Electrochemical cholesterol biosensor as well as preparation method and application thereof

Abstract

The electrochemical cholesterol biosensor comprises a flexible substrate and a three-electrode system formed on the flexible substrate, and the three-electrode system comprises a working electrode, a counter electrode and a reference electrode which are respectively provided with a detection end and an electric connection end; the working electrode is constructed as follows: a detection end of the working electrode comprises an electrode base layer as well as artificial nano-enzyme and cholesterol oxidase which are modified on the surface of the electrode base layer; the artificial nano-enzyme is of a binary alloy hollow cage structure which has catalase activity and is composed of first precious metal and second precious metal. The invention further discloses a preparation method and application of the electrochemical cholesterol biosensor. According to the invention, non-invasive, real-time and continuous monitoring of the cholesterol concentration of sweat can be realized.

Description

Technical Field

[0001] This invention pertains to electrochemical biosensors, specifically to an electrochemical cholesterol biosensor that can be used for monitoring cholesterol in sweat. Background Technology

[0002] Cholesterol, a derivative of cyclopentanoperhydrophenanthrene, is a major steroidal compound in mammals and a crucial physiological indicator in clinical biochemical testing. Clinically, total blood cholesterol levels are typically used as the gold standard for detection. Normal physiological levels range from 3.1 to 6.7 mmol / L, remaining in a dynamic equilibrium with changes in lifestyle. High blood cholesterol levels can easily lead to various chronic diseases such as hyperlipidemia, atherosclerosis, and fatty liver; low levels may indicate underlying diseases or malnutrition. Therefore, dynamic monitoring of cholesterol levels has significant clinical importance in health management.

[0003] Currently, clinical cholesterol testing is performed using large-scale fully automated biochemical analyzers based on the chemiluminescence method. These analyzers use blood as the test sample, but the collection of such samples is often invasive, and they can usually only detect cholesterol levels at a specific point in time. If continuous changes in cholesterol levels are required, frequent blood sampling is necessary, which is very unfavorable for monitoring real-time changes in cholesterol concentration.

[0004] Electrochemical biosensors, due to their fast detection speed, high specificity, high sensitivity, ease of operation, and miniaturization, possess unique advantages in health management compared to other biosensors. Furthermore, their unique advantages in wearable devices hold promise for application in clinical testing to achieve dynamic monitoring of cholesterol levels. Based on the different biosensitive elements within the sensor, electrochemical biosensors can be categorized into electrochemical enzyme sensors, electrochemical immunosensors, electrochemical DNA sensors, and cellular electrode sensors. Electrochemical cholesterol biosensors are predominantly electrochemical enzyme sensors and electrochemical immunosensors. Among these, enzyme-based cholesterol biosensors are one of the most common types of electrochemical cholesterol sensors. These sensors typically utilize the cholesterol generated during an enzyme reaction... Through testing The signal generated by electro-oxidation at the electrode indicates cholesterol concentration. This is due to the electrocatalytic oxidation at the pure electrode potential. Because of their low efficiency and susceptibility to interference from other biomolecules, biosensors with only a single cholesterol oxidase have poor sensitivity.

[0005] To improve sensor sensitivity, natural catalases such as horseradish peroxidase are often used to modify the electrodes, utilizing their ability to react with generated catalases. The signal is amplified by highly efficient electroreduction catalysis. However, natural hydrogen peroxide bioenzymes are greatly affected by the environment, are easily deactivated, and have poor stability. Therefore, their use is very limited, and they cannot achieve dynamic monitoring of trace biomarkers. Summary of the Invention

[0006] In view of this, the present invention proposes an electrochemical cholesterol biosensor that enables non-invasive, real-time, and continuous monitoring of cholesterol concentration in sweat. Furthermore, a method for preparing this electrochemical cholesterol biosensor is also disclosed.

[0007] The first aspect of the present invention discloses an electrochemical cholesterol biosensor, comprising a flexible substrate and a three-electrode system formed on the flexible substrate, wherein the three-electrode system comprises a working electrode, a counter electrode, and a reference electrode, each having a detection end and an electrical connection end; The working electrode is constructed such that its detection end includes an electrode base layer and artificial nanozymes and cholesterol oxidases modified on its surface; the artificial nanozymes are binary alloy hollow cage structures with catalase activity and composed of a first noble metal and a second noble metal.

[0008] Optionally, the first noble metal is palladium; the second noble metal is iridium or platinum.

[0009] Optionally, the molar ratio of the first noble metal to the second noble metal is 5:1 to 1:1.

[0010] Optionally, the reference electrode is a silver / silver chloride electrode; the electrode substrate layers of both the counter electrode and the working electrode are carbon electrodes.

[0011] Optionally, the detection end of the working electrode may further include a protective outer membrane covering the surface of the artificial nanozyme and cholesterol oxidase.

[0012] Optionally, the material of the protective outer membrane is selected from chitosan, glutaraldehyde, and perfluorosulfonic acid, or any one or more of these.

[0013] Optionally, the electrochemical cholesterol biosensor also includes an insulating layer partially covering the three-electrode system.

[0014] Optionally, the surface of the electrode substrate layer of the working electrode has an in-situ generated porous graphene microstructure, which has multiple catalytic active sites for immobilizing enzymes.

[0015] The second aspect of this invention discloses a method for preparing the electrochemical cholesterol biosensor described in the first aspect of this invention, characterized by comprising the following steps: Preparation of artificial nanoenzyme dispersions with hollow cage structures of noble metal alloys; Electrode substrates for the counter electrode, reference electrode, and working electrode of the three-electrode system were fabricated on a flexible substrate. The artificial nanozyme dispersion was coated onto the detection end surface of the electrode substrate layer of the working electrode and then dried. The cholesterol oxidase solution was coated onto the detection end surface of the dried electrode substrate and then dried. The electrochemical cholesterol biosensor was obtained after drying.

[0016] Optionally, the preparation of the artificial nanoenzyme dispersion with a hollow cage structure specifically includes: Provide the first precious metal seeds; A second noble metal particle is grown on the surface of a first noble metal seed using a directional growth process, forming a core-shell structured noble metal alloy nanoparticle. The noble metal alloy nanoparticles are etched using a directional etching process to selectively remove some of the noble metal alloy particles inside the core-shell structure, thereby obtaining a binary alloy hollow cage structure composed of a first noble metal and a second noble metal.

[0017] Optionally, the electrode substrate layer on which the counter electrode, reference electrode, and working electrode of the three-electrode system are respectively fabricated on the flexible substrate includes: Laser-induced graphene technology was used to fabricate the electrode base layer of the working electrode on a flexible substrate on which the counter electrode had been prepared.

[0018] A third aspect of the present invention uses the electrochemical cholesterol biosensor described in the first aspect for non-invasive, real-time or continuous monitoring of cholesterol concentration in sweat.

[0019] The present invention has the following beneficial effects: This invention prepares artificial nanozymes with a binary alloy hollow cage structure, achieving a larger specific surface area and adsorption capacity. This effectively increases the contact sites between the enzyme and the electrode surface, improving the electron transport efficiency between the enzyme and the electrode. Simultaneously, this artificial nanozyme, as an artificial catalase, exhibits stable hydrogen peroxide catalytic efficiency and can also serve as a highly efficient catalyst to enhance the electron generation rate in the catalytic decomposition of hydrogen peroxide. Furthermore, this artificial nanozyme material possesses good biocompatibility, effectively maintaining protein molecular activity and promoting electron transport between the protein active site and the electrode surface.

[0020] The electrochemical cholesterol biosensor provided in this invention achieves specific detection by modifying the electrode base layer of the working electrode with artificial nanozymes and cholesterol oxidase having a binary alloy hollow cage structure. Furthermore, based on the high sensitivity, tolerance, and stability of the electrochemical cholesterol biosensor, real-time, continuous, and non-invasive monitoring of sweat cholesterol can be achieved. Simultaneously, due to its flexibility and small structure, it also has excellent potential for applications in portable and wearable devices.

[0021] The electrochemical cholesterol biosensor provided in this invention can also generate a porous graphene micro-three-dimensional structure in situ on the surface of the working electrode through laser-induced graphene technology. By increasing the specific surface area of ​​the electrode, the contact sites between the enzyme and the electrode surface are effectively increased, thereby further improving the electron transport efficiency between the enzyme and the electrode.

[0022] In the fabrication process of the electrochemical cholesterol biosensor, if screen printing technology is used to print the electrode base layer of the sensor working electrode, the process is simple, low-cost, and easy to achieve low-variance mass production; if laser-induced graphene technology is used to prepare the electrode base layer of the sensor working electrode, it has advantages such as high conductivity, customizable morphology, and low-cost preparation. Attached Figure Description

[0023] Figure 1 A schematic diagram of the overall structure of the cholesterol biosensor; Figure 2 A schematic diagram of the cross-sectional structure of the working electrode of a cholesterol biosensor; Figure 3 A schematic diagram of the synthesis of artificial nanozymes with a binary alloy hollow cage structure; Figure 4 A schematic diagram of the cholesterol biosensor fabrication process; Figure 5 A schematic diagram of a cholesterol biosensor testing system and its working principle; Figure 6 A schematic diagram showing the performance comparison of the cholesterol biosensor prepared in Example 1 and the comparative example in a PBS environment; Figure 7 A schematic diagram showing the performance comparison of the cholesterol biosensors prepared in Examples 1 and 2 and the comparative example in a PBS environment; Figure 8 This is a schematic diagram illustrating the performance of a cholesterol biosensor in an artificial sweat environment. Detailed Implementation

[0024] Current clinical methods for detecting cholesterol all require invasive blood sampling and are typically single-point tests that can only reflect cholesterol levels at a specific moment and cannot track changes in cholesterol levels over a continuous period. This makes personalized health tracking difficult and complicates the early prevention and long-term dynamic management of chronic diseases such as atherosclerotic cardiovascular disease.

[0025] Sweat, due to its ease of non-invasive collection from the skin, is considered an important tool for health monitoring through in-situ electrochemical analysis. Scholars have proposed wearable detection methods for various analytes in sweat, such as glucose, estradiol, and uric acid. The applicant has discovered a correlation between sweat and blood cholesterol, suggesting sweat as a potential alternative blood sample for monitoring cholesterol concentration changes. However, limitations stemming from the extremely low molecular level of cholesterol in sweat (its concentration is approximately one-thousandth of that in blood, on the micromolar scale) and the instability of existing electrochemical enzyme sensors (low electron generation rate and low electron transport efficiency) prevent most cholesterol biosensors from achieving dynamic monitoring of trace biomarkers based on sweat. Therefore, addressing the instability of electrochemical enzyme sensors and improving their continuous trace detection capabilities is a key challenge for applying wearable electrochemical sensing to in-situ real-time sweat monitoring.

[0026] In view of this, this invention first proposes an artificial nanozyme with a binary alloy hollow cage structure, and then uses this artificial nanozyme to fabricate an electrochemical sweat cholesterol sensor for monitoring cholesterol in sweat, achieving online continuous non-invasive cholesterol monitoring. Compared to natural catalase, which is greatly affected by the environment, easily deactivated, and unsuitable for acidic environments such as sweat, the artificial nanozyme proposed in this invention has better stability, is less affected by harsh environments, and is easy to artificially modify to improve catalytic activity. Therefore, this invention uses this artificial nanozyme to replace natural catalase in a cholesterol biosensor, which not only improves the sensor's tolerance and stability but also effectively enhances its sensitivity, enabling cholesterol detection under trace conditions.

[0027] This invention provides a high-performance electrochemical cholesterol biosensor (hereinafter referred to as "cholesterol biosensor") for monitoring cholesterol in sweat, employing a three-electrode system. As shown in Figure 1, the cholesterol biosensor includes a flexible substrate 100, a three-electrode system formed on the flexible substrate, and an insulating layer 300 partially covering the surface of the three-electrode system. The three-electrode system includes a working electrode 210 (WE), a reference electrode 220 (RE), and a counter electrode 230 (CE), arranged in parallel with the working electrode 210 located in the middle. The two ends of each electrode constitute a detection end and an electrical connection end, respectively. The detection ends of the three electrodes together form a detection area 201, and the electrical connection ends of the three electrodes are respectively connected to different input ports of a circuit board, forming an electrical connection portion 202. Within the detection area, the detection end of the working electrode 210 is circular, while the detection ends of the reference electrode 220 and the counter electrode 230 are arc-shaped, together surrounding the detection end of the working electrode 210, and the three are arranged concentrically.

[0028] It is worth noting that the three-electrode system used in this invention can eliminate the significant error interference caused by polarization current in the electrode potential. This electrode system adds a reference electrode to the ordinary two-electrode system (working electrode and counter electrode) to stabilize the working electrode. The reference electrode is calibrated to a standard potential in the electrochemical system and can serve as a comparison standard for the potential electrodes to help eliminate the influence of the potential electrodes on the measurement results, thereby improving accuracy.

[0029] In the three-electrode system, the reference electrode can be a silver / silver chloride electrode with an internal solution of 5% potassium chloride or sodium chloride. The counter electrode can be a carbon electrode. The base layer of the working electrode can be made of carbon material, prepared by screen printing and laser-induced graphene (LIG) processes, respectively.

[0030] In a three-electrode system, the working electrode is the primary sensor. This invention uses an artificial nanozyme-cholesterol oxidase cascade enzyme system as the sensitive material for the working electrode. Figure 2 As shown, the working electrode 210 mainly consists of an electrode base layer 211, enzyme material formed on the surface of the electrode base layer 211, and a protective outer membrane 214. This invention improves the ability of cholesterol redox decomposition to generate electrons and the transfer efficiency of electrons generated on the electrode surface by loading highly catalytically active enzyme material onto the surface of the working electrode 210 at a certain ratio (e.g., 0.1-0.5 mg per square millimeter). The enzyme material includes artificial nanoenzymes 212 and cholesterol oxidase 213 with a binary alloy hollow cage structure.

[0031] A protective outer membrane 214 can also be fabricated on the surface of the electrode base layer 211 to cover the enzyme material, which can slow down the rate of enzyme loss during long-term use, thereby enabling the sensor to continuously monitor cholesterol levels at different levels. The protective outer membrane 214 can be made from any material such as chitosan, glutaraldehyde, or perfluorosulfonic acid.

[0032] Furthermore, the present invention can also modify the three-dimensional structure of the electrode base layer 211 of the working electrode 210 by directly irradiating carbon-based materials (such as paper fibers or polyimide) with a laser to carbonize the surface and generate porous graphene in situ, thereby constructing a porous graphene microscopic three-dimensional structure (hereinafter referred to as "porous three-dimensional structure") on the surface of the electrode base layer 211. This porous three-dimensional structure increases the specific surface area of ​​the electrode base layer 211, enabling it to provide more catalytic active sites, thereby significantly enhancing its enzyme immobilization ability.

[0033] It is worth noting that nanozymes with hollow cage structures are one of the core sensing materials for cholesterol biosensors, and can be prepared through directional growth and directional etching processes. For example... Figure 3 As shown, a second noble metal particle (e.g., iridium, platinum) is grown on the surface of a first noble metal seed (e.g., palladium). The resulting alloy nanoparticles have a core-shell structure with the first noble metal as the core and the second noble metal as the shell. The core structure is then etched away, causing some of the first noble metal and some of the second noble metal particles encased on the surface of the first noble metal to detach, forming nanoparticles with a binary alloy hollow cage structure. Due to the inherent hydrogen peroxide catalytic activity of noble metal elements, and the unique three-dimensional spatial structure, the nanoparticles have a larger specific surface area, exposing more catalytically active sites, thus enhancing catalytic activity. When applied to this cholesterol biosensor, it synergistically promotes cholesterol oxidase, improving the electron generation efficiency in cholesterol decomposition and further enhancing sensor performance.

[0034] It should also be noted that, furthermore, the present invention can also employ laser-induced graphene technology, using a carbon dioxide laser to induce carbon-based materials (such as paper fibers or polyimide) in the electrode base layer 211 to carbonize and generate a porous three-dimensional graphene microstructure in situ on its surface. This endows the electrode base layer 211 of the working electrode 210 with a three-dimensional porous structure, and exposes more catalytic active sites that bind to enzymes, thereby increasing the electron transfer pathways generated on the electrode surface, improving electron transfer efficiency, and further enhancing the sensor performance.

[0035] The flexible substrate 1 of this cholesterol biosensor can be made of flexible thin film materials such as polyimide (PI). The insulating layer 3 is positioned between the detection area and the electrical connection portion on the surface of the three-electrode system, covering the area of ​​each electrode except for the electrical connection and detection ends, and serves to protect the electrodes. Specifically, the insulating layer can be screen-printed using materials such as photocurable insulating varnish.

[0036] Combination Figure 5 As shown, the enzyme-based cholesterol biosensor uses cholesterol oxidase as the biosensitive element. The reaction process of the system is demonstrated through the reaction equation of cholesterol oxidase interacting with cholesterol, including: The reaction that occurs at the anode of the electrolytic cell: The reaction that occurs at the cathode of the electrolytic cell: Cholesterol biosensors typically utilize cholesterol generated during enzymatic reactions. The output signal from electro-oxidation at the electrode indicates cholesterol concentration. This invention utilizes artificial catalase modified onto the electrode to accelerate the oxidation process. The electroreduction catalytic efficiency is greatly improved, thereby significantly enhancing the sensitivity of the sensor.

[0037] It is worth noting that, because the redox center of cholesterol oxidase (i.e., flavin adenine dinucleotide, FAD) is deeply embedded within its three-dimensional structure, the electron transport efficiency between the enzyme and the electrode is very limited under conditions where only this enzyme exists during the catalytic reaction. This invention improves the sensor's sensing capability by adding an electron transport medium between the electrode and cholesterol oxidase, connecting the electrode interface and the enzyme's redox center, or creating more electron transport sites.

[0038] In a specific embodiment 1, the electrode base layer of the working electrode of the cholesterol biosensor is fabricated using a screen printing process, and a nanozyme-cholesterol oxidase cascade enzyme system is used as the sensitive membrane of the working electrode. The fabrication of this cholesterol biosensor mainly includes two parts: the fabrication of a nanozyme with a hollow cage structure and the fabrication of the cholesterol biosensor itself. Part 1: Preparation of artificial nanozymes with hollow cage structure.

[0039] In this embodiment, the cholesterol biosensor uses an artificial catalase, specifically a palladium-iridium alloy nanozyme with a binary alloy hollow cage structure. It is worth noting that, besides palladium-iridium alloy nanoparticles, other artificial nanozymes with catalase activity, such as palladium-platinum alloy nanoparticles, can also be used in this system. This embodiment uses palladium-iridium alloy nanoparticles as an example; the specific fabrication process is as follows: Synthesis of palladium seeds: 20-40 mg of polyvinylpyrrolidone, 10-25 mg of L-ascorbic acid, 140-160 mg of potassium bromide, and 1-4 mL of deionized water were mixed in a glass flask with magnetic stirring. After complete dissolution, the flask was placed in an oil bath preheated to 80 °C, and magnetic stirring was continued for 10 min. Subsequently, 0.5-1.5 mL of an aqueous solution containing 10-16 mg of sodium tetrachloropalladate was added to the mixture. The reaction solution was maintained at 80 °C for 3 h, and then cooled to room temperature. Tetrahydrofuran was then added to collect the solid product. The precipitate was centrifuged, washed three times with deionized water, and the resulting palladium seeds were redispersed in ethylene glycol solution to form a palladium seed ethylene glycol solution for later use.

[0040] Synthesis of core-shell palladium-iridium alloy nanoparticles: 100-150 mg polyvinylpyrrolidone, 140-160 mg potassium bromide, 50-80 mg L-ascorbic acid, 3-6 mL palladium seed glycol solution, and 4-7 mL ethylene glycol were mixed in a flask. The mixture was preheated to 110 °C and held for 10 min, then further heated to 195 °C. 14-20 mL of a 1 mg / mL sodium chloroiridate hexahydrate glycol solution was added to the reaction solution at a rate of 1.0 mL / h. After complete addition of the sodium chloroiridate hexahydrate glycol solution, the mixture was allowed to stand for 1 h and then cooled to room temperature. Dihydrofuran was then added to collect and precipitate the nanoparticles. After centrifugation, the nanoparticles were washed three times with deionized water and then dispersed with deionized water to obtain an aqueous suspension of core-shell palladium-iridium alloy nanoparticles for further use.

[0041] Synthesis of Palladium-Iridium Alloy Nanoparticles with Hollow Cage Structure: 20-40 mg of polyvinylpyrrolidone, 140-160 mg of potassium bromide, 0.1-0.3 mL of hydrochloric acid, 40-50 mg of ferric chloride hexahydrate, and 3-5 mL of deionized water were mixed to obtain a reaction system solution. 1-3 mL of an aqueous suspension of core-shell Palladium-Iridium alloy nanoparticles was added to the reaction system solution. After heating at 80 ℃ for 3 h, the etched Palladium-Iridium alloy nanoparticles with a binary alloy hollow cage structure were collected. The precipitate was washed with ethanol, centrifuged, washed three times with deionized water, and redispersed in deionized water to obtain a nanoenzyme dispersion for subsequent use.

[0042] The nanoenzyme alloy particles prepared based on the above scheme were tested after preparation. The results showed that the molar ratio of palladium to iridium in the nanoenzyme with good catalytic activity was 5:1 to 1:1.

[0043] Part Two: Preparation of Cholesterol Biosensors

[0044] In this embodiment, screen printing is used to fabricate the three-electrode system. The specific process is as follows: First, a suitable electrode pattern is designed using AutoCAD software. Then, a corresponding screen printing stencil is customized according to the three-layer electrode drawing. The screen-printed electrodes are then fabricated on a flexible substrate using the stencil, squeegee, and other tools. Carbon paste material can be used for the base layer of the counter electrode and the working electrode, while a 5% silver / silver chloride mixed paste is used for the reference electrode. The total area of ​​the detection end of the working electrode is approximately 31.4 mm². 2 .

[0045] Combination Figure 4 As shown, after pretreatment of the electrode base layer at the detection end of the working electrode, nanozymes and cholesterol oxidases with binary alloy hollow cage structures can be layer-by-layer modified onto the surface of the pretreated electrode base layer (also known as the carbon electrode) and then immobilized to fabricate the desired cholesterol biosensor. The specific steps are as follows: S11: Pretreatment of the surface of the screen-printed electrode base layer, including rinsing with anhydrous ethanol and drying in a nitrogen stream; S12: Dilute the nanozyme dispersion obtained in the first part with 5% chitosan saturated aqueous solution at a volume ratio of 1:1, stir evenly, and obtain nanozyme-chitosan aqueous solution with binary alloy hollow cage structure; S13: Drop 50-150 μl of nano-enzyme-chitosan aqueous solution onto the surface of the base electrode, spread it evenly, and place it in a drying oven at 120℃ for 1 h; S14: After removing the dried electrode and allowing it to return to room temperature, drop 5-15 μl of 5 mg / ml cholesterol oxidase solution onto the surface of the base electrode and spread it evenly. Place it in a 4℃ environment to dry for 24 h. S15: Take out the dried electrode, drop 5-15 μl of 5% chitosan saturated aqueous solution onto the surface of the base electrode, spread it evenly, and dry it at 4℃ for 24 h; at this point, the cholesterol biosensor preparation is complete.

[0046] It is worth noting that, considering that a drying temperature of 120°C would inactivate cholesterol oxidase, the nanozyme should be added first, followed by the cholesterol oxidase, in the above process. Furthermore, to better protect the cholesterol oxidase and extend the sensor's lifespan, the modification process of the cholesterol oxidase can be performed at an ambient temperature of 2-6°C (e.g., 4°C). Moreover, after the cholesterol biosensor is prepared, it can also be stored at 2-6°C (e.g., 4°C).

[0047] In a specific embodiment 2, laser-induced graphene (LIG) technology was used to fabricate the electrode base layer of the working electrode of the cholesterol biosensor, and a nanozyme-cholesterol oxidase cascade enzyme system was used as the sensitive membrane of the working electrode. The fabrication of this cholesterol biosensor also includes two parts: the fabrication of the nanozyme with a binary alloy hollow cage structure and the fabrication of the cholesterol biosensor. The fabrication of the nanozyme is the same in both parts. The main difference between Embodiment 2 and Embodiment 1 lies in the fabrication of the working electrode.

[0048] In Example 2, a three-electrode system was fabricated by combining screen printing and laser-induced graphene technology.

[0049] The specific process is as follows: First, a suitable electrode pattern is designed using AutoCAD software. Then, a corresponding screen printing stencil is customized according to the reference electrode layer drawing. Using the screen printing stencil, squeegee, and other tools, a 5% silver / silver chloride reference electrode is brushed onto the flexible polyimide film material. Next, the flexible polyimide film material with the brushed reference electrode is positioned and fixed on the laser engraving table of a CO2 laser cutting machine. A CO2 laser is then used to induce the fabrication of LIG electrodes according to the pattern of the working electrode and counter electrode drawings. The laser settings of the CO2 laser cutting machine are: power 50%-70%, speed 50%-70%, PPI 600-1000.

[0050] After preparing the base layers for the reference electrode, counter electrode, and working electrode, the following processes are also included: S21: Pretreatment of the screen-printed LIG electrode, including rinsing with anhydrous ethanol and drying in a nitrogen stream; S22: Use a plasma cleaner to perform surface hydrophilic treatment on the LIG electrode to improve its hydrophilicity for subsequent enzyme modification; S23: Dilute the hollow cage structure nanoenzyme dispersion obtained in the first part with 5% chitosan saturated aqueous solution at a volume ratio of 1:1, and stir until homogeneous; S24: Add 10 μl of hollow cage structure nanozyme-chitosan aqueous solution to the working electrode area, spread it evenly, place it in a drying oven at 60-80℃ for 5 min, repeat this step 8-12 times, so that the nanozyme can be evenly distributed in the three-dimensional structure network of LIG electrode. S25: After removing the dried electrode and allowing it to return to room temperature, add 5-15 μl of 5 mg / ml cholesterol oxidase solution to the working electrode area, spread it evenly, and dry it at 4℃ for 24 h. S26: Remove the dried electrode, add 5-15 μl of 5% chitosan saturated aqueous solution to the working electrode area, spread evenly, and dry at 4℃ for 24 h; at this point, the cholesterol biosensor preparation is complete. The prepared sensor needs to be stored at 4℃.

[0051] It should be noted that laser-induced graphene technology is primarily used to fabricate the electrode base layer in the working electrode. However, considering that the counter electrode is also made of carbon material, to simplify the process, the electrode base layers in both the counter electrode and the working electrode can be fabricated simultaneously. In other embodiments, screen printing can also be used to fabricate the counter electrode.

[0052] After the cholesterol biosensors prepared in Examples 1 and 2 were completed, their performance was tested using the Shanghai Chenhua Chi660E electrochemical workstation. The test methods and results are as follows: like Figure 5 As shown, the working electrode, counter electrode, and reference electrode of the cholesterol biosensor are first connected to the WE, CE, and RE alligator clips of the electrochemical workstation, respectively, and then fixed in the electrolytic cell with a built-in rod rotor (a 50 ml electrolytic cell system was used in the example test), thus completing the construction of the electrochemical testing system.

[0053] Based on the established electrochemical testing system, the electrochemical testing mode is as follows: The performance of the cholesterol biosensor with modified nanozymes prepared in Example 1 and the cholesterol biosensor without modified nanozymes (as a comparative example of Example 1, the preparation process was basically the same as in Example 1, except that the nanozyme modification step was omitted) was compared in a PBS environment. Of the two sensors, the one prepared in Example 1 was modified with both nanozymes and biozymes, while the comparative example was modified only with biozymes.

[0054] Add an appropriate amount of 1M PBS buffer (cholesterol-free) to the electrolytic cell, then completely immerse the detection areas of both sensors in the PBS buffer and perform IT testing using an electrochemical workstation. Using the chronoamperometric method, after removing the polarization curve (data from the polarization curve can be omitted using Chi660E software), add a certain amount of cholesterol solution of known concentration (e.g., 12.9mM) to the PBS buffer dropwise at 150 s, 300 s, 450 s, 600 s, 750 s, 900 s, and 1050 s using a pipette, increasing the cholesterol concentration in the environmental system by 1.3 μM each time. Figure 6 The IT curve shown.

[0055] Figure 6 The T1 curves show that the cholesterol biosensor modified with nanozymes having a binary alloy hollow cage structure significantly increases the current change values ​​corresponding to different cholesterol concentrations compared to the cholesterol biosensor without nanozyme modification. This indicates that the sensitivity of the modified cholesterol biosensor is greatly improved, proving that modifying nanozymes with hollow cage structures can effectively improve the electron generation rate and electron transfer efficiency, making it suitable for preparing high-performance cholesterol biosensors.

[0056] Furthermore, the cholesterol biosensors prepared in Examples 1 and 2, respectively, and the cholesterol biosensor without modified nanozyme (as a comparative example of Example 1, the preparation process was basically the same as in Example 1, except that the nanozyme modification step was omitted) were subjected to performance testing in a PBS environment.

[0057] Specifically, an appropriate amount of PBS buffer was added to the electrolytic cell, and the detection area of ​​the sensor was completely immersed in the PBS buffer. The electrolytic cell chamber was then sealed with a sealing film, and an IT test was performed using an electrochemical workstation. During the hypoxic condition test, nitrogen gas was continuously introduced into the solution to purge the oxygen from the electrolytic cell. Using the chronoamperometry method, after removing the polarization curve, a certain amount of cholesterol solution of known concentration was added dropwise to the PBS buffer at 100 s, 200 s, 300 s, 400 s, 500 s, 600 s, and 700 s using a pipette. This increased the cholesterol concentration in the environmental system by 1.3 μM / L each time, obtaining the sensor's IT curve. A linear fitting curve was then constructed to show the relationship between the current response value and the cholesterol concentration, as shown in the figure. Figure 7 As shown.

[0058] Figure 7The curves showing the relationship between the current response value and cholesterol concentration in Example 2 indicate that the cholesterol biosensor with LIG electrode as the electrode base layer in Example 2 has a higher current response change compared to the cholesterol biosensor with screen-printed electrode as the electrode base layer in Example 1. However, both current responses are higher than those of the cholesterol biosensor without modified nanozymes. This demonstrates that the cholesterol biosensor with LIG electrode as the electrode base layer manufactured in this invention can further improve electron transport efficiency and obtain a higher sensitivity response by changing the microscopic three-dimensional structure of the electrode.

[0059] Furthermore, the performance of the cholesterol biosensor with screen-printed electrodes as the electrode base layer in Example 1 was tested in an artificial sweat environment. Specifically, an appropriate amount of artificial sweat was added to an electrolytic cell, and then the detection area of ​​the sensor was completely immersed in the artificial sweat. An electrochemical workstation was used for IT testing. Similarly, using the chronoamperometry method, after removing the polarization curve, a certain amount of cholesterol solution of known concentration was added dropwise with a pipette at 150 s, 300 s, 450 s, 600 s, 750 s, 900 s, and 1050 s, respectively, to obtain... Figure 8 The IT curve shown.

[0060] Figure 8 The T1 curve shows that the current value output by the cholesterol biosensor in artificial sweat changes with the cholesterol concentration, proving that it can detect cholesterol levels in sweat, has strong anti-interference ability, and has the potential to be applied to sweat cholesterol monitoring.

[0061] In summary, this invention explores and optimizes the structure and preparation method of a high-sensitivity cholesterol biosensor. First, based on the working principle of electrochemical enzyme sensors, it focuses on improving nanomaterials, studying the influence of highly catalytically active artificial nanozymes with a binary alloy hollow cage structure on the performance of the electrochemical enzyme sensor. Furthermore, this invention also explores the modification of the electrode surface structure, studying the influence of a graphene electrode base layer with a porous graphene microstructure on the performance of the electrochemical enzyme sensor, further enhancing its high sensitivity. Moreover, this invention experimentally verifies that the composite nanomaterial layer (simultaneously modified with artificial nanozymes and cholesterol oxidase) and the three-dimensional porous electrode structure can effectively improve the electron generation rate and electron transfer efficiency between the electrode and the enzyme during the reaction, thereby enhancing the detection sensitivity of the cholesterol sensor and achieving a significant breakthrough in the continuous detection of trace levels of cholesterol in sweat using electrochemical sensors.

[0062] Finally, it should be noted that although the embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art, guided by this specification, can make many other forms without departing from the scope of protection of the claims of the present invention, and all of these are within the scope of protection of the present invention.

Claims

1. An electrochemical cholesterol biosensor, characterized in that, The invention includes a flexible substrate and a three-electrode system formed on the flexible substrate, wherein the three-electrode system includes a working electrode, a counter electrode, and a reference electrode, each having a detection end and an electrical connection end; The working electrode is constructed such that its detection end includes an electrode base layer and artificial nanozymes and cholesterol oxidases modified on its surface; the artificial nanozymes are binary alloy hollow cage structures with catalase activity and composed of a first noble metal and a second noble metal.

2. The electrochemical cholesterol biosensor as described in claim 1, characterized in that, The first noble metal is palladium; the second noble metal is iridium or platinum; the molar ratio of the first noble metal to the second noble metal is 5:1 to 1:

1.

3. The electrochemical cholesterol biosensor as described in claim 1, characterized in that, The reference electrode is a silver / silver chloride electrode; the electrode substrates of both the counter electrode and the working electrode are carbon electrodes.

4. The electrochemical cholesterol biosensor as described in claim 1, characterized in that, The detection end of the working electrode also includes a protective outer membrane covering the surface of the artificial nanozyme and cholesterol oxidase; the material of the protective outer membrane is selected from any one or more of chitosan, glutaraldehyde, and perfluorosulfonic acid.

5. The electrochemical cholesterol biosensor as described in claim 1, characterized in that, It also includes an insulating layer that partially covers the three-electrode system.

6. The electrochemical cholesterol biosensor according to any one of claims 1 to 5, characterized in that, The surface of the electrode substrate layer of the working electrode has an in-situ generated porous graphene microstructure, which has multiple catalytic active sites for immobilizing enzymes.

7. A method for preparing an electrochemical cholesterol biosensor as described in any one of claims 1 to 6, characterized in that, Includes the following steps: Preparation of artificial nanoenzyme dispersions with hollow cage structures of noble metal alloys; Electrode substrates for the counter electrode, reference electrode, and working electrode of the three-electrode system were fabricated on a flexible substrate. The artificial nanozyme dispersion was coated onto the detection end surface of the electrode substrate layer of the working electrode and then dried. The cholesterol oxidase solution was coated onto the detection end surface of the dried electrode substrate and then dried. The electrochemical cholesterol biosensor was obtained after drying.

8. The preparation method according to claim 7, characterized in that, The preparation of the artificial nanoenzyme dispersion with a hollow cage structure specifically includes: Provide the first precious metal seeds; A second noble metal particle is grown on the surface of a first noble metal seed using a directional growth process, forming a core-shell structured noble metal alloy nanoparticle. The noble metal alloy nanoparticles are etched using a directional etching process to selectively remove some of the noble metal alloy particles inside the core-shell structure, thereby obtaining a binary alloy hollow cage structure composed of a first noble metal and a second noble metal.

9. The preparation method according to claim 7, characterized in that, The electrode substrate layer on which the counter electrode, reference electrode, and working electrode of the three-electrode system are respectively fabricated on a flexible substrate includes: Laser-induced graphene technology was used to fabricate the electrode base layer of the working electrode on a flexible substrate on which the counter electrode had been prepared.

10. The electrochemical cholesterol biosensor according to any one of claims 1-6 is used for non-invasive, real-time or continuous monitoring of cholesterol concentration in sweat.

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