °C for 2 h, then add 150 μL of AuNS nanoparticles and react at 37 °C for 1 h; (c) Take 21 μL of freshly prepared 50 mM Tollen's reagent and mix with 150 μL of AuNS nanoparticles, then incubate at 37 °C for 1 h; (d) Take 3 μL of 100 mM benzylamine solution, incubate at 37 °C for 2 h, add 21 μL of freshly prepared 50 mM Tollen's reagent and 150 μL of AuNS nanoparticles, mix well and react at 37 °C for 1 h; (e) 0.5 μL of 100 μg / mL MAO-B solution, incubate at 37 °C for 2 h, add 21 μL of freshly prepared 50 mM Tollen's reagent and 150 μL of AuNS nanoparticles and react at 37 °C for 1 h; (f) Take 3 μL of 100 mM benzylamine solution and 0.5 μL of 100 μg / mL MAO-B, incubate at 37 °C for 2 h, then add 21 μL of freshly prepared 50 mM Tollen's reagent and 150 μL of AuNS nanoparticles and react at 37 °C for 1 h. After the reaction was completed, the absorption spectra of the above-prepared solutions were collected using a UV-visible spectrophotometer, and the results are as Figure 2 shown.

[0056] Figure 2 UV-visible absorption spectral peak shift and solution color change diagram for the feasibility of the colorimetric method based on AuNS nanoparticles in detecting MAO-B; among them, Figure A is the UV-visible absorption spectral peak shift diagram for the feasibility of the colorimetric method based on AuNS nanoparticles in detecting MAO-B, and Figure B is the solution color change diagram of systems a - f. From Figure 2It can be seen that MAO-B has a decisive influence on the formation of AuNS@Ag nanoparticles. Curves a - e and pictures a - e show that in the absence of any one of MAO-B, benzylamine or Tollen's reagent, there is no obvious shift in the LSPR peak and no visual color change. However, in curve f and picture f, that is, only when all these components are present in the solution, there is a significant blue shift in the LSPR peak, accompanied by a color change of the solution from blue to yellow. These results indicate that MAO-B catalyzes the formation of benzaldehyde from benzylamine, leading to the deposition of silver onto the AuNS nanoparticles.

[0057] Example 3 Optimization of the conditions for colorimetric detection of MAO-B based on AuNS nanoparticles

[0058] To obtain a good MAO-B concentration response, three experimental parameters were optimized: the concentration of Tollen's reagent, the concentration of the substrate benzylamine, and the silver deposition time.

[0059] 1. Optimization of the Tollen's reagent concentration: Take 3 μL of 100 mM benzylamine solution and 0.5 μL of 100 μg / mL MAO-B reaction solution respectively, add 96.5 μL of deionized water and incubate at 37 °C for 2 h. Then continue to add 150 μL of AuNS solution and different volumes of 50 mM Tollen's reagent (0 μL, 0.6 μL, 1.5 μL, 3 μL, 12 μL, 21 μL, 30 μL, corresponding to the final concentration of Tollen's reagent in the system being 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 2 mM, 3.5 mM, 5 mM respectively), and continue to react at 37 °C in a water bath for 1 h. After the reaction is completed, collect the absorption spectrum using a UV-visible spectrophotometer. The results are shown in Figure 4 A as shown. In the figure, Δλ = λ 1 −λ 0 where λ 0 is the wavelength of the LSPR absorption peak of the reaction solution when the concentration of Tollen's reagent is 0 mM, and λ 1 is the wavelength of the LSPR absorption peak of the reaction solution corresponding to the concentration of Tollen's reagent from 0.1 to 5 mM.

[0060] 2. Optimize the concentration of the substrate benzylamine for MAO-B. Take 0.5 μL of 100 μg / mL MAO-B and different volumes of 100 mM benzylamine, namely 0 μL, 0.1 μL, 0.5 μL, 1 μL, 3 μL, 5 μL, and 10 μL (corresponding to the final concentrations of benzylamine in the system being 0 mM, 0.1 mM, 0.5 mM, 1 mM, 3 mM, 5 mM, and 10 mM) respectively. After mixing, add deionized water to ensure the reaction volume is 100 μL, and incubate at 37 °C for 2 h. After the reaction, continue to add 150 μL of AuNS solution, 18 μL of 50 mM Tollen's reagent, and 32 μL of deionized water, and then continue the reaction at 37 °C in a water bath for 1 h. After the reaction is completed, collect the absorption spectrum using a UV-visible spectrophotometer. The results are shown in Figure 4 as shown in B in the figure. In the figure, Δλ = λ 1 −λ 0 , where λ 0 is the LSPR absorption peak wavelength of the system when the final concentration of benzylamine is 0 mM, and λ 1 is the LSPR absorption peak wavelength of the corresponding systems when the final concentration of benzylamine is 0.1 - 10 mM.

[0061] 3. Optimize the silver deposition time. Take 3 μL of 100 mM benzylamine solution and 0.5 μL of the reaction solution of 100 μg / mL MAO-B, add 96.5 μL of deionized water, and incubate at 37 °C for 2 h. Then continue to add 150 μL of AuNS solution, 18 μL of 50 mM Tollen's reagent, and 32 μL of deionized water, and place it in a 37 °C water bath to react for different times, 0 min, 5 min, 15 min, 30 min, 60 min, and 90 min. After that, collect the absorption spectrum using a UV-visible spectrophotometer. The results are shown in Figure 4 as shown in C in the figure. In the figure, Δλ = λ 1 −λ 0 , where λ 0 is the LSPR absorption peak wavelength of the reaction solution when the reaction time is 0 min, and λ 1 is the LSPR absorption peak wavelength of the corresponding reaction solutions when the reaction time is 5 - 90 min.

[0062] Figure 4 is the diagram of the change in the LSPR absorption peak displacement of AuNS nanoparticles in the system under different conditions when the concentration of MAO-B is 0.5 μg / mL; among them, A is the diagram of the change in the LSPR absorption peak displacement of AuNS nanoparticles in the system with the Tollen's reagent concentration of 0.1 mM - 5 mM, B is the diagram of the change in the LSPR absorption peak displacement of AuNS nanoparticles in the system with the substrate benzylamine concentration of 0.1 mM - 10 mM, and C is the diagram of the change in the LSPR absorption peak displacement of AuNS nanoparticles in the system with the in-situ silver deposition time on the AuNS surface of 0 min - 90 min. From Figure 4It can be seen that in Figure A, as the concentration of the Tollen's reagent increases, the blue shift (Δλ) of the LSPR peak of the AuNS nanoparticles gradually increases, and Δλ has reached a stable plateau at a Tollen's reagent concentration of 3 mM. Figure B shows that when the concentration of benzylamine is 3 mM, the value of Δλ is the largest. Figure C shows that the optimal time for silver deposition is selected as 60 min, at which time Δλ reaches a plateau. Therefore, the optimal reaction conditions are a Tollen's reagent concentration of 3 mM, a benzylamine concentration of 3 mM, and a silver deposition time of 60 min.

[0063] Example 4 Detection of the target MAO-B based on AuNS nanoparticles in the colorimetric mode

[0064] Based on the optimal reaction conditions in Example 2, different concentrations of MAO-B were detected. First, MAO-B catalyzes the oxidative deamination of the substrate benzylamine. Take 3 μL of 100 mM benzylamine and a series of different volumes of 0 μL, 0.1 μL, 0.2 μL, 0.5 μL, 1 μL, 2 μL, 3 μL, 5 μL, 7 μL, 9 μL, 10 μL (corresponding to the final concentrations of MAO-B in the system being 0 μg / mL, 0.01 μg / mL, 0.02 μg / mL, 0.05 μg / mL, 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.5 μg / mL, 0.7 μg / mL, 0.9 μg / mL, 1 μg / mL) of 10 μg / mL MAO-B, mix them, add deionized water to a total reaction volume of 100 μL, and react in a 37 °C water bath for 2 h. After the first reaction is completed, continue to add 150 μL of AuNS nanoparticles, 18 μL of 50 mM Tollen's reagent and 32 μL of deionized water to the system, and continue to react at 37 °C for 1 h. After the reaction is completed, use a UV-visible spectrophotometer to collect the absorption spectrum (LSPR), and the results are as Figures 5 - 7 shown. Figure 5 It is a graph of the absorption spectrum and the corresponding solution color change of the colorimetric method based on AuNS nanoparticles for detecting the target MAO-B in the range of 0.01 - 1 μg / mL; among them, A is the absorption spectrum of the colorimetric method based on AuNS nanoparticles for detecting the target MAO-B in the range of 0.01 - 1 μg / mL, and B is the graph of the solution color change of the colorimetric method based on AuNS nanoparticles for detecting the target MAO-B in the range of 0.01 - 1 μg / mL. As Figure 5 can be seen, the ultraviolet spectrum can realize the quantitative analysis of the MAO-B concentration. As the MAO-B concentration gradually increases, the LSPR absorption peak gradually blue-shifts from 600 nm to 520 nm, and it is observed that the solution also changes from blue to purple and finally to yellow, and the concentration change can be visually recognized by the naked eye.

[0065] Figure 6Figure showing the relationship between the degree of blue shift (Δλ) of the LSPR absorption peak and the concentration of MAO-B. It can be seen that as the concentration of MAO-B increases from 0.01 μg / mL to 1 μg / mL, the value of Δλ gradually increases from 8 nm to 80 nm. Figure 6 As can be seen, as the concentration of MAO-B increases from 0.01 μg / mL to 1 μg / mL, the value of Δλ gradually increases from 8 nm to 80 nm.

[0066] Figure 7 Figure showing the linear relationship between the degree of blue shift (Δλ) of the LSPR absorption peak and MAO-B. It can be known that the value of Δλ shows a good linear relationship with the logarithm of the concentration of MAO-B. The calculated linear equation is Δλ = 78.8346 + 34.9486 logC, where Δλ represents the displacement of the LSPR absorption peak of AuNS nanoparticles, C represents the concentration of MAO-B, and R Figure 7 is 0.9934. In 2 and Figure 6 and Figure 7 Δλ = λ 1 −λ 0 where λ 0 is the wavelength of the LSPR absorption peak of the reaction solution when the concentration of MAO-B is 0 μg / mL, and λ 1 is the wavelength of the LSPR absorption peak of the corresponding system when the concentration of MAO-B is 0.01 - 1 μg / mL.

[0067] Example 5 Selectivity analysis of colorimetric detection of MAO-B based on AuNS nanoparticles

[0068] The target monoamine oxidase MAO-B was replaced with 10 μg / mL bovine serum albumin (BSA), 10 μg / mL glucose oxidase (GOX), 10 μg / mL horseradish peroxidase (HRP), and 0.4 U / mL alkaline phosphatase (ALP) respectively to evaluate the selectivity for MAO-B in the colorimetric mode. 3 μL of 100 mM benzylamine solution was reacted with deionized water, 10 μg / mL BSA, 10 μg / mL GOX, 10 μg / mL HRP, 0.4 U / mL ALP in a 37 °C water bath for 2 h, and then 150 μL of AuNS solution, 18 μL of 50 mM Tollen reagent, and 32 μL of deionized water were added to the system and reacted for another 1 h under the condition of 37 °C water bath. The results are as Figure 8 shown. In the figure, Δλ = λ 1 −λ 0 where λ 0 is the wavelength of the LSPR absorption peak of the system without adding biomolecules to participate in the reaction, and λ 1 is the wavelength of the LSPR absorption peak of the corresponding system in the presence of different biomolecules.

[0069] Figure 8High selectivity graph for the detection of MAO-B based on AuNS nanoparticles in the colorimetric mode. Among them, A is the graph of Δλ values of MAO-B, BSA, GOX, HRP, and ALP, and B is the graph of the color change of the reaction solution of MAO-B, BSA, GOX, HRP, and ALP. From Figure 8 it can be seen that the presence of MAO-B causes an approximately 80 nm blue shift in the LSPR absorption peak, and the solution changes significantly from blue to yellow. However, the introduction of other common biomolecules results in negligible LSPR displacement and color change. Thus, it can be seen that the colorimetric method based on AuNS nanoparticles has unique selectivity for the detection of MAO-B.

[0070] Feasibility study on the detection of MAO-B based on AuNS nanoparticles in the dark-field mode

[0071] The scattering spectra and dark-field images of the AuNS nanoparticles and AuNS@Ag nanoparticles prepared in Example 1 were collected respectively. After 100 μL of AuNS nanoparticles were electrostatically adsorbed in the PDMS channel of the amino-functionalized glass slide prepared in Example 1 for 15 min, the excess AuNS nanoparticles were removed with deionized water. The glass slide was placed on the sample stage of a dark-field microscope, and the dark-field image and the corresponding scattering spectrum were recorded with a dark-field microscope and a scattering spectrometer respectively. After 100 μL of AuNS@Ag nanoparticles were electrostatically adsorbed in the PDMS channel of the amino-functionalized glass slide for 15 min, the excess AuNS@Ag nanoparticles were removed with deionized water. The glass slide was placed on the sample stage of a dark-field microscope, and the dark-field image and the corresponding scattering spectrum were recorded with a dark-field microscope and a scattering spectrometer respectively. The results are as Figures 9 - 10 shown.

[0072] Figure 9 Scattering spectra of AuNS nanoparticles and AuNS@Ag nanoparticles in the dark-field mode. Figure 10 Scattering color maps of AuNS nanoparticles and AuNS@Ag nanoparticles in the dark-field mode. Among them, A is the scattering color map of AuNS nanoparticles in the dark-field mode, and B is the scattering color map of AuNS@Ag nanoparticles in the dark-field mode. From Figures 9 - 10 it can be seen that when there is no MAO-B, the scattering wavelength of AuNS nanoparticles is 682 nm, showing red in the dark field; when MAO-B is present, the scattering wavelength of the formed AuNS@Ag nanoparticles is 507 nm, showing blue in the dark field. The obvious blue shift of the scattering spectrum and the significant dark-field color change verify the feasibility of analyzing MAO-B in this mode.

[0073] Example 7 Detection of different concentrations of MAO-B based on AuNS nanoparticles in the dark-field mode

[0074] After the feasibility verification of Implementation Case 6, dark-field detection was performed on MAO-B at different concentrations. After mixing 3 μL of 1 mM benzylamine solution with a series of MAO-B at different concentrations, the final concentrations of MAO-B in the system were 0 ng / mL, 0.5 ng / mL, 1 ng / mL, 3 ng / mL, 5 ng / mL, 7 ng / mL, 10 ng / mL, and 20 ng / mL. The reaction was carried out in a 37 °C water bath for 2 h, and then 150 μL of AuNS nanoparticles, 18 μL of 0.5 mM Tollen's reagent, and 32 μL of deionized water were added to the system, and the reaction was continued at 37 °C for 1 h. Then, 100 μL of the above reaction solution was added to the PDMS channel of the amino-functionalized glass slide prepared in Example 1 for electrostatic adsorption for 15 min, and the excess nanoparticles were removed with deionized water. The glass slide was placed on the sample stage of a dark-field microscope, and the dark-field image and the corresponding scattering spectrum were recorded with a dark-field microscope and a scattering spectrometer, respectively. The results are as Figures 11 - 13 shown. In the figure, Δλ = λ 1 − λ 0 , where λ 0 is the scattering peak wavelength of the system when the final concentration of MAO-B is 0 ng / mL, and λ 1 is the scattering peak wavelength of the system when the final concentration of MAO-B is 0.5 - 20 ng / mL.

[0075] Figure 11 is the change diagram of the scattering spectrum and the corresponding scattering light color of the system with the concentration of the detection target MAO-B. Among them, A is the change diagram of the scattering spectrum of the system with the concentration of the detection target MAO-B, and B is the change diagram of the scattering light of the system with the concentration of the detection target MAO-B. As Figure 11 can be seen, as the concentration of MAO-B increases from 0.5 ng / mL to 20 ng / mL, the scattering spectrum peak gradually blue-shifts from 662 nm to 537 nm, and the scattering light color changes from red to orange and finally to green with the increase in concentration. Figure 12 is the function relationship diagram of the blue-shift degree (Δλ) of the scattering peak with the concentration of MAO-B, Figure 13 is the linear relationship diagram of the blue-shift degree (Δλ) of the scattering peak with the concentration of MAO-B. As Figures 12 - 13 can be seen, Δλ increases from 20 nm to 160 nm, and Δλ shows a linear relationship with the logarithm of the MAO-B concentration. The calculated linear equation is Δλ = 44.8895 + 88.1904log C, where Δλ represents the displacement of the scattering spectrum wavelength of AuNS, C represents the concentration of MAO-B, and R 2 = 0.9939.

[0076] Example 8 Selectivity Analysis of Detecting MAO-B Based on AuNS Nanoparticles in Dark-Field Mode

[0077] The target monoamine oxidase MAO-B was replaced with deionized water, 2 μg / mL bovine serum albumin (BSA), 1 μg / mL glucose oxidase (GOX), 10 μg / mL horseradish peroxidase (HRP), and 0.04 U / mL alkaline phosphatase (ALP) to evaluate the selectivity for MAO-B in dark-field mode. Take 3 μL of 1 mM benzylamine solution and react with 2 μg / mL BSA, 1 μg / mL GOX, 10 μg / mL HRP, and 0.04 U / mL ALP in a 37 °C water bath for 2 h. Then, continue to add 150 μL of AuNS solution, 18 μL of 0.5 mM Tollen's reagent, and 32 μL of deionized water to the system and continue to react for 1 h under 37 °C water bath conditions. Then, take 100 μL of the above reaction solution and add it to the PDMS channel of the amino-functionalized glass slide prepared in Example 1. After electrostatic adsorption for 15 min, remove the excess nanoparticles with deionized water. Place the glass slide on the sample stage of a dark-field microscope and record the dark-field image and the corresponding scattering spectrum with a dark-field microscope and a scattering spectrometer, respectively. The results are as Figure 14 shown. Figure 14 It is a high-selectivity graph for detecting MAO-B based on AuNS nanoparticles in dark-field mode. In the figure, Δλ = λ 1 −λ 0 , where λ 0 is the scattering peak wavelength of the system in the absence of biomolecules, and λ 1 is the scattering peak wavelength of the corresponding system in the presence of different biomolecules. As Figure 14 can be seen, only the presence of MAO-B can cause an approximately 160 nm blue shift in the scattering peak value, and an obvious change in the color of the scattered light from red to green. However, after adding BSA, GOX, HRP, and ALP, there is no obvious change in the scattering peak shift and the color of the scattered light.

[0078] Spiked recovery detection of MAO-B in serum samples in Example 8

[0079] To promote the application of this method in the field of clinical disease diagnosis, serum samples from healthy donors at the Southeast University Hospital were used. The standard addition method was adopted to detect the concentration of MAO-B, and the spike recovery experiments were carried out under two sensing modes of ultraviolet absorption and dark-field microscopy. The MAO-B content in the serum samples was detected by colorimetry. Different concentrations of MAO-B (the final concentration of MAO-B in the system was 0.05 μg / mL, 0.5 μg / mL, 1 μg / mL) were injected into the serum samples diluted 100 times and 3 μL of 100 mM benzylamine, and then incubated at 37 °C for 2 h. Then, 150 μL of AuNS nanoparticles and 18 μL of 50 mM Tollen's reagent were added to the system, and deionized water was added to make the total reaction volume 300 μL. Incubation continued at 37 °C for 1 h, and the resulting sample was used to collect the absorption spectrum with a UV-visible spectrophotometer. The MAO-B content in the serum samples was detected by the dark-field method. Different concentrations of MAO-B (the final concentration of MAO-B in the system was 0.5 ng / mL, 5 ng / mL, 10 ng / mL) were injected into the serum samples diluted 100 times and 3 μL of 1 mM benzylamine, and then incubated at 37 °C for 2 h. Then, 150 μL of AuNS nanoparticles and 18 μL of 0.5 mM Tollen's reagent were added to the system, and deionized water was added to ensure the total reaction volume was 300 μL. Incubation continued at 37 °C for 1 h. After the reaction was completed, 100 μL of the reaction solution was added to the PDMS channel of the amino-functionalized glass slide prepared in Example 1 and electrostatically adsorbed for 15 min, and then the excess nanoparticles were removed with deionized water. The glass slide was placed on the sample stage of the dark-field microscope, and the dark-field image and the corresponding scattering spectrum were recorded with a dark-field microscope and a scattering spectrometer, respectively. The dark-field microscope measured the absorption spectrum and the scattering spectrum. The experimental results are shown in Table 1.

[0080] Table 1

[0081]

[0082] As can be seen from Table 1, the recoveries of the actual samples detected by the two analysis modes varied between 91.74% and 96.11%, and the relative standard deviations were all within 5%. These results indicate acceptable recoveries and relative standard deviations, suggesting that the proposed sensing strategy has good performance and can be used to detect MAO-B in serum samples.

[0083] In the colorimetric detection mode, the blue shift value of the absorption peak shows a good linear relationship with the MAO-B concentration in the range of 0.01 - 1 μg / mL. The blue shift value of the LSPR absorption peak can reach about 80 nm, accompanied by an obvious change in the solution color from blue to yellow. Different concentrations of MAO-B can be identified by the naked eye according to the obvious color change of the solution. Due to the advantages of high sensitivity, high analysis precision for single particles, and low imaging background, dark-field microscopy has widely become a single-particle imaging analysis tool. Then, we adopted a dark-field scattering analysis strategy based on the established colorimetric method to improve the detection sensitivity. In the dark-field analysis mode, the scattering peak displacement value shows a good linear relationship with the MAO-B concentration in the range of 0.5 - 20 ng / mL. The blue shift value of the scattering peak can reach about 160 nm, accompanied by an obvious change in the scattered light color from red to green. The degree of peak blue shift during the scattering method detection is twice that of the absorption peak blue shift in the colorimetric method. The detection sensitivity of the dark-field method is about 200 times higher than that of the colorimetric method, significantly improving the analysis precision. The combined use of colorimetry and the dark-field method can first perform a preliminary quantification of MAO-B based on the multi-color response induced by the target. In addition, the dark-field analysis method shows a larger scattering spectral peak displacement, further improving the detection sensitivity.

Claims

1. A method for qualitatively and quantitatively detecting the concentration of MAO-B, characterized in that, it comprises the following steps: (1) Mix MAO-B solutions with different concentrations with a substrate benzylamine solution respectively, and incubate in a water bath to obtain a reaction solution, wherein the concentration of the benzylamine solution is 0.1 - 10 mM; (2) Add an AuNS nanoparticle solution and a Tollen's reagent to the above reaction solution, and continue the reaction in a water bath to obtain a post-reaction solution, wherein the concentration of the Tollen's reagent is 0.1 - 5 mM; (3) Establish the correspondence between the color change of the post-reaction solution and the concentration of MAO-B in the post-reaction solution; (4) Perform quantitative analysis and detection on the post-reaction solution at room temperature using an ultraviolet-visible absorption spectrum and a dark-field spectrometer respectively. Establish a corresponding standard curve for the relationship between the LSPR peak shift value Δλ obtained by ultraviolet-visible absorption spectrum detection and the concentration of MAO-B, and establish a corresponding standard curve for the relationship between the blue shift value Δλ of the scattering peak and the concentration of MAO-B when detected by a dark-field spectrometer; (5) Replace the MAO-B solutions with different concentrations in step (1) with the test sample, and repeat the operations in steps (1)-(2); (6) Qualitatively analyze MAO-B in the test sample and preliminarily quantify the concentration of MAO-B according to the color of the post-reaction solution of the test sample based on the correspondence in step (3); (7) Perform quantitative analysis and detection on the post-reaction solution of the test sample using an ultraviolet-visible absorption spectrometer and a dark-field spectrometer respectively. According to the obtained LSPR peak shift value Δλ and the blue shift value Δλ of the scattering peak, look up the concentration of MAO-B in the test sample on the standard curves in step (4); The preparation of the AuNS nanoparticle solution includes the following steps: Under vigorous stirring, 15 mL of 1% citrate solution is added to 100 mL of boiling 1 mM HAuCl 4 solution. After boiling for 15 minutes, while keeping the solution volume stable, the solution is cooled and filtered through a 0.22 μm nitrocellulose membrane. 500 μL of the above gold seed solution is added to 50 mL of 0.25 mM HAuCl containing 50 μL of 1 M HCl 4 solution. After stirring evenly, 1 mL of 0.6 mM AgNO 3 and 250 μL of 100 mM ascorbic acid are added simultaneously. The solution is stirred for 30 seconds, and then 500 μL of 10 mM sodium dodecyl sulfate (SDS) solution is continuously added, and the reaction continues at 25 °C for 5 min. The nucleation is stopped by centrifugal washing at a centrifugal force of 5000 g for 15 min in a centrifuge tube. The solution is redispersed in 50 mL of deionized water to obtain the AuNS nanoparticle solution.

2. The method for qualitatively and quantitatively detecting the concentration of MAO-B according to claim 1, characterized in that, in step (1), the water bath temperature is 36.5 - 37.5 °C, and the incubation time is 1.5 - 2.5 h.

3. The method for qualitatively and quantitatively detecting the concentration of MAO-B according to claim 1, characterized in that, in step (2), the particle size of the AuNS nanoparticles is 46.6 nm, and the Zeta potential is -38.2 mV.

4. The method for qualitatively and quantitatively detecting the concentration of MAO-B according to claim 1, characterized in that, in step (2), the temperature of the continued water bath reaction is 36.5 - 37.5 °C, and the reaction time is 1.5 - 2 h.

5. The method for qualitatively and quantitatively detecting the concentration of MAO-B according to claim 1, characterized in that, when analyzed and detected by an ultraviolet-visible absorption spectrometer, the concentration of MAO-B is 0.01 - 1 μg / mL, and when analyzed and detected by a dark-field spectrometer, the concentration of MAO-B is 0.5 - 20 ng / mL.

6. Application of the method for qualitatively and quantitatively detecting the concentration of MAO-B according to any one of claims 1 - 5 in the field of biosensing.

7. The application according to claim 6, characterized in that, the biological sample in the application of the method for qualitatively and quantitatively detecting the concentration of MAO-B in the field of biosensing is serum.

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