An enzyme activity optimization feedback device based on a passive resonant cavity and a working method thereof

Abstract

The application provides an enzyme activity optimization feedback device based on a passive resonant cavity and a working method thereof, and belongs to the field of enzyme reaction control devices. The device comprises a tunable passive resonant cavity, an electrochemical sensing device and a microfluidic system. A dynamic optical length interval controlled by a piezoelectric ceramic translation stage is arranged in the passive resonant cavity, the electrochemical sensing device is used for real-time monitoring of enzyme product concentration and high-throughput screening, and the microfluidic system is used for controlling the input and output of the enzyme reaction system. The application dynamically adjusts the optical length of the passive resonant cavity by using piezoelectric materials, so as to realize real-time matching with the vibration mode of the active site of enzyme molecules, realize adaptive optimization of enzyme activity, and break through the limitation of static regulation of enzyme immobilization technology; the microfluidic system is combined with the electrochemical sensor to realize real-time monitoring of enzyme product concentration and high-throughput screening, and realize the closed-loop sensing feedback of "detection-regulation-reoptimization".

Description

Technical Field

[0001] This invention relates to the field of enzyme reaction control device technology, and more particularly to an enzyme activity optimization feedback device based on a passive resonant cavity and its working method. Background Technology

[0002] Traditional enzyme catalysis relies on chemical modification of active sites to enhance enzyme reusability and stability. However, this also presents challenges such as enzyme conformational distortion, reduced accessibility to active sites, and high modification costs. In particular, metal ion induction can lead to metal ion contamination. Therefore, novel activity optimization strategies are needed to overcome the bottleneck of the inability to synergistically optimize enzyme catalytic efficiency and stability in traditional technologies.

[0003] As an emerging tool for regulating molecular energy states, strong vibrational coupling can control chemical reactions by directly altering the molecular energy structure. Under dark conditions, resonant coupling with molecular vibrational transitions can be achieved by adjusting the optical modes of a passive resonant cavity (Fabry-Perot cavity, FP microcavity), thereby changing the reaction energy barrier by modulating the vibrational modes. The coupling mechanism between the passive resonant cavity and molecular vibrational modes provides a novel, catalyst-free, and "green" approach to enzyme activity regulation.

[0004] Current research has proposed applications in passive resonant cavities, such as enhancing α-trypsin activity, regulating the decomposition of ethyl p-nitrophenylacetate, and increasing the ATP hydrolysis rate. However, the detection of the regulatory effect in most reactions requires the recovery of reaction products, which suffers from drawbacks such as low product recovery rate and low reproducibility of optimization effects. To improve upon these methods, the structure of the passive resonant cavity must be modified to achieve a closed-loop intelligent feedback mechanism for optimizing enzyme activity through an integrated "regulation-reaction-detection" system. Summary of the Invention

[0005] To overcome the shortcomings of existing technologies, this invention provides an enzyme activity optimization feedback device based on a passive resonant cavity and its working method.

[0006] The present invention is achieved through the following technical solution: an enzyme activity optimization feedback device based on a passive resonant cavity, comprising a tunable passive resonant cavity and an electrochemical sensing feedback device;

[0007] The tunable passive resonant cavity includes a passive resonant cavity and a piezoelectric ceramic displacement controller;

[0008] The passive resonant cavity comprises, from the inside out, a set of MYLAR film gaskets, two CaF2 windows, two gaskets, and a stainless steel infrared liquid cell. The MYLAR film gaskets are one or more PET transparent polyester sheets with a thickness of 2.5 μm. The opposing surfaces of the two CaF2 windows are the modified surfaces. After forming a gold film on the modified surfaces using a vacuum ion sputtering instrument, a polymethyl methacrylate (PMMA) solution is spin-coated, and then a layer of immobilized carrier ZIF-8 is modified. The upper CaF2 window has a first through hole and a second through hole at its edge. The modified surfaces of the two CaF2 windows are opposite each other and separated by the MYLAR film gaskets. The upper CaF2 window is tightly attached to the upper fixed substrate of the stainless steel infrared liquid cell through the gaskets. The first and second through holes have the same radius as the injection holes of the upper fixed substrate of the stainless steel infrared liquid cell and are aligned with each other. The lower CaF2 window is tightly attached to the lower fixed substrate of the stainless steel infrared liquid cell through the gaskets.

[0009] The passive resonant cavity is fixed to the piezoelectric ceramic displacement controller by bolts. The piezoelectric ceramic displacement controller controls the high voltage drive circuit to cause the piezoelectric ceramic to produce electrostriction. The piezoelectric ceramic displacement controller adjusts the optical length of the passive resonant cavity through electrostriction.

[0010] Electrochemical sensing feedback devices include microfluidic systems and electrochemical sensing systems;

[0011] The microfluidic system includes a dual-channel microfluidic chip, channel in and channel out. The two ends of channel in are A and B, respectively, and the two ends of channel out are C and D, respectively. The A end of channel in is the sample inlet of the dual-channel microfluidic chip. The B end of channel in is connected to the first through-hole of the passive resonant cavity. The C end of channel out is connected to the second through-hole of the passive resonant cavity. The D end of channel out is connected to the electrochemical sensing system.

[0012] The electrochemical sensing system comprises a three-electrode detection system assembled with a specific glassy carbon electrode as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, and connected to the D end of the microfluidic system channel out; the surface of the glassy carbon electrode is functionalized.

[0013] As a preferred option, the thickness of the MYLAR film gasket is less than or equal to the optical length of the passive resonant cavity.

[0014] As a preferred option, the CaF2 window has a transmission wavelength range of 0.256 ~ 10 μm and a refractive index N. 633 =1.45.

[0015] Furthermore, after forming a gold film on the modified surface using a vacuum ion sputtering instrument, the parameters for the vacuum ion sputtering instrument are: 5 ~ 6 Pa, 5 mA, 60 s.

[0016] Furthermore, the concentration of the polymethyl methacrylate (PMMA) solution is 10% (w / w).

[0017] A method for operating an enzyme activity optimization feedback device based on a passive resonant cavity includes the following steps:

[0018] Step S1: Assemble the tunable passive resonant cavity:

[0019] Two CaF2 windows are positioned with their modified surfaces facing each other, separated by a MYLAR film gasket. The upper CaF2 window is tightly attached to the upper fixed substrate of the stainless steel infrared liquid cell via a gasket. The first and second through holes have the same radius as the injection holes of the upper fixed substrate of the stainless steel infrared liquid cell and are aligned with each other. The lower CaF2 window is tightly attached to the lower fixed substrate of the stainless steel infrared liquid cell via a gasket. The passive resonant cavity is fixed to the piezoelectric ceramic displacement controller by bolts, forming a hollow passive resonant cavity. The cavity is filled by inputting 10 μL of the catalytic substrate-enzyme reaction system through the injection hole and the first through hole on the stainless steel infrared liquid cell.

[0020] Step S2, Optical length of the tunable passive resonant cavity:

[0021] The high-voltage drive circuit controls the piezoelectric ceramic displacement controller to generate electrostriction. The piezoelectric ceramic displacement controller adjusts the optical length of the passive resonant cavity through electrostriction: 6 μm corresponds to the C=O vibration of the amide I band (~1650 cm). -1 The 8.4 μm corresponds to the vibrational frequency of the PO bond, and the 28.2 μm passive resonant cavity can resonate with the 13th mode of the carbonyl (C=O) stretching vibration; the 6.05 μm corresponds to the 5th mode of the OH stretching vibration of the water molecule, and the 25.4 μm corresponds to the 21st mode of the OH stretching vibration of the water molecule.

[0022] Step S3, Optical Length Inspection:

[0023] The actual optical length of the passive resonant cavity is verified by calculation using infrared transmission spectra. The calculation formula can be expressed as:

[0024]

[0025]

[0026] in L , m , n and v Representing cavity pitch and cavity mold respectively m = 1, 2, 3, ...), and the refractive index and wavenumber of the medium; FSR Free spectral range refers to when m When = 1, the average distance between two adjacent peaks in the infrared spectrum;L 1 This represents the actual distance between the two gold surfaces in a first-order cavity resonator; while when L = L 1 At that time, the optimal condition for strong vibration coupling is met;

[0027] Step S4: Establishment of an intelligent feedback system:

[0028] The microfluidic system connects a tunable passive resonant cavity in series with an electrochemical sensing system to form an intelligent feedback system;

[0029] The microfluidic system's channel in controls the flow rate from 0.5 to 10 μL / min. Terminal A of channel in is the sample inlet of the dual-channel microfluidic chip. Terminal B of channel in connects to the first through-hole of the passive resonant cavity, allowing the input of enzyme solution and enzyme substrate. These are immobilized by the Y-type mixer of the microfluidic system, forming the site for the enzyme catalytic reaction. A micro heater at 25–50 °C is integrated, and the pH range of the reaction buffer is controlled from 4 to 7. Terminal C of channel out connects to the second through-hole of the passive resonant cavity, outputting the enzyme catalytic product. Terminal D of channel out connects to the electrochemical sensing system.

[0030] The electrochemical sensing system comprises a three-electrode detection system assembled with a specific glassy carbon electrode as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, and connected to the D end of the microfluidic system channel out;

[0031] Step S5: Electrochemical detection and analysis of enzyme activity regulation mechanisms:

[0032] A three-electrode detection system was assembled using a specific glassy carbon electrode as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, and connected to the D-terminus of the microfluidic system channel out. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were performed using the three-electrode system in PBS with pH 4–7. Utilizing the structural differences between the enzyme-catalyzed substrate and product, the potential differences and electron transfer rates during redox reactions on the electrode surface lead to different peak positions. The substrate concentration affects the current signal value. By detecting the peak position (voltage) and magnitude (current), the concentration of catalyzed products under different enzyme activities was determined, establishing a quantitative relationship between the substrate degradation rate v and the coupling strength g, and analyzing the regulatory mechanism of strong coupling on enzyme activity.

[0033] As a preferred option, the DPV parameters in step S5 are set as follows: scan range -0.2~0.8 V, pulse amplitude 0.05 V, and pulse width 0.05 s.

[0034] As a preferred embodiment, the enzyme solution and enzyme catalytic substrate in step S4 are laccase solution and catalytic substrate 2,4-DCP, respectively.

[0035] Furthermore, the surface of the glassy carbon electrode was polished with alumina slurry and washed alternately with ethanol and ultrapure water; 3-7 μL of composite material solution was rapidly drop-coated onto the polished electrode surface to form an electron transfer interface; after drying, it was covered with PSBMA ion exchange resin to improve the sensor's anti-fouling ability and construct a specific electrochemical sensor; the composite material consisted of multi-walled carbon nanotubes (MWCNTs) and COF-366-Zn dispersed in PBS, and ultrasonic treatment was used to obtain a uniformly dispersed composite suspension.

[0036] Furthermore, COF-366-Zn was obtained by imine condensation reaction, using tetraaminophenylporphyrin zinc and aldehyde compounds as precursors, and ultrasonically treated with THF / DMF / o-DCB / n-BuOH until completely dissolved. The mixture was then heated in an oil bath at 120 °C. After the reaction was completed, the solid product was recovered by centrifugation and washed sequentially with THF, DMF and methanol to remove unreacted monomers and solvent residues. The product was then vacuum dried to obtain powdered hollow microspheres COF-366-Zn.

[0037] By employing the above technical solutions, this invention has the following beneficial effects compared to existing technologies:

[0038] (1) This invention utilizes piezoelectric materials to dynamically adjust the optical cavity length of the FP microcavity. When the optical length of the passive resonant cavity is matched with the vibration mode of the enzyme active site in real time, the enzyme activity can be "adaptively" optimized without the need for a quantum cascade emitter to provide energy. The resonant coupling mechanism breaks through the limitations of static regulation in enzyme immobilization technology.

[0039] (2) This invention achieves automated control of the enzyme catalysis process by precisely controlling the input, reaction and output of enzyme and substrate through a microfluidic system; and it is used in conjunction with an electrochemical sensor to monitor the product concentration in real time for high-throughput screening, realizing a closed-loop sensing feedback of "detection-regulation-re-optimization", integrating enzyme activity optimization and product detection on the same platform, and has the potential for intelligent sensing.

[0040] Additional aspects and advantages of the invention will become apparent in the following description or may be learned by practice of the invention. Attached Figure Description

[0041] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0042] Figure 1 Schematic diagram of a passive resonant cavity enzyme activity optimization feedback device;

[0043] Figure 2 Schematic diagram of the modification principle of the electrochemical sensor;

[0044] Figure 3A physical diagram of the assembly components for a passive resonant cavity;

[0045] Figure 4 This is a schematic diagram of a passive resonant cavity assembly.

[0046] Figure 5 The diagram shows the coupling effect of the passive resonant cavity at different optical lengths. Detailed Implementation

[0047] To better understand the above-mentioned objectives, features, and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features described in these embodiments can be combined with each other.

[0048] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and therefore the scope of protection of the invention is not limited to the specific embodiments disclosed below.

[0049] The following is combined with Figures 1 to 3 The enzyme activity optimization feedback device based on a passive resonant cavity and its working method according to embodiments of the present invention will be described in detail.

[0050] like Figure 1 , Figure 2 As shown, this invention proposes an enzyme activity optimization feedback device based on a passive resonant cavity, comprising a tunable passive resonant cavity and an electrochemical sensing feedback device.

[0051] A tunable passive resonant cavity is created by fixing a passive resonant cavity (Fabry-Perot cavity, FP microcavity) mirror on a piezoelectric ceramic translation stage, precisely controlling the optical length of the passive resonant cavity to any fixed position within the range of 5 to 35 μm. A tunable passive resonant cavity includes the passive resonant cavity and a piezoelectric ceramic displacement controller.

[0052] like Figure 1 As shown, the passive resonant cavity, from the inside out, includes one set of MYLAR film gaskets, two CaF2 windows, two gaskets, and one stainless steel infrared liquid cell. The MYLAR film gaskets are one or more PET transparent polyester sheets with a thickness of 2.5 μm (total thickness ≤ optical length), selected according to the optical length of the passive resonant cavity. The transmission wavelength range of the CaF2 windows is between 0.256 and 10 μm, and the refractive index N0 is... 633= 1.45. The opposing surfaces of the two CaF2 windows are the modified surfaces. A gold film is formed on the modified surfaces using a vacuum ion sputtering instrument with the following parameters: 5 ~ 6 Pa, 5 mA, 60 s. Then, a 10% (w / w) polymethyl methacrylate (PMMA) solution is spin-coated onto the surface. A smooth, stable, and reusable "gold" reflective layer is obtained, which is then modified with a ZIF-8 immobilized support. A MYLAR membrane gasket is installed between the opposing modified surfaces of the two CaF2 windows, and together with the infrared liquid cell and the gasket, they form a passive resonant cavity. The MYLAR membrane gasket and the windows form an intermediate layer, serving as the site for enzyme catalysis. The upper CaF2 window has a first through-hole and a second through-hole at its edge for subsequent assembly (e.g., ...). Figure 3 The modified surfaces of the two CaF2 windows face each other and are separated by a MYLAR film gasket. The upper CaF2 window is tightly attached to the upper fixed substrate of the stainless steel infrared liquid tank through the gasket. The first and second through holes have the same radius as the injection holes of the upper fixed substrate of the stainless steel infrared liquid tank and are aligned with the center. The lower CaF2 window is tightly attached to the lower fixed substrate of the stainless steel infrared liquid tank through the gasket.

[0053] The passive resonant cavity is fixed to the piezoelectric ceramic displacement controller by bolts. The high voltage drive circuit is precisely controlled to make the piezoelectric ceramic electrostrictive. The piezoelectric ceramic displacement controller adjusts the optical length of the passive resonant cavity through electrostriction.

[0054] Electrochemical sensing feedback devices include microfluidic systems and electrochemical sensing systems;

[0055] The microfluidic system includes a dual-channel microfluidic chip, an in channel, and an out channel. The two ends of the in channel are A and B, respectively, and the two ends of the out channel are C and D, respectively. The A end of the in channel is the sample inlet of the dual-channel microfluidic chip. The B end of the in channel is connected to the first through-hole of the passive resonant cavity. For the laccase solution-catalyst 2,4-DCP reaction system, laccase solution and enzyme catalytic substrate 2,4-DCP are input and immobilized through the Y-type mixer of the microfluidic system, forming the site for enzyme catalytic reaction. A micro heater with an integrated temperature of 25-50 °C is also included. The C end of the out channel is connected to the second through-hole of the passive resonant cavity, outputting the enzyme catalytic product. The D end of the out channel is connected to the electrochemical sensing system.

[0056] The electrochemical sensing system comprises a three-electrode detection system assembled with a specific glassy carbon electrode as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, connected to the D-terminus of the microfluidic system channel out. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were performed in PBS using this three-electrode system. Utilizing the structural differences between the substrate and product catalyzed by laccase, the potential differences and electron transfer rates during redox reactions at the electrode surface lead to different peak positions. The substrate concentration affects the current signal value. By detecting the peak position (voltage) and magnitude (current), the concentration of catalyzed products under different laccase activities was determined, establishing a quantitative relationship between substrate degradation rate (v) and coupling strength (g), and analyzing the regulatory mechanism of strong coupling on enzyme activity.

[0057] The enzyme solution can be a variety of biological enzymes capable of degrading organic pollutants (such as laccase, urease, phosphotriesterase, horseradish peroxidase, biphenyl dehydrogenase, biphenyl meta-product hydrolase, mucoconazole dehalogenase, fluoroacetate dehalogenase, flavin adenine dinucleotide-dependent nitroso synthase, and glutathione transferase). Based on different enzyme solutions and catalytic substrates, corresponding specific working electrodes are constructed for subsequent steps.

[0058] For the laccase solution-catalyst 2,4-DCP reaction system, the surface of the glassy carbon electrode is coated with a composite material and covered with PSBMA ion exchange resin; the composite material is a composite suspension of multi-walled carbon nanotubes (MWCNTs) and COF-366-Zn dispersed in PBS.

[0059] A method for operating an enzyme activity optimization feedback device based on a passive resonant cavity includes the following steps:

[0060] Step S1: Assemble the tunable passive resonant cavity:

[0061] Two CaF2 windows are positioned with their modified surfaces facing each other, separated by a MYLAR film gasket. The upper CaF2 window is tightly attached to the upper fixed substrate of the stainless steel infrared liquid cell via a gasket. The first and second through holes have the same radius as the injection holes of the upper fixed substrate of the stainless steel infrared liquid cell and are aligned with each other. The lower CaF2 window is tightly attached to the lower fixed substrate of the stainless steel infrared liquid cell via a gasket. The passive resonant cavity is fixed to the piezoelectric ceramic displacement controller by bolts, forming a hollow passive resonant cavity. The cavity is filled by inputting 10 μL of the catalytic substrate-enzyme reaction system through the injection hole and the first through hole on the stainless steel infrared liquid cell.

[0062] Step S2, Optical length of the tunable passive resonant cavity:

[0063] The piezoelectric ceramic displacement controller uses precise control of a high-voltage drive circuit to induce electrostriction in the piezoelectric ceramic displacement controller. This electrostriction adjusts the optical length of the passive resonant cavity: 6 μm corresponds to the C=O vibration of the amide I band (~1650 cm). -1 The vibrational frequency of the PO bond is 8.4 μm, and the vibrational frequency of the Cu-O bond in the laccase active site is ~550 cm⁻¹. -1 The passive resonant cavity of 28.2 μm can resonate with the 13th mode of the carbonyl (C=O) stretching vibration; 6.05 μm corresponds to the 5th mode of the OH stretching vibration of water molecules; and 25.4 μm corresponds to the 21st mode of the OH stretching vibration of water molecules.

[0064] Step S3, Optical Length Inspection:

[0065] To ensure repeatability and accuracy, a small step voltage and step time interval are selected to achieve linear displacement. It should be noted that the actual optical length of the passive resonant cavity is verified by calculation using infrared transmission spectra; the calculation formula can be expressed as:

[0066]

[0067]

[0068] in L , m , n and v Representing cavity pitch and cavity mold respectively m = 1, 2, 3, ...), and the refractive index and wavenumber of the medium; FSR Free spectral range refers to when m When = 1, the average distance between two adjacent peaks in the infrared spectrum; L 1 This represents the actual distance between the two gold surfaces in a first-order cavity resonator; while when L = L 1 At that time, the optimal condition for strong vibration coupling is met;

[0069] Figure 4 These images characterize the coupling effect of passive resonant cavities at different optical lengths, showing how a tunable FP microcavity can achieve strong vibrational coupling with different chemical bonds (OH, OD, C=O). The passive resonant cavity with a length L = 25.4 μm can resonate with the 21st mode of the OH stretching vibration. The FTIR spectrum of the reactive microcavity is visible at 3454 cm⁻¹. -1The nearby resonance peak splits into two new polarization states; the passive resonant cavity with a cavity length of L=28.2 μm can resonate with the 13th mode of the carbonyl (C=O) stretching vibration; the FTIR spectrum of the passive resonant cavity after detecting the injected enzyme solution is visible at 1785 cm⁻¹. -1 The spectral characteristics of the nearby resonance peak splitting into two new polarization states satisfy the Jaynes-Cummings (JC) model, indicating a strong coupling between the cavity mode and molecular vibrations. This suggests that the cavity length can be dynamically changed using tunable techniques, thereby enabling real-time control of the coupling strength.

[0070] Step S4: Establishment of an intelligent feedback system:

[0071] The microfluidic system connects a tunable passive resonant cavity in series with an electrochemical sensing system to form an intelligent feedback system;

[0072] The microfluidic system's channel in controls the flow rate from 0.5 to 10 μL / min. Terminal A of channel in is the sample inlet of the dual-channel microfluidic chip. Terminal B of channel in connects to the first through-hole of the passive resonant cavity. For the laccase solution-catalyst 2,4-DCP reaction system, laccase solution and catalyst 2,4-DCP are input and immobilized through the Y-type mixer of the microfluidic system, forming the site of the enzyme catalytic reaction. A micro heater at 25-50℃ is integrated, and the pH range of the reaction buffer is controlled from 4 to 7. Terminal C of channel out connects to the second through-hole of the passive resonant cavity, outputting the enzyme catalytic product phenol. Terminal D of channel out connects to the electrochemical sensing system.

[0073] The electrochemical sensing system comprises a three-electrode detection system assembled with a specific glassy carbon electrode as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, connected to the D-terminus of the microfluidic system channel out. The surface of the glassy carbon electrode is polished with alumina slurry and washed alternately with ethanol and ultrapure water. 3-7 μL of a composite material solution is rapidly drop-coated onto the polished electrode surface to form an electron transfer interface. After drying, it is coated with PSBMA ion exchange resin to improve the sensor's anti-fouling ability, thus constructing a specific electrochemical sensor (such as...). Figure 5 The composite material consists of multi-walled carbon nanotubes (MWCNTs) and COF-366-Zn dispersed in PBS, which are then ultrasonically treated to obtain a uniformly dispersed composite suspension.

[0074] Step S5: Electrochemical detection and analysis of enzyme activity regulation mechanisms:

[0075] A three-electrode detection system was assembled using a glassy carbon electrode of a specific electrochemical sensor as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, and connected to the D-terminus of the microfluidic system channel out. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were conducted using the three-electrode system in PBS with pH ranges of 4–7, targeting the laccase solution-catalyst 2,4-DCP reaction system. Utilizing the structural differences between the laccase-catalyst substrate and product, the potential differences and electron transfer rates during redox reactions at the electrode surface lead to different peak positions. The substrate concentration affects the current signal value. By detecting the peak position (voltage) and magnitude (current), the concentration of catalyzed products under different enzyme activities was determined, establishing a quantitative relationship between the substrate degradation rate v and the coupling strength g, and analyzing the regulatory mechanism of strong coupling on enzyme activity.

[0076] In the description of this invention, the term "a plurality of" refers to two or more. Unless otherwise explicitly defined, the terms "upper," "lower," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. The terms "connection," "installation," "fixing," etc., should be interpreted broadly. For example, "connection" can be a fixed connection, a detachable connection, or an integral connection; it can be a direct connection or an indirect connection through an intermediate medium. For those skilled in the art, the specific meaning of the above terms in this invention can be understood according to the specific circumstances.

[0077] In the description of this specification, the terms "one embodiment," "some embodiments," "specific embodiment," etc., refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0078] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An enzyme activity optimization feedback device based on a passive resonant cavity, comprising a tunable passive resonant cavity and an electrochemical sensing feedback device, characterized in that... , The tunable passive resonant cavity includes a passive resonant cavity and a piezoelectric ceramic displacement controller; The passive resonant cavity comprises, from the inside out, a set of MYLAR film gaskets, two CaF2 windows, two gaskets, and a stainless steel infrared liquid pool. The MYLAR film gaskets are one or more PET transparent polyester films with a thickness of 2.5 μm. The opposite surfaces of the two CaF2 windows are the modified surfaces. After forming a gold film on the modified surfaces using a vacuum ion sputtering instrument, a polymethyl methacrylate (PMMA) solution is spin-coated, and then a layer of immobilized carrier ZIF-8 is modified. The upper CaF2 window has a first through hole and a second through hole at its edge. The modified surfaces of the two CaF2 windows are opposite each other and separated by the MYLAR film gaskets. The upper CaF2 window is tightly attached to the upper fixed substrate of the stainless steel infrared liquid pool through the gaskets. The first and second through holes have the same radius as the injection holes of the upper fixed substrate of the stainless steel infrared liquid pool and are aligned with each other. The lower CaF2 window is tightly attached to the lower fixed substrate of the stainless steel infrared liquid pool through the gaskets. The passive resonant cavity is fixed to the piezoelectric ceramic displacement controller by bolts. The piezoelectric ceramic displacement controller controls the high voltage drive circuit to cause the piezoelectric ceramic to produce electrostriction. The piezoelectric ceramic displacement controller adjusts the optical length of the passive resonant cavity through electrostriction. The electrochemical sensing feedback device includes a microfluidic system and an electrochemical sensing system; The microfluidic system includes a dual-channel microfluidic chip, channel in and channel out. The two ends of channel in are A and B, respectively, and the two ends of channel out are C and D, respectively. The A end of channel in is the sample inlet of the dual-channel microfluidic chip. The B end of channel in is connected to the first through-hole of the passive resonant cavity. The C end of channel out is connected to the second through-hole of the passive resonant cavity. The D end of channel out is connected to the electrochemical sensing system. The electrochemical sensing system comprises a three-electrode detection system assembled with a specific glassy carbon electrode as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, and connected to the D end of the microfluidic system channel out; the surface of the glassy carbon electrode is functionalized.

2. The enzyme activity optimization feedback device based on a passive resonant cavity according to claim 1, characterized in that... The thickness of the MYLAR film gasket is less than or equal to the optical length of the passive resonant cavity.

3. The enzyme activity optimization feedback device based on a passive resonant cavity according to claim 2, characterized in that... The transmission wavelength range of the CaF2 window is between 0.256 and 10 μm, and the refractive index N is... 633 = 1.

45.

4. The enzyme activity optimization feedback device based on a passive resonant cavity according to claim 3, characterized in that... After the gold film is formed on the modified surface, the parameters of the vacuum ion sputtering instrument are: 5 ~ 6 Pa, 5 mA, 60 s.

5. The enzyme activity optimization feedback device based on a passive resonant cavity according to claim 4, characterized in that... The concentration of the polymethyl methacrylate (PMMA) solution is 10% (w / w).

6. The working method of the enzyme activity optimization feedback device based on a passive resonant cavity as described in claim 5, characterized in that... Specifically, it includes the following steps: Step S1: Assemble the tunable passive resonant cavity: Two CaF2 windows are positioned with their modified surfaces facing each other, separated by a MYLAR film gasket. The upper CaF2 window is tightly attached to the upper fixed substrate of the stainless steel infrared liquid cell via a gasket. The first and second through holes have the same radius as the injection holes of the upper fixed substrate of the stainless steel infrared liquid cell and are aligned with each other. The lower CaF2 window is tightly attached to the lower fixed substrate of the stainless steel infrared liquid cell via a gasket. The passive resonant cavity is fixed to the piezoelectric ceramic displacement controller by bolts, forming a hollow passive resonant cavity. The cavity is filled by inputting 10 μL of the catalytic substrate-enzyme reaction system through the injection hole and the first through hole on the stainless steel infrared liquid cell. Step S2, Optical length of the tunable passive resonant cavity: The piezoelectric ceramic displacement controller induces electrostriction by controlling a high-voltage drive circuit. This electrostriction adjusts the optical length of the passive resonant cavity: 6 μm corresponds to the C=O vibration of the amide I band (~1650 cm⁻¹). -1 ), 8.4 μm corresponds to the vibrational frequency of the PO bond, and 18.2 μm corresponds to the vibrational frequency of the Cu-O bond in the laccase active site. Dynamic frequency (~550 cm) -1 The passive resonant cavity at 28.2 μm corresponds to the 13th mode of carbonyl (C=O) stretching vibration; 6.05 μm corresponds to the 5th mode of water molecule OH stretching vibration; and 25.4 μm corresponds to the 21st mode of water molecule OH stretching vibration. Step S3, Optical Length Inspection: The actual optical length of the passive resonant cavity is verified by calculation using infrared transmission spectra. The calculation formula can be expressed as: ， ， in L , m , n and v Representing cavity pitch and cavity mold respectively m = 1, 2, 3, ...), and the refractive index and wavenumber of the medium; FSR Free spectral range refers to when m When = 1, the average distance between two adjacent peaks in the infrared spectrum; L 1 This represents the actual distance between the two gold surfaces in a first-order cavity resonator; while when L = L 1 At that time, the optimal condition for strong vibration coupling is met; Step S4: Establishment of an intelligent feedback system: The microfluidic system connects a tunable passive resonant cavity in series with an electrochemical sensing system to form an intelligent feedback system; The microfluidic system's channel in controls the flow rate from 0.5 to 10 μL / min. Terminal A of channel in is the sample inlet of the dual-channel microfluidic chip. Terminal B of channel in connects to the first through-hole of the passive resonant cavity, allowing the input of enzyme solution and enzyme substrate. These are immobilized by the Y-type mixer of the microfluidic system, forming the site for the enzyme catalytic reaction. A micro heater at 25-50 °C is integrated, and the pH range of the reaction buffer is controlled from 4 to 7. Terminal C of channel out connects to the second through-hole of the passive resonant cavity, outputting the enzyme catalytic product. Terminal D of channel out connects to the electrochemical sensing system. The electrochemical sensing system comprises a three-electrode detection system assembled with a glassy carbon electrode of a specific electrochemical sensor as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, and connected to the D end of the microfluidic system channel out; Step S5: Electrochemical detection and analysis of enzyme activity regulation mechanisms: A three-electrode detection system was assembled using a specific glassy carbon electrode as the working electrode, Pt as the counter electrode, and Ag / AgCl as the reference electrode, and connected to the D-terminus of the microfluidic system channel out. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) experiments were performed using the three-electrode system in PBS with pH 4–7. Utilizing the structural differences between the enzyme-catalyzed substrate and product, the potential differences and electron transfer rates during redox reactions at the electrode surface lead to different peak positions. The substrate concentration affects the current signal value. By detecting the peak position (voltage) and magnitude (current), the concentration of catalyzed products under different enzyme activities was determined, establishing a quantitative relationship between the substrate degradation rate v and the coupling strength g, and analyzing the regulatory mechanism of strong coupling on enzyme activity.

7. The working method of the enzyme activity optimization feedback device based on a passive resonant cavity according to claim 6, characterized in that... The DPV parameters in step S5 are set as follows: scan range -0.2~0.8 V, pulse amplitude 0.05 V, and pulse width 0.05 s.

8. The working method of the enzyme activity optimization feedback device based on a passive resonant cavity according to claim 6, characterized in that... In step S4, the enzyme solution and the enzyme catalytic substrate are laccase solution and catalytic substrate 2,4-DCP, respectively.

9. The enzyme activity optimization feedback device based on a passive resonant cavity according to claim 8, characterized in that... The surface of the glassy carbon electrode is polished with alumina slurry and washed alternately with ethanol and ultrapure water; 3 ~ 7 μL of composite material solution is rapidly drop-coated onto the polished electrode surface to form an electron transfer interface; After drying, the sensor is coated with PSBMA ion exchange resin to improve its anti-fouling ability and construct a specific electrochemical sensor. The composite material consists of multi-walled carbon nanotubes (MWCNTs) and COF-366-Zn dispersed in PBS and ultrasonically treated to obtain a uniformly dispersed composite suspension.

10. The operating method of the enzyme activity optimization feedback device based on a passive resonant cavity according to claim 9, characterized in that... The COF-366-Zn was obtained by imine condensation reaction, using tetraaminophenylporphyrin zinc and aldehyde compounds as precursors, and ultrasonically treated with THF / DMF / o-DCB / n-BuOH until completely dissolved. The mixture was then heated in an oil bath at 120 °C. After the reaction was completed, the solid product was recovered by centrifugation and washed sequentially with THF, DMF and methanol to remove unreacted monomers and solvent residues. The product was then vacuum dried to obtain powdered hollow microspheres COF-366-Zn.

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