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CN-121450417-B - Enzyme activity optimization feedback device based on passive resonant cavity and working method thereof

CN121450417BCN 121450417 BCN121450417 BCN 121450417BCN-121450417-B

Abstract

The invention provides an enzyme activity optimization feedback device based on a passive resonant cavity and a working method thereof, belonging to the field of enzyme reaction control devices. Including tunable passive resonators, electrochemical sensing devices, and microfluidic systems. The passive resonant cavity is internally provided with an optical length dynamic interval controlled by the piezoelectric ceramic translation stage, the electrochemical sensing device monitors the concentration of an enzyme product in real time and performs high-flux screening, and the microfluidic system controls the input and output of an enzyme reaction system. The invention dynamically adjusts the optical length of the passive resonant cavity by utilizing the piezoelectric material to match the vibration mode of the active site of the enzyme molecule in real time, realizes the self-adaptive optimization of the enzyme activity, breaks through the limitation of static regulation of an enzyme immobilization technology, combines a microfluidic system with an electrochemical sensor, monitors the concentration of an enzyme product in real time to carry out high-flux screening, and realizes closed-loop sensing feedback of detection, regulation and re-optimization.

Inventors

  • LI NA
  • DING CAIFENG

Assignees

  • 青岛科技大学

Dates

Publication Date
20260508
Application Date
20251106

Claims (10)

  1. 1. An enzyme activity optimizing feedback device based on a passive resonant cavity comprises a tunable passive resonant cavity body and an electrochemical sensing feedback device, and is characterized in that, The tunable passive resonant cavity comprises a passive resonant cavity and a piezoelectric ceramic displacement controller; The passive resonant cavity sequentially comprises 1 group of MYLAR film gaskets, 2 CaF 2 window sheets, 2 gaskets and 1 stainless steel infrared liquid pool from inside to outside, wherein the MYLAR film gaskets are 1 or more PET transparent polyester films with the thickness of 2.5 mu m, the opposite surfaces of the 2 CaF 2 window sheets are modified surfaces, after a gold film is formed on the modified surfaces by using a vacuum ion sputtering instrument, polymethyl methacrylate (PMMA) solution is spin-coated by using a spin-coating instrument, a layer of immobilized carrier ZIF-8 is modified, the edge positions of the CaF 2 window sheets on the upper layer are respectively provided with a first through hole and a second through hole, the modified surfaces of the 2 CaF 2 window sheets are opposite, the middle is separated by the MYLAR film gaskets, the upper CaF 2 window sheets are tightly attached to an upper fixed substrate of the stainless steel infrared liquid pool through the gaskets, the first through holes and the second through holes are identical in radius and aligned with the upper fixed substrate of the stainless steel infrared liquid pool, and the lower CaF 2 window sheets are tightly attached to the fixed through the stainless steel perforation substrate; The passive resonant cavity is fixed on the piezoelectric ceramic displacement controller through a bolt, the piezoelectric ceramic displacement controller enables piezoelectric ceramic to generate electrostriction through controlling the high-voltage driving circuit, and the piezoelectric ceramic displacement controller adjusts the optical length of the passive resonant cavity through electrostriction; The electrochemical sensing feedback device comprises a microfluidic system and an electrochemical sensing system; The microfluidic system comprises a two-channel microfluidic chip, a channel in and a channel out, wherein the two ends of the channel in are an end A and an end B respectively, the two ends of the channel out are an end C and an end D respectively, the end A of the channel in is a sample injection end of the two-channel microfluidic chip, the end B of the channel in is connected with a first through hole of a passive resonant cavity, the end C of the channel out is connected with a second through hole of the passive resonant cavity, and the end D of the channel out is connected with an electrochemical sensing system; The electrochemical sensing system comprises a three-electrode detection system assembled by taking a specific glassy carbon electrode as a working electrode, pt as a counter electrode and Ag/AgCl as a reference electrode, and is connected with a D end of a microfluidic system channel out, and the surface of the glassy carbon electrode is subjected to functional modification.
  2. 2. The passive resonator-based enzyme activity optimization feedback device of claim 1 wherein the thickness of the MYLAR film gasket is less than or equal to the optical length of the passive resonator.
  3. 3. The passive resonator-based enzyme activity optimization feedback device according to claim 2, wherein the transmission wavelength range of the CaF 2 window is between 0.256 μm and 10 μm, and the refractive index N 633 =1.45.
  4. 4. The passive resonant cavity-based enzyme activity optimization feedback device according to claim 3, wherein after the vacuum ion sputtering instrument forms a gold film on the modified surface, parameters of the vacuum ion sputtering instrument are 5-6 Pa,5 mA and 60 s.
  5. 5. The passive resonator-based enzyme activity optimization feedback device of claim 4 wherein the concentration of polymethyl methacrylate PMMA solution is 10% (w/w).
  6. 6. The method of operation of a passive resonator based enzyme activity optimization feedback apparatus of claim 5, comprising the steps of: Step S1, assembling a tunable passive resonant cavity: The modified surfaces of the 2 CaF 2 window sheets are opposite, the middle is separated by a MYLAR film gasket, the upper CaF 2 window sheet is tightly attached to an upper fixed substrate of the stainless steel infrared liquid tank through a gasket, the first through hole and the second through hole are identical in radius and aligned in circle center with the injection hole of the upper fixed substrate of the stainless steel infrared liquid tank, the lower CaF 2 window sheet is tightly attached to a lower fixed substrate of the stainless steel infrared liquid tank through the gasket; step S2, tunable passive resonant cavity optical length: the piezoelectric ceramic displacement controller enables the piezoelectric ceramic displacement controller to generate electrostriction by controlling the high-voltage driving circuit, and adjusts the optical length of the passive resonant cavity by electrostriction, wherein the optical length of the piezoelectric ceramic displacement controller is 6 mu m corresponding to C=O vibration of an amide I band (1650 cm -1 ), 8.4 mu m corresponding to the vibration frequency of a P-O bond and 18.2 mu m corresponding to the vibration of a laccase active center Cu-O bond A passive resonant cavity with a dynamic frequency of (550 cm -1 ), a 13 th order mode of stretching vibration of a carbonyl (C=O), a 5 th order mode of stretching vibration of water molecules O-H, and a 21 st order mode of stretching vibration of water molecules O-H, wherein the dynamic frequency is 28.2 mu m; step S3, optical length inspection: The actual optical length of the passive resonant cavity is checked by infrared transmission spectrum calculation, and the calculation formula can be expressed as follows: , , Wherein L, m, n and v represent the cavity distance, cavity mode (m=1, 2, 3.), medium refractive index and wave number, respectively, the FSR free spectral range refers to the average distance between two adjacent peaks in the infrared spectrogram when m=1, L 1 represents the actual distance between two gold surfaces in the first-order cavity subfilm resonant cavity, and the optimal condition for strong coupling of vibration is satisfied when l=l 1 ; step S4, establishing an intelligent feedback system: The micro-fluidic system connects the tunable passive resonant cavity with the electrochemical sensing system in series to form an intelligent feedback system; the channel in of the microfluidic system controls the flow rate to be 0.5-10 mu L/min, the end A of the channel in is a sample injection end of a double-channel microfluidic chip, the end B of the channel in is connected with a first through hole of a passive resonant cavity, enzyme solution and enzyme catalysis substrate are input, the channel in is immobilized through a Y-shaped mixer of the microfluidic system and connected to form an enzyme catalysis reaction generating place, a micro heater of 25-50 ℃ is integrated, the pH range of a reaction buffer solution is controlled to be 4-7, the end C of a channel out is connected with a second through hole of the passive resonant cavity, enzyme catalysis products are output, and the end D of the channel out is connected with an electrochemical sensing system; The electrochemical sensing system comprises a three-electrode detection system assembled by taking a glassy carbon electrode of a specific electrochemical sensor as a working electrode, pt as a counter electrode and Ag/AgCl as a reference electrode, and is connected with a D end of a microfluidic system channel out; Step S5, electrochemical detection and enzyme activity regulation mechanism analysis: A three-electrode detection system is assembled by taking a specific glassy carbon electrode as a working electrode, taking Pt as a counter electrode and taking Ag/AgCl as a reference electrode and is connected with the D end of a microfluidic system channel out, cyclic voltammetry CV and differential pulse voltammetry DPV experiments are carried out in PBS with pH value of 4-7 by utilizing the three-electrode system, the difference of potential and electron transfer rate and the like when oxidation-reduction reaction occurs on the surface of the electrode are utilized to cause the difference of peak positions by utilizing the difference of enzyme catalytic substrates and products in structure, the concentration of the substrates influences current signal values, the concentration of the products is generated by catalysis under different enzyme activities through the peak position (voltage) and the size (current), the quantitative relation between the degradation rate v of the substrates and the coupling strength g is established, and a regulation mechanism of strong coupling on the enzyme activity is analyzed.
  7. 7. The method of claim 6, wherein the DPV parameter in step S5 is set to a scan range of-0.2-0.8V, a pulse width of 0.05V, and a pulse width of 0.05S.
  8. 8. The method of claim 6, wherein the enzyme solution and the enzyme catalytic substrate in the step S4 are laccase solution and catalytic substrate 2,4-DCP, respectively.
  9. 9. The enzyme activity optimization feedback device based on the passive resonant cavity is characterized in that the surface of a glassy carbon electrode is polished by alumina slurry, ethanol and ultrapure water are used for alternately washing, 3-7 mu L of composite material solution is quickly dripped on the polished electrode surface to form an electron transfer interface, PSBMA ion exchange resin is covered after drying to improve the pollution resistance of the sensor, a specific electrochemical sensor is constructed, the composite material is formed by dispersing MWCNTs and COF-366-Zn of a multi-wall carbon nano tube in PBS, and uniformly dispersed composite suspension is obtained by ultrasonic treatment.
  10. 10. The working method of the passive resonant cavity-based enzyme activity optimization feedback device is characterized in that the COF-366-Zn is subjected to imine condensation reaction, zinc tetraminobenzene porphyrin and aldehyde-based compounds are used as precursors, THF/DMF/o-DCB/n-BuOH is used for ultrasonic treatment until complete dissolution, 120 ℃ oil bath heating is carried out, solid products are centrifugally recovered after the reaction is finished, THF, DMF and methanol are sequentially used for washing, unreacted monomers and solvent residues are removed, and powdered hollow microspheres COF-366-Zn are obtained through vacuum drying.

Description

Enzyme activity optimization feedback device based on passive resonant cavity and working method thereof Technical Field The invention relates to the technical field of enzyme reaction control devices, in particular to an enzyme activity optimization feedback device based on a passive resonant cavity and a working method thereof. Background Traditional enzyme catalysis relies on chemical modification of active sites to enhance reusability and stability of the enzyme, but the problems of enzyme conformation distortion, reduced accessibility of the active sites, high modification cost and the like are faced, and particularly, metal ion pollution is generated by metal ion induction. Therefore, a new activity optimization strategy needs to be developed to break through the bottleneck that the enzyme catalytic efficiency and stability cannot be optimized synergistically in the conventional technology. As an emerging tool for modulating molecular energy states, vibration strong coupling can control chemical reactions by directly changing molecular energy patterns. The resonance coupling with the molecular vibration transition can be realized by adjusting the optical mode of a passive resonant cavity (Fabry-Perot cavity, F-P microcavity) under the dark condition, and the reaction energy barrier is changed by adjusting the vibration mode. The coupling mechanism of the passive resonant cavity and the molecular vibration mode provides a brand new 'green' idea without catalyst intervention for enzyme activity regulation and control. In the current research, the application of enhancing the activity of alpha-trypsin, regulating and controlling the p-nitrophenylacetate decomposition reaction, improving the ATP hydrolysis rate and the like in a passive resonant cavity has been proposed, but the regulation and control effect detection of most reactions needs to recover reaction products and has the defects of low product recovery rate, low optimization effect reproducibility and the like. The structure of the passive resonant cavity must be improved to make improvement on the basis of the structure, and the integrated regulation, reaction and detection realizes closed loop intelligent feedback of the enzyme activity optimizing effect. Disclosure of Invention In order to make up for the defects of the prior art, the invention provides an enzyme activity optimization feedback device based on a passive resonant cavity and a working method thereof. The invention is realized by the following technical scheme that the enzyme activity optimizing feedback device based on the passive resonant cavity comprises a tunable passive resonant cavity and an electrochemical sensing feedback device; the tunable passive resonant cavity comprises a passive resonant cavity and a piezoelectric ceramic displacement controller; The passive resonant cavity sequentially comprises 1 group of MYLAR film gaskets, 2 CaF 2 window sheets, 2 gaskets and 1 stainless steel infrared liquid pool from inside to outside, wherein the MYLAR film gaskets are 1 or more PET transparent polyester films with the thickness of 2.5 mu m, the opposite surfaces of the 2 CaF 2 window sheets are modified surfaces, after a gold film is formed on the modified surfaces by using a vacuum ion sputtering instrument, polymethyl methacrylate (PMMA) solution is spin-coated by using a spin-coating instrument, a layer of immobilized carrier ZIF-8 is modified, the edge positions of the CaF 2 window sheets on the upper layer are respectively provided with a first through hole and a second through hole, the modified surfaces of the 2 CaF 2 window sheets are opposite, the middle of the two CaF 2 window sheets are separated by the MYLAR film gaskets, the upper CaF 2 window sheets are tightly attached to an upper fixed substrate of the stainless steel infrared liquid pool through the gaskets, the first through holes and the second through holes are aligned with the radius of the upper fixed substrate of the stainless steel infrared liquid pool, and the lower CaF 2 window sheets are tightly attached to the lower fixed substrate of the stainless steel infrared liquid pool through the gaskets; the passive resonant cavity is fixed on the piezoelectric ceramic displacement controller through a bolt, the piezoelectric ceramic displacement controller enables piezoelectric ceramic to generate electrostriction through controlling the high-voltage driving circuit, and the piezoelectric ceramic displacement controller adjusts the optical length of the passive resonant cavity through electrostriction; The electrochemical sensing feedback device comprises a microfluidic system and an electrochemical sensing system; The microfluidic system comprises a two-channel microfluidic chip, a channel in and a channel out, wherein the two ends of the channel in are an end A and an end B respectively, the two ends of the channel out are an end C and an end D respectively, the end A of the channel in is a sample inje