An enzyme-electron mediator micro-nano composite for biosensing and a preparation method and application thereof
By employing dynamic confinement techniques involving pre-loading and secondary shrinkage, the problem of spatial distance regulation between enzymes and electron mediators at the nanoscale was solved, achieving efficient and stable enzyme-mediator synergistic immobilization, improving the sensitivity and lifespan of biosensors, and making them suitable for various enzyme-mediator systems.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGDONG TRANSTEK MEDICAL ELECTRONICS CO LTD
- Filing Date
- 2026-04-30
- Publication Date
- 2026-08-04
AI Technical Summary
Existing technologies struggle to precisely control the spatial distance and ratio between enzymes and electron mediators at the nanoscale, resulting in limited improvements in electron transfer efficiency. Furthermore, the carriers have poor versatility and are cumbersome to prepare, making it impossible to achieve low-load, high-efficiency co-immobilization of enzymes and mediators.
A two-step dynamic confinement technique involving pre-loading and secondary shrinkage is employed. First, the enzyme and mediator are co-loaded in a mild carrier precursor. Then, the carrier is shrunk to the target size through physical or chemical processes to form a confined cavity structure, ensuring that the average effective electron transfer distance between the enzyme and the mediator is less than 5 nm.
This method achieves high-efficiency enzyme activity loading, improves catalytic efficiency and stability, reduces electron mediator spillover, and enhances sensor sensitivity and lifetime. It is also applicable to a variety of enzyme-mediator systems, thus improving the method's versatility and reproducibility.
Smart Images
Figure CN122214327B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of biosensor technology, and in particular to an enzyme-electron mediator micro / nano composite for biosensing, its preparation method, and its application. Background Technology
[0002] The catalytic reactions of dehydrogenases (such as glucose dehydrogenase, GDH) are completely independent of molecular oxygen. Their natural coenzymes (such as NAD(P)+ / NAD(P)H or PQQ / FAD) accept electrons in the reaction and must then transfer these electrons to a downstream artificial electron mediator, which in turn transfers them to the electrode. The efficiency of this reaction pathway directly determines the sensitivity, response speed, and signal-to-noise ratio of the entire sensor.
[0003] Electrochemical biosensors based on dehydrogenases utilize artificial electron mediators to replace oxygen, effectively overcoming the dependence of first-generation electrochemical sensors on dissolved oxygen and becoming an important platform for highly selective detection of biomolecules. Electron mediators, including quinone compounds (2,6-dichlorophenolindophenol, 2,3-dichloronaphthoquinone) and aromatic amine compounds (dimethylaniline, nitrosoaniline), show potential applications in the electron transport chains of oxidoreductases such as glucose dehydrogenase due to their tunable redox potentials.
[0004] To address the issue of electron mediator loss, existing technologies often employ a co-immobilization strategy where enzymes and mediators are co-encapsulated in carriers such as hydrogels or polymer membranes. However, these methods lack sufficient ability to regulate the microscopic spatial structure. The random distribution and loose binding of enzymes and electron mediators within the gel network make it difficult to precisely control the effective interaction distance, resulting in limited improvement in electron transfer efficiency. Furthermore, the porous structure of the gel cannot completely suppress the long-term leakage of small molecule mediators.
[0005] Current electrochemical biosensors based on dehydrogenases and electron mediators face inherent contradictions: while increasing the amount of mediator can improve electron transfer efficiency, it can also inhibit enzyme activity and increase background noise; traditional physical embedding methods struggle to achieve efficient and directional electron transfer at the molecular scale. Therefore, precisely controlling the spatial distance and ratio between the enzyme and the mediator at the nanoscale to achieve low-load, high-efficiency synergistic immobilization is a key technical challenge that urgently needs to be overcome in this field.
[0006] While confined supports such as mesoporous silica, metal-organic frameworks, metal nanocages, and polymer microspheres can provide space for immobilization, their cavity sizes are statically unadjustable. This necessitates the separate synthesis of supports with corresponding pore sizes for different enzymes and mediators, resulting in poor versatility and cumbersome preparation processes. Furthermore, static supports exhibit loose encapsulation during liquid-phase loading, retaining significant spatial spacing, and cannot be optimized for in-situ compression of the enzyme-mediator distance after loading, making it difficult to achieve optimal electron transfer efficiency. Summary of the Invention
[0007] To address the shortcomings of existing technologies and overcome the deficiencies of static confinement, this invention proposes a two-step dynamic confinement technique involving pre-loading and secondary shrinkage. The core of this technique lies in the following: First, the enzyme and mediator are pre-loaded into a large, mild carrier precursor. At this stage, the carrier primarily serves to protect and spatially confine the enzyme, preventing damage to enzyme activity during subsequent processing and preventing random diffusion of the enzyme and mediator. Subsequently, a controlled physical or chemical process causes the carrier to shrink uniformly to the target size.
[0008] This invention provides an enzyme-electron mediator micro / nano complex for biosensing, comprising a dehydrogenase, an electron mediator, and a carrier having a confined cavity structure. The dehydrogenase and the electron mediator are loaded and confined within the confined cavity of the carrier. The carrier is shrinkable, and the particle size of the shrunken carrier ranges from 20 to 200 nm.
[0009] Furthermore, the electron mediator is one or more of 2,6-dichlorophenolindophenol, 2,3-dichloronaphthoquinone, dimethylaniline, and nitrosoaniline.
[0010] Furthermore, the average effective electron transfer distance between the electron mediator and the dehydrogenase is less than 5 nm.
[0011] Furthermore, the carrier is any one of polylactic acid-glycolic acid copolymer, polyacrylic acid, polyvinyl alcohol, polyacrylic acid-co-acrylamide hydrogel copolymer, and poly(N-isopropylacrylamide) and its copolymers. The carrier is a thermoresponsive, osmotically responsive, or pH-responsive polymer.
[0012] Furthermore, the carrier has a hollow structure and a cavity wall with channels. The dehydrogenase and electron mediator are confined within the cavity of the micro-nano complex. The reaction substrate enters the confined space through the channels of the cavity wall to undergo the reaction. The confined space provides a dedicated microenvironment for the dehydrogenase-catalyzed reaction and avoids random diffusion of the enzyme and mediator, ensuring the directionality of electron transfer.
[0013] Furthermore, the molar ratio of the dehydrogenase to the electron mediator is 1:(1-50).
[0014] This invention provides a method for preparing the micro / nano composite, comprising the following steps:
[0015] S1: Dehydrogenase and electron mediator are encapsulated in a carrier precursor with a cavity size larger than the effective electron transfer distance to form a pre-encapsulated complex. S2: Stimulate the carrier precursor obtained in S1 to shrink the carrier cavity size to the effective particle size for electron transfer. Electron mediator molecules in the cavity are distributed around the active center of the dehydrogenase, forming reaction microunits.
[0016] Furthermore, the stimulus described in step S2 is a physical or chemical stimulus, including one or more of the following: thermally induced contraction, osmotic pressure-driven contraction, solvent exchange-induced contraction, photoinduced contraction, and secondary chemical crosslinking contraction.
[0017] The present invention also provides a bioelectrochemical test strip sensor, comprising a working electrode and a counter electrode, wherein the working electrode is a silver electrode and the surface of the working electrode is modified with the aforementioned micro-nano composite.
[0018] The present invention also provides an application of the electrochemical test strip sensor in any one of blood glucose detection, uric acid detection and lactic acid detection.
[0019] The micro-nano composite is modified on the detection surface of the working electrode, serving as the core sensitive layer for electrochemical detection. The counter electrode is only used to form a closed electrochemical circuit and has no sensitive layer modification. The sensor relies on the efficient electron transfer characteristics of the micro-nano composite to achieve high sensitivity and high stability in detection.
[0020] In summary, compared with the prior art, the present invention achieves the following technical effects: (1) The present invention performs the two key steps of enzyme-electron mediator mixing and secondary contraction in separate steps, ensuring high enzyme activity load and locking the average distance of enzyme-mediator pair within the optimal transmission distance.
[0021] (2) The present invention improves the catalytic efficiency of dehydrogenase in confined structure. The response current generated by unit enzyme catalysis is significantly higher than that of traditional methods. Fewer dehydrogenases can be used when the detection current amplitude requirement remains unchanged.
[0022] (3) The present invention improves the stability of enzymes through confined encapsulation technology, which is beneficial to extend the service life of the sensor, while reducing the leakage of electron mediators and reducing their signal interference on the electrode surface.
[0023] (4) The dynamic confinement structure obtained by the present invention not only has all the advantages of traditional confinement structures, but also realizes active and precise control of electron transfer distance. Moreover, a set of shrinkage processes can be adapted to a variety of different enzyme-mediator systems, which greatly improves the universality and reproducibility of the method and is suitable for the preparation of various dehydrogenase-based biosensors. Attached Figure Description
[0024] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.
[0025] Figure 1 The images are transmission electron microscope images of enzyme-loaded shrinkable confined microspheres prepared by thermal shrinkage method in Example 1 of the present invention. (A) shows the morphology of the microspheres before shrinkage, and (B) shows the morphology of the microspheres after shrinkage. Figure 2 The electrochemical response curves of the enzyme-loaded shrinkable confined microsphere system prepared by the thermo-shrinkage method in Example 1 of this invention are shown in the glucose concentration range of 2-22M. Figure 3 The electrochemical response curves of the enzyme-loaded shrinkable confined microsphere system prepared by the osmotic pressure-driven method in Example 2 of this invention are shown in the glucose concentration range of 2-22 mM. Figure 4 In Example 2 of this invention, fluorescence intensity and motion state before and after contraction were detected by fluorescence resonance energy transfer method. (A) shows the fluorescence intensity and motion state before contraction, and (B) shows the fluorescence intensity and motion state after contraction. Detailed Implementation
[0026] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.
[0027] The average effective electron transfer distance (AEL) test method determines whether the carrier microspheres have shrunk by detecting changes in the ratio of donor to acceptor fluorescence intensity. The core principle is that when the spatial distance between the donor and acceptor is less than 10 nanometers, the energy transfer efficiency increases significantly, manifested as enhanced acceptor fluorescence and weakened donor fluorescence. A significantly increased acceptor / donor ratio indicates that the carrier microspheres have entered a shrinkage state.
[0028] Fluorescence resonance energy transfer (FRET) assay: Microspheres labeled with donor and acceptor fluorescent groups are dispersed in a buffer solution and added to a sample cell. Fluorescence intensities of the donor and acceptor emission channels are collected separately. First, fluorescence intensity is recorded in the uncontracted state, and the spatial relative position of the enzyme protein is calculated based on the acceptor / donor fluorescence ratio. Then, a contraction trigger is added to induce microsphere contraction. After contraction, fluorescence intensity is measured again, and the acceptor / donor fluorescence ratio is calculated. If the ratio significantly increases from its pre-contraction value to above 2, accompanied by restricted spatial movement, it can be determined that the carrier microspheres are in a contracted state and the relative distance between the enzyme protein has decreased.
[0029] Example 1: Preparation of enzyme-loaded shrinkable confined microspheres by thermo-induced shrinkage method (I) Preparation method Pre-coating process: A W / O / W dual emulsion-solvent evaporation method was employed. Glucose dehydrogenase (GDH) and 2,3-dichloronaphthoquinone were dissolved in the inner aqueous phase. Polylactic acid-glycolic acid copolymer (PLGA) was dissolved in dichloromethane as the oil phase. An emulsion was formed in the outer aqueous phase containing stabilizers, and the solvent was evaporated overnight with stirring to form an emulsion as shown in the figure. Figure 1 The PLGA cavity microspheres shown in Figure A have an initial particle size D1 of approximately 120 nm and are internally loaded with GDH and electron mediators.
[0030] Microsphere shrinkage process: The above microsphere suspension was heat-treated in a 50°C water bath for 10 minutes. The glass transition temperature (Tg) of PLGA is approximately 45°C. At temperatures above Tg, polymer chain segment movement intensifies, and the microspheres shrink uniformly. By controlling the temperature and time, microspheres are formed as shown in the diagram. Figure 1 Figure B shows a shrunken microsphere with a particle size of D2 45 nm. During the shrinkage process, the enzyme inside the microsphere is protected by the polymer shell, and the enzyme activity retention rate is greater than 95% after shrinkage.
[0031] (II) Test Results The reduced internal cavity volume of the shrunken microspheres forces electron mediator molecules to orbit the GDH active center more tightly, shortening the calculated average effective electron transfer distance from approximately 25 nm initially to approximately 4 nm. The shrunken enzyme-loaded microspheres are mixed with other excipients and spotted onto a silver electrode to form a GDH electrochemical test strip. Test strips were prepared in equal proportions using unshrunken enzyme-loaded microspheres and free enzyme without microspheres as two control groups for sensitivity comparison testing. Figure 2 As shown, in the 2-22 mM range test, the test strip with the shrunken microspheres showed a response sensitivity that was more than 60% higher than the other two control groups, indicating that this method effectively improves the transmission efficiency between GDH and the electronic medium.
[0032] Example 2: Preparation of Polyvinyl Alcohol (PVA) Shrinkable Confined Microspheres by Osmotic Pressure Driven Shrinkage Method (I) Preparation method Pre-loading process: The reverse emulsification method was used to mix and emulsify an aqueous solution containing GDH and 2,6-dichlorophenolindophenol with a DMSO solution of PVA, and then pour it into ethanol to precipitate and crosslink PVA to form microspheres with an initial particle size D1 of about 150 nm.
[0033] Particle size shrinkage process: Microspheres were collected by centrifugation from the storage buffer and resuspended in a series of progressively increasing concentrations of polyethylene glycol (PEG-6000) aqueous solutions (10%, 20%, 30% w / v). After equilibration for 10 minutes at each step, particle size changes were monitored using a dynamic light scattering instrument. The external high osmotic pressure drove water out of the microspheres, achieving controlled shrinkage. When the target particle size D2 was reached at approximately 40 nm, the PEG solution was removed by centrifugation, and the microspheres were resuspended in the detection buffer.
[0034] (II) Test Results This method is carried out entirely at room temperature and in an aqueous phase, achieving an activity retention rate of 98%, and providing better protection for enzyme activity than the heating method. Figure 3 Electrochemical testing results showed that all systems exhibited good linearity within the glucose concentration range of 2–22 mM. However, the current response of the confined microsphere system after shrinkage was significantly higher than that of the unshrinked system and the free enzyme system, with a sensitivity increase of approximately 40–60%. These results indicate that by selecting the PEG concentration, this method can achieve precise control of the confinement distance under mild conditions and significantly improve electrochemical response performance, making it suitable for constructing highly sensitive enzyme electrochemical detection systems.
[0035] Figure 4 In the figure, A represents the state before contraction as detected by fluorescence resonance energy transfer (FRET): Fluorescence intensity analysis showed that the emission intensity of the acceptor channel fluctuated between 210 and 300 units, with an average intensity of approximately 260 units; the emission intensity of the donor channel remained between 840 and 900 units, with an average intensity of approximately 870 units. The calculated ratio of acceptor fluorescence intensity to donor fluorescence intensity was approximately 0.30, indicating spatial separation between the donor and acceptor, and low FRET efficiency. Simultaneously recorded spatial location data showed that the enzyme protein fluorescent labeling shifted in the plane from -40 nm to +55 nm along the X-axis and from -48 nm to +43 nm along the Y-axis, with an estimated root mean square deviation of approximately 35 nm. These large-scale positional fluctuations indicate that the enzyme protein exists in monomeric form, experiencing relatively low resistance, and exhibits a diffusion-like movement pattern within a certain range.
[0036] Figure 4In the figure, B represents the state after shrinkage as detected by fluorescence resonance energy transfer (FRET): After the carrier microspheres shrank, fluorescence intensity analysis showed that the emission intensity of the acceptor channel significantly increased to an arbitrary range of 908 to 998 units, with an average intensity of approximately 960 units; the emission intensity of the donor channel decreased to an arbitrary range of 347 to 400 units, with an average intensity of approximately 370 units. The acceptor / donor fluorescence intensity ratio increased to approximately 2.62, an improvement of approximately 8.7 times compared to the pre-shrinkage state, indicating that the spatial distance between the donor and acceptor was shortened to within 10 nanometers, and the FRET efficiency was at a high level. Simultaneous spatial position monitoring data showed that the displacement range in the X-axis direction narrowed to -4.0 nanometers to +5.5 nanometers, and the displacement range in the Y-axis direction narrowed to -4.3 nanometers to +4.2 nanometers, with the root mean square deviation estimated to decrease to approximately 3 nanometers, a reduction of approximately one order of magnitude compared to the pre-shrinkage state. These restricted motion characteristics indicate that the carrier shrinkage significantly increased the resistance to enzyme protein movement, leading to a substantial decrease in the diffusion coefficient.
[0037] This embodiment confirms that by simultaneously monitoring fluorescence resonance energy transfer efficiency and spatial diffusion behavior, the restricted motion state of enzyme proteins can be effectively distinguished. The high FRET efficiency and small-scale restricted motion characteristics after contraction indicate that the enzyme and electron mediator in the restricted space form a close-range match.
[0038] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.
Claims
1. An enzyme-electron mediator micro / nano complex for biosensing, characterized in that, The micro / nano composite comprises a dehydrogenase, an electron mediator, and a carrier with a confined cavity structure. The dehydrogenase and the electron mediator are loaded and confined within the confined cavity of the carrier. The carrier is contractible, and the average effective electron transfer distance between the electron mediator and the dehydrogenase is less than 5 nm. The electron mediator is any one of 2,6-dichlorophenolindophenol, 2,3-dichloronaphthoquinone, dimethylaniline, and nitrosoaniline. The carrier is any one of polylactic acid-glycolic acid copolymer, polyacrylic acid, polyvinyl alcohol, polyacrylic acid-co-acrylamide hydrogel copolymer, and poly(N-isopropylacrylamide) and its copolymers. The preparation method of the micro / nano composite includes the following steps: S1: Dehydrogenase and electron mediator are encapsulated in a carrier precursor with a cavity size larger than the effective electron transfer distance to form a pre-encapsulated complex; S2: Stimulate the carrier precursor obtained in S1 to shrink the carrier cavity size to the effective electron transfer distance. Electron mediator molecules in the cavity are distributed around the active center of the dehydrogenase to form reaction micro-units. The stimulation is either thermally induced contraction or osmotic pressure driven contraction.
2. The micro / nano composite according to claim 1, characterized in that, The carrier has a hollow structure and a cavity wall with channels.
3. The micro / nano composite according to claim 1, characterized in that, The molar ratio of the dehydrogenase to the electron mediator is 1:(1-50).
4. A bioelectrochemical test strip sensor, characterized in that, It includes a working electrode and a counter electrode, wherein the working electrode is a silver electrode and the surface of the working electrode is modified with the micro-nano composite as described in any one of claims 1-3.
5. The application of the electrochemical test strip sensor according to claim 4 in any one of blood glucose detection, uric acid detection and lactic acid detection for non-diagnostic purposes.