Plasmon imaging-based real-time monitoring system and method for nano-enzyme polymer degradation kinetics

Abstract

The invention discloses a real-time monitoring system and method for nano-enzyme degradation polymer dynamics based on plasmon imaging, and the system comprises a monochromatic laser emission module which is used for generating excitation light with a fixed wavelength, and adjusting a light path to excite surface plasmon resonance; the sample reaction module comprises a plasmon resonance gold sheet of which the surface is modified with a polymer molecular layer, and a sample pool for accommodating a reaction solution; the optical detection module is used for collecting and detecting scattered light signals from the sample reaction module and carrying out image acquisition; wherein the monochromatic laser emission module, the sample reaction module and the optical detection module are sequentially connected according to a light path; the invention provides a new important research means for researching the local change of the single nano enzyme and the interaction of biomolecules.

Description

Technical Field

[0001] This invention relates to the field of biochemical reaction monitoring technology, specifically to a real-time monitoring system and method for the degradation kinetics of polymers by nanozymes based on plasmon imaging. Background Technology

[0002] The application of nanozymes in the degradation of organic pollutants (such as dyes, antibiotics, and polymers) is a current research hotspot. Currently, existing techniques for monitoring catalytic degradation kinetics include Local Surface Plasmon Resonance (LSPR) sensing based on noble metal nanoparticles. This technique utilizes the LSPR properties of noble metal nanoparticles (such as gold and silver), whose resonance peak positions are extremely sensitive to the refractive index of the surrounding dielectric environment. By monitoring changes in the intensity or color of scattered light from individual nanoparticles during the catalytic reaction, the chemical reactions occurring on their surface can be inferred. This technique has been used to monitor some catalytic reactions, such as the decomposition of hydrogen peroxide catalyzed by platinum nanoparticles. However, the LSPR signal reflects the overall change in refractive index around the noble metal nanoparticle, rather than the generation or consumption of specific chemical substances. When the refractive index differences among degradation reactants, products, and intermediates are not significant, the signal changes are weak, making accurate differentiation and quantification difficult, resulting in insufficient specificity.

[0003] In summary, the current technology lacks a method that can perform highly specific, high spatiotemporal resolution, real-time, in-situ analysis of the important process of nanozyme degradation of organic polymers at the single-particle (plasmotropic nanozyme complex) level. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a real-time monitoring system and method for the degradation kinetics of polymers by nanozymes based on plasmon imaging, which solves the problem that existing technologies cannot study the degradation kinetics of nanozymes in real time, in situ, visualized and quantitatively at the single-particle level.

[0005] Technical Solution: The present invention discloses a real-time monitoring system for the degradation kinetics of polymers by nanoenzymes based on plasmon imaging, comprising: a monochromatic laser emission module for generating excitation light of a fixed wavelength and adjusting the optical path to excite surface plasmon resonance; a sample reaction module including a plasmon resonance gold sheet with a polymer molecular layer on its surface and a sample cell for containing the reaction solution; and an optical detection module for collecting and detecting the scattered light signal from the sample reaction module and performing image acquisition; wherein the monochromatic laser emission module, the sample reaction module, and the optical detection module are connected sequentially according to the optical path.

[0006] Furthermore, the monochromatic laser emission module includes: a monochromatic laser source, optical adjustment components, a high numerical aperture optical microscope objective lens, and a refractive index matching immersion oil; the optical adjustment components include a collimating lens, a p-polarizer, a condenser lens, and a semi-transparent mirror, used to adjust the beam direction, polarization state, and focusing position; a plasmonic resonance gold sheet with a polymer molecular layer on its surface is used to simulate the degradation reaction interface; the optical detection module includes a reflector and an image acquisition device, used to receive and record the scattered light signal.

[0007] Furthermore, the excitation wavelength of the monochromatic laser emitting module is 680 nm.

[0008] Furthermore, a glass substrate with a 2 nm chromium layer and a 47 nm gold layer was sequentially sputtered onto the plasmonic resonance gold sheet.

[0009] Furthermore, the polymer molecular layer is a polybutylene terephthalate (PBAT) film, which is applied to the surface of the gold sheet by spin coating.

[0010] Furthermore, the sample cell is made of polydimethylsiloxane (PDMS) material, which adheres to the surface of the gold sheet to form a closed reaction space.

[0011] Furthermore, the image acquisition device is a complementary metal-oxide-semiconductor (CMOS) camera with a shooting speed of no less than 5 frames per second.

[0012] The real-time monitoring method for polymer degradation kinetics based on plasmon imaging of nanoenzymes according to the present invention includes the following steps in its implementation: (1) Place the gold sheet modified with polymer molecular layer in the sample cell and add the reaction solution containing nanozyme; (2) The surface of the gold sheet is irradiated by a monochromatic laser emission module to excite surface plasmon resonance; (3) Acquire the scattered light image in real time through the optical detection module to obtain the plasmon intensity signal; (4) Calculate the positional change of the nanozyme during the reaction process based on the exponential decay relationship between plasmon intensity and nanozyme-gold sheet distance; (5) Based on the position change data, the reaction kinetics process is analyzed by the free energy distribution.

[0013] Furthermore, in step (4), the relationship between the plasmon intensity I and the distance z is as follows: I=I0e -z / L Where I0 is the SPR intensity at z=0, with a value of 200, and L is the decay constant, with a value of 100; the thermal fluctuation of the single-gold nanozyme along the z-direction is determined by the intensity of the delayed plasmon resonance image. Δz=100*(log(I0)-log(It )) Among them, I t Let t be the SPR signal intensity; by measuring the change in SPR intensity signal during the decomposition of polymer molecular layers by the single gold nanozyme, the position information of the single gold nanozyme in the z-direction is obtained.

[0014] Furthermore, in step (5), the free energy distribution is calculated using the following formula: The free energy distribution for different states is calculated using the relationship between the free energy G(z) and the probability distribution P(z): G(z) = -k B T log[AP(z)] Where A is a constant obtained by normalizing P(z), and k B Let be the Boltzmann constant and T be the temperature. The kinetics of the interaction between the single-gold nanozyme and the polymer molecular layer can be analyzed using the free energy distribution diagram. The reaction process can be analyzed by the energy and number of different stable states. Furthermore, the nanozyme is a complex formed by linking gold nanoparticles with Rhizopus oryzae lipase ROL via 11-mercaptoundecanoic acid 11-MUA.

[0015] Beneficial Effects: Compared with the prior art, the present invention has the following significant advantages: The present invention converts the SPR intensity change of a single nanozyme into information about the distance between the single nanozyme and the gold sheet surface, further obtaining the free energy information of the system during the reaction process and analyzing the kinetic process; The present invention applies a plasmonic resonance imaging module to the polymer degradation process of a single nanozyme, and obtains the ability to detect the reaction process between the single nanozyme and the polymer molecules modified on the gold sheet surface by directly detecting the SPR intensity change of the single nanozyme, providing a new and important research method for studying the local changes of single nanozymes and biomolecular interactions. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the structure of the present invention; Figure 2 These are plasmon images of the interaction between polymer molecular layers and enzyme molecules at different time points, as per the present invention. Figure 3 This is a schematic diagram illustrating the interaction process between the polymer molecular layer and enzyme molecules in this invention; Figure 4 A is a plasmon image of a single gold nanoparticle of the present invention; Figure 4 B represents the difference in optical intensity along the transverse line at different time points during the interaction between a single gold nanoparticle and the polymer molecular layer in this invention. Figure 4 C is a pseudo-color image of the optical intensity along the transverse line in real time during the interaction between a single gold nanoparticle and the polymer molecular layer of the present invention. Figure 5 A is a pseudo-color image of plasmon resonance during the initial stage of the interaction between a single gold nanozyme particle and the polymer molecular layer of the present invention. Figure 5 B is a pseudo-color image of plasmon resonance during the mid-term interaction between a single gold nanozyme particle and the polymer molecular layer of the present invention. Figure 5 C is a pseudo-color image of plasmon resonance during the later stage of the interaction between a single gold nanozyme particle and the polymer molecular layer in this invention. Figure 5 D represents the time-dependent change in plasmon intensity during the interaction between a single gold nanoparticle and the polymer molecular layer in this invention. Figure 6 The value of Δz is the interaction between a single gold nanoparticle and the polymer molecular layer in this invention. Figure 7 A represents the Δz value and its corresponding state in the first stage of the interaction between a single gold nanoparticle and the polymer molecular layer in this invention. Figure 7 B represents the probability distribution of Δz values ​​in the first stage of the interaction between a single gold nanoparticle and the polymer molecular layer in this invention; Figure 7 C represents the free energy spectrum of the first stage of the interaction between a single gold nanozyme particle and the polymer molecular layer in this invention; Figure 8 A represents the Δz value and its corresponding state in the second stage of the interaction between a single gold nanoparticle and the polymer molecular layer in this invention. Figure 8 B represents the probability distribution of Δz values ​​in the second stage of the interaction between a single gold nanoparticle and the polymer molecular layer in this invention. Figure 8 C represents the free energy spectrum of the second stage of the interaction between a single gold nanozyme particle and the polymer molecular layer in this invention. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings.

[0018] like Figure 1 As shown in the figure, this invention provides a real-time monitoring system for the polymer degradation kinetics of nanoenzymes based on plasmon imaging, comprising: Monochrome laser emission module: Monochrome laser source 11, used to provide excitation light of a fixed wavelength; Optical adjustment assembly 12, including collimating lens 121, p-polarizer 122, condenser lens 123 and semi-transparent mirror 124, used to adjust the optical path direction, polarization state and focusing position; High numerical aperture optical microscope objective lens 13, used to amplify the optical path signal; Refractive index matching immersion oil 14, used to improve the optical path coupling efficiency.

[0019] Sample reaction module: a plasmon resonance gold sheet 21 with a polymer molecular layer on its surface; a sample cell 22 for containing the reaction solution. Optical detection module: a reflector 31 for adjusting the direction of the light path; an image acquisition unit 32 for receiving and recording the scattered light image. The monochromatic laser emission module, sample reaction module, and optical detection module are sequentially connected by optical paths to form a complete real-time monitoring system.

[0020] Among them, the gold sheet preparation: a 2 nm chromium layer and a 47 nm gold layer are sequentially sputtered on a glass substrate using magnetron sputtering technology to form a plasmonic resonance substrate.

[0021] Polymer modification: A uniform polymer molecular layer, such as polybutylene terephthalate (PBAT), is formed on the surface of the gold sheet by spin coating.

[0022] Nanozyme preparation: Gold nanoparticles are combined with 11-mercaptoundecanoic acid (11-MUA), and then activated by EDC / NHS to connect with enzyme molecules (such as Rhizopus oryzae lipase ROL) to form a single-particle nanozyme complex.

[0023] Image acquisition: A high-speed, low-noise CMOS camera is used, with a shooting speed of no less than 5 frames per second to ensure real-time dynamic capture.

[0024] A real-time monitoring method for the polymer degradation kinetics of nanozymes based on plasmon imaging, the system implementation of which includes the following steps: (1) Place the gold sheet modified with polymer molecular layer in the sample cell and add the reaction solution containing nanozyme; (2) The surface of the gold sheet is irradiated by a monochromatic laser emission module to excite surface plasmon resonance; (3) Acquire the scattered light image in real time through the optical detection module to obtain the plasmon intensity signal; (4) Calculate the positional change of the nanozyme during the reaction process based on the exponential decay relationship between plasmon intensity and nanozyme-gold sheet distance; (5) Based on the position change data, the reaction kinetics process is analyzed by the free energy distribution.

[0025] Among them, the plasmon intensity I and the distance z between the nanozyme and the gold sheet surface satisfy an exponential decay relationship: The relationship between plasmon intensity I and distance z is as follows: I=I0e -z / L Where I0 is the SPR intensity at z=0, with a value of 200, and L is the decay constant, with a value of 100; the thermal fluctuation of the single-gold nanozyme along the z-direction is determined by the intensity of the delayed plasmon resonance image. Δz=100*(log(I0)-log(It )) Among them, I t Let t be the SPR signal intensity; by measuring the change in SPR intensity signal during the decomposition of polymer molecular layers by the single gold nanozyme, the position information of the single gold nanozyme in the z-direction is obtained.

[0026] Calculate the free energy distribution: Using the relationship between the free energy G(z) and the probability distribution P(z), calculate the free energy distribution for different states. G(z) = -k B T log[AP(z)] Where A is a constant obtained by normalizing P(z), and k B Here, denoted by Boltzmann's constant, and T represents temperature. The kinetics of the interaction between single-gold nanozymes and polymer molecular layers can be analyzed using free energy distribution diagrams. The reaction process can be analyzed by examining the energy and number of different stable states.

[0027] Example 1

[0028] This embodiment establishes a real-time monitoring system for surface plasmon resonance imaging of the polymer degradation process by single-gold nanoenzymes. The system includes a monochromatic laser emission module, a sample reaction module, and an optical detection module.

[0029] Monochromatic laser emission module: This system introduces a monochromatic laser source into plasmonic imaging as the excitation source for a gold-film glass slide exhibiting plasmonic resonance. In this embodiment, a 680 nm monochromatic red laser is selected as the excitation source. By controlling the intensity of the light source, the optimal scattering image and signal of a single particle are obtained. The monochromatic light passes through a p-polarizer capable of generating specific polarized light and exciting surface plasmonic resonance, and a 60x high numerical aperture optical magnifying lens. It then passes through a refractive index-matched immersion oil to irradiate the surface of the gold-film glass slide.

[0030] Sample reaction module: The sample reaction module is constructed using a gold glass slide and a polydimethylsiloxane (PDMS) sample cell, with phosphate-buffered saline (PBS) containing gold nanozymes added. First, a gold-filmed glass slide is obtained by sequentially sputtering 2 nm thick chromium and 47 nm thick gold onto a 22 mm × 22 mm glass slide using magnetron sputtering. The gold-filmed glass slide is rinsed with ultrapure water and dried with nitrogen before use. Polybutylene terephthalate (PBAT) is dissolved in N,N-dimethylformamide (DMF). PBAT is then spun onto the gold glass slide surface using a spin coater at 1000 rpm for 5 seconds. This ensures the formation of a stable and uniform PBAT molecular layer on the gold film surface. During the experiment, a sample cell made of polydimethylsiloxane (PDMS) was attached to the surface of a gold film glass slide to construct a sample cell that can hold the solution and perform gold nanozyme imaging detection. Before use, it needs to be rinsed with ethanol and deionized water in sequence and then dried with nitrogen gas.

[0031] Optical detection module: In terms of detectors, a complementary metal-oxide-semiconductor high-speed low-noise camera (CMOS) is used as an image acquisition device to directly detect the scattering intensity value and image changes on the surface of the gold film glass, which can greatly improve the temporal and spatial resolution of optical imaging.

[0032] After determining the optimal incident light intensity and resonance angle, an image acquisition device was used to record the surface plasmon images of the polymer molecular layer modified with gold film glass under the action of enzyme molecules, and the images were transmitted to a computer for signal extraction and analysis using image processing software such as Matlab and ImageJ.

[0033] The dynamic interaction between the matrix polymer molecular layer and the degrading enzyme was monitored using surface plasmon resonance imaging (SPR). A schematic diagram of the experimental setup is shown below. Figure 1 As shown in the figure. The experiment used SPR microscopy to capture images of the interaction between polymer PBAT and Rhizopus oryzae lipase molecules (ROL) over 10 hours, as shown in the figure. Figure 2 Experiments revealed that the PBAT membrane ruptured and exhibited spatial heterogeneity as the interaction time increased.

[0034] Example 2

[0035] The following example, using the interaction between Au / ROL single-gold nanozyme and PBAT molecular layer, explores the system and method involved in this invention. The specific experimental scheme is as follows: Preparation of single-gold nanozymes: Gold nanoparticles were combined with 11-mercaptoundecanoic acid (11-MUA), and then activated with a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) before being linked to enzyme molecules: 300 μL of 1 mM gold nanoparticles (Au NP) with a diameter of 150 nm were added to an ethanol solution of 100 μL of 1 mM 11-mercaptoundecanoic acid (11-MUA), and stirred overnight at 4 °C. The carboxyl groups on the surface were activated by adding a mixed solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) in a molar ratio of 4:1 prepared with PBS at pH=6. Then, 15 μL of 1 mg / mL ROL solution was added and the mixture was stirred at 4 °C for 12 hours to obtain Au NP@ROL single gold nanozyme.

[0036] SPR images and intensities of gold nanozymes on PBAT-modified gold glass slides: First, a PDMS cell was used as the sample cell, rinsed with ethanol and deionized water, and dried with nitrogen. It was then placed on a PBAT-modified gold glass slide, and 200 μL of PBS solution was added. The SPR optical path and receiving angle were adjusted to the optimal imaging state, and 50 μL of Au NP@ROL gold nanozyme solution was added. Simultaneously, SPR image acquisition began at a camera speed of 5 frames per second for 600 seconds. ROL is an enzyme molecule with relatively low accessibility to its active site. Figure 3 A schematic diagram illustrating the polymer degradation process. (Example) Figure 4 This invention acquired SPR images and intensity signal changes during the degradation of polymers by a single gold nanozyme. It also obtained the positional information of the single gold nanozyme in the z-direction. The results show that the device involved in this invention can accurately and effectively monitor the dynamic interaction between the gold nanozyme and the polymer molecular layer by measuring changes in SPR intensity.

[0037] The imaging film was a glass slide sequentially sputtered with 2 nm chromium and 47 nm gold. A polymer-modified gold glass slide was obtained via spin coating. A monochromatic LED laser was introduced into the imaging system as the excitation source for plasmon imaging. A p-polarizer and a semi-transparent mirror were used to adjust the direction of the incident light. A sample cell made of polydimethylsiloxane (PDMS) was adhered to the pre-cleaned gold film glass slide surface to construct a sample cell capable of single-gold nanozyme imaging. Scattered light generated by the resonant excitation of a single gold nanoparticle was collected through a 60x magnification optical microscope objective with a numerical aperture of 1.49. A matching immersion oil with a refractive index of 1.51 was filled between the objective and the gold slide. Figure 4-5As shown, real-time SPR images and intensity signal changes over time of single-gold nanozymes can be obtained, capturing the dynamic process of the dynamic interaction between gold nanozymes and polymer molecular layers. It can be observed that the degradation rate of the polymer molecular layer by the nanozymes varies, and the degradation sites tend to become more complex.

[0038] The intensity changes of the SPR image of the single-gold nanozyme were converted into the distance z between the single-gold nanozyme and the gold substrate surface. Based on the fact that the plasmon pattern intensity (I) decreases exponentially with the distance (z) between the single-gold nanozyme and the substrate:

[0039] I=I0 e -z / L

[0040] Where I0 is the SPR intensity at z=0, with a value of 200, and L is the decay constant, with a value of 100; the thermal fluctuation of the single-gold nanozyme along the z-direction is determined by the intensity of the delayed plasmon resonance image.

[0041] Δz=100*(log(I0)-log(I t ))

[0042] Among them, I t Let be the SPR signal intensity at time t; such as Figure 6 As shown, the position of the gold nanozyme in the z-direction was obtained by measuring the change in SPR intensity signal during the degradation of the polymer molecular layer by the gold nanozyme. It was found that the distance between the gold nanozyme and the substrate decreased over time, successfully capturing the degradation process of the polymer molecular layer. The figure shows that the interaction states between the nanozyme and the polymer molecular layer increased from two to three or even more. We simply divided the initial degradation process into two stages for analysis.

[0043] Calculate the free energy distribution: Using the relationship between the free energy G(z) and the probability distribution P(z), calculate the free energy distribution for different states.

[0044] G(z) = -k B T log[AP(z)]

[0045] Where A is a constant obtained by normalizing P(z), and k B Let T be the Boltzmann constant and T be the temperature. Figure 7-8As shown, in the first stage, there are two stable states, with State 2 being more stable. This is because State 2 represents the state where the system tends towards the binding of the single gold nanozyme with the polymer molecules. That is, the interaction between the nanozyme and the polymer molecular layer tends to shift towards a binding configuration more prone to degradation. In the second stage, the probability distribution range of the z-shift decreases, and there are three or more stable states. Among them, State 2' is the most stable, and the appearance of State 1' indicates that the nanozyme gradually degrades from the surface of the polymer molecular chain to deeper layers. In summary, the nanozyme continuously degrades the polymer, shortening the polymer molecular chain. Subsequently, the binding sites between the nanozyme and the polymer molecular chain become more complex. It was also found that the rate at which the nanozyme approaches the gold sheet slows down, meaning the rate at which the nanozyme penetrates the polymer molecular layer decreases.

[0046] This result demonstrates that the device can accurately and rapidly determine the kinetics of polymer molecular layer degradation by single-gold nanozymes.

Claims

1. A system for real-time monitoring of nanoscale enzyme degradation polymer dynamics based on plasmonic imaging, characterized in that, The system comprises: a monochromatic laser emission module for generating excitation light of a fixed wavelength and adjusting the light path to excite surface plasmon resonance; a sample reaction module comprising a plasmonic gold sheet with a polymer molecular layer on the surface and a sample cell for containing a reaction solution; an optical detection module for collecting and detecting scattered light signals from the sample reaction module and performing image acquisition; wherein the monochromatic laser emission module, the sample reaction module and the optical detection module are sequentially connected in the light path.

2. The system for real-time monitoring of nanoscale enzyme degradation kinetics based on plasmonic imaging according to claim 1, wherein, The monochromatic laser emission module comprises a monochromatic laser light source (11), an optical adjustment assembly (12), a high numerical aperture optical microscopic magnifying objective lens (13) and a refractive index matching mirror oil (14); the optical adjustment assembly (12) comprises a collimating lens (121), a p-polarizer (122), a condenser lens (123) and a semi-transparent half-mirror (124) for adjusting the direction, polarization state and focusing position of the light beam; the plasmonic gold sheet with a polymer molecular layer on the surface is used to simulate the interface of a degradation reaction; the optical detection module comprises a mirror (31) and an image acquisition device (32) for receiving and recording scattered light signals.

3. The system for real-time monitoring of nanoscale enzyme degradation kinetics based on plasmonic imaging according to claim 1, wherein, The excitation light wavelength of the monochromatic laser emission module is 680 nm.

4. The system for real-time monitoring of nanoscale enzyme degradation kinetics based on plasmonic imaging according to claim 1, wherein, The plasmonic gold sheet is a glass substrate sputtered with a 2 nm chromium layer and a 47 nm gold layer in sequence.

5. The system for real-time monitoring of nanoscale enzyme degradation kinetics based on plasmonic imaging according to claim 1, wherein, The polymer molecular layer is a polybutylene adipate terephthalate (PBAT) film modified on the surface of the gold sheet by a glue spinning and spin coating method.

6. The system for real-time monitoring of nanoscale enzyme degradation kinetics based on plasmonic imaging according to claim 1, wherein, The sample cell is made of polydimethylsiloxane (PDMS) and adheres to the surface of the gold sheet to form a sealed reaction space.

7. A method for real-time monitoring of the kinetics of polymeric degradation by nanoenzymes based on plasmonic imaging, characterized by, The system is implemented according to any one of claims 1-6, comprising the following steps: (1) placing the gold sheet modified with a polymer molecular layer in the sample cell and adding a reaction solution containing nanoscale enzymes; (2) irradiating the surface of the gold sheet by the monochromatic laser emission module to excite surface plasmon resonance; (3) acquiring scattered light images in real time by the optical detection module to obtain plasmonic intensity signals; (4) calculating the position change of the nanoscale enzymes during the reaction process according to the exponential decay relationship between the plasmonic intensity and the distance between the nanoscale enzymes and the gold sheet; (5) analyzing the reaction kinetics process based on the position change data through free energy distribution.

8. The method of real-time monitoring of nanoscale enzyme degradation kinetics based on plasmonic imaging according to claim 7, wherein, In step (4), the relationship between the plasmonic intensity I and the distance z is: I = I0 e -z / L ; Wherein, I0 is the SPR intensity at z=0, the value is 200, L is the decay constant, the value is 100; the thermal fluctuation of single nanoszyme along the z direction is determined by the time-delay plasmonic image intensity: Δz=100*(log(I0)-log(I t )); where I t SPR signal intensity at time t; by measuring the change in SPR intensity signal during the process of polymer molecular layer on single gold nanoszyme, the position information of single gold nanoszyme in z direction is obtained.

9. The method of real-time monitoring of nanoscale enzyme degradation kinetics based on plasmonic imaging according to claim 7, wherein, In step (5), the free energy distribution is calculated by the following equation: The free energy distribution of different states is calculated by the relationship between the free energy G(z) and the probability distribution P(z): G(z) = -k B T log[AP(z)]; where A is a constant obtained by normalizing P(z), k B is the Boltzmann constant, and T is the temperature. The dynamics of the interaction between the single nanoszyme and the polymer molecular layer were analyzed by free energy distribution images; the reaction process was analyzed by the energy and number of different stable states.

10. The method of real-time monitoring of nanoscale enzyme degradation kinetics based on plasmonic imaging according to claim 7, wherein, The nanoscale enzyme is a complex of gold nanoparticles and Rhizopus oryzae lipase (ROL) connected by 11-mercapto-undecanoic acid (11-MUA).

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