Preparation method and application of renewable biosensor for detecting preparation completion degree of carbohydrate-containing drugs

Abstract

The invention relates to the technical field of detection for judging drug preparation completeness by detecting HMF content, in particular to a renewable biosensor for detecting preparation of carbohydrate-containing drugs, which comprises a biosensor core, a composite coated film electrode assembly, an environment adaptation module, a signal conversion module and a data transmission module, the biosensor core comprises a mesoporous SiO2 protective shell and a Fe-Zr double metal coated GOase immobilized enzyme; the composite coating film electrode assembly comprises a multi-layer gradient composite coating film, a glassy carbon working electrode, a saturated calomel reference electrode and a platinum wire counter electrode; the environment adaptation module comprises a stir-frying scene adaptation unit and a cooking scene adaptation unit, and is internally provided with a high-temperature cooling and heat insulation device. GOase is fixed by a Fe-Zr bimetal frame through a biosensor core, and a mesoporous SiO2 protective shell and a multi-layer gradient composite coating film are matched, so that the immobilized enzyme effectively resists a high-temperature and high-humidity environment in the processing process, and meanwhile, the detection sensitivity and accuracy are ensured.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of drug preparation detection, in particular to a preparation method and application of a renewable biosensor for detecting the preparation completion degree of a sugar-containing drug. BACKGROUND

[0002] Traditional Chinese medicine (TCM), as a unique health resource and cultural treasure in China, plays an irreplaceable role in disease prevention, treatment and health care. The World Health Organization has included TCM in the global traditional medicine development strategy several times, and has put forward higher requirements for the quality standards of Chinese herbal medicines and processed decoction pieces, promoting the continuous rise in demand for high-quality and effective TCM products worldwide.

[0003] Traditional Chinese medicine processing is the core link for TCM to exert precise efficacy, and the processing quality of sugar-containing Chinese herbal medicines (such as rehmannia, cornus, radix polygoni multiflori, ophiopogon, asparagus, astragalus and licorice) directly determines their clinical efficacy. Such Chinese herbal medicines need to be processed through steaming, frying and other processes to gradually hydrolyze and dehydrate internal glucose, fructose and polysaccharides into 5-hydroxymethylfurfural (HMF). If the HMF content is too low, the processing is insufficient, and the conversion of effective components is insufficient, resulting in weak efficacy. If the content is too high, it may be accompanied by component degradation, posing potential safety risks. Therefore, as the "golden indicator" of the processing completion degree of sugar-containing Chinese herbal medicines, the precise monitoring of HMF concentration is the key to ensuring the uniformity and safety of TCM quality.

[0004] Current detection technologies for HMF in the process of traditional Chinese medicine processing can be divided into offline detection and online detection systems. The offline detection method represented by high-performance liquid chromatography and ultra-high-performance liquid chromatography-mass spectrometry can achieve a sensitivity of 0.01 μmol / L, with good accuracy and repeatability. However, it needs to go through complex pretreatment processes such as sample sampling, solvent extraction and centrifugal purification, and the single detection time is as long as 1-2 hours, which cannot provide real-time feedback for process parameter adjustment, often leading to quality risks of over-processing or under-processing, and seriously lagging behind the actual production rhythm.

[0005] The representative of online detection is mainly an electrochemical sensor, which becomes a research hotspot for real-time detection on site due to its rapid response and strong portability. In recent years, the enzyme electrode sensor developed for traditional Chinese medicine matrix uses the specific catalytic action of galactose oxidase to significantly improve the selectivity of HMF detection, but the existing technology still faces three major problems: first, the enzyme stability is poor, galactose oxidase (GOase) is easy to inactivate in the high-temperature (80-260℃) and high-humidity environment of processing, and the enzyme activity retention rate is low; second, the anti-interference ability is weak, the components such as polysaccharide and polyphenol in the traditional Chinese medicine matrix are easy to combine with the enzyme active center or interfere with the electrochemical signal, resulting in large detection error; third, the scene adaptability and economy are insufficient, most sensors can only adapt to a single processing technology, and the core components are not renewable, the single use cost is high, which is difficult to meet the continuous quality control demand of industrial production line.

[0006] The above technical problems directly restrict the standardization development of sugar-containing drug preparation process. On the one hand, the lack of real-time monitoring means leads to the dependence of processing quality on the experience of operators, and the HMF content of different batches of products is significantly different; on the other hand, the existing technology cannot balance accurate detection and efficient production, and it is difficult to meet the strict requirements of internationalization of traditional Chinese medicine on quality stability. Therefore, it is urgent to develop an HMF detection technology with real-time, high-temperature resistance, strong anti-interference, renewability and multi-scene adaptation to solve the problems of quality control lag, high cost and poor stability in the preparation process of sugar-containing drugs, and provide key technical support for the standardization and intelligent upgrading of drug preparation process. SUMMARY

[0007] The present application aims to provide a preparation method and application of a biosensor with reproducible characteristics and high-temperature cooling and heat insulation design by detecting HMF threshold to judge the completion degree of sugar-containing drug preparation, so as to solve the technical problems of poor enzyme stability, weak anti-interference ability, insufficient scene adaptability and non-renewability in the existing detection technology.

[0008] To achieve the above purpose, the present application is implemented by the following technical scheme: a renewable biosensor for detecting the preparation of sugar-containing drugs, which is composed of a biosensor core, a composite coated film electrode assembly, an environment adaptation module, a signal conversion module and a data transmission module;

[0009] The biosensor core is a composite structure of Fe-Zr@GOase immobilized enzyme and mesoporous SiO2 protective shell, which is prepared by one-pot method and interfacial polymerization process, specifically as follows:

[0010] Fe-Zr@GOase immobilized enzyme is constructed by one-pot method, FeCl3·6H2O and ZrCl4 are weighed, Fe 3+ and Zr 4+molar ratio of 1:(1-10) in PBS buffer solution, and stirred magnetically for 30 min until completely dissolved; GOase was added, and the mass ratio of GOase to bimetal was controlled at 1:(1-6), and stirring was continued for 60 min to form a uniform mixture; the mixture was transferred to a reaction kettle, and reacted at room temperature for 6-48 h, and then centrifuged at 12,000 rpm for 30 min to collect the precipitate, which was washed with deionized water for 3 times, and freeze-dried at -80℃ for 4 h to obtain Fe-Zr@GOase immobilized enzyme powder. The immobilized enzyme has a particle size of 1-100 μm, a pore size of 2-800 nm, an enzyme activity retention rate of ≥85% at 15-80℃, and a surface resistivity of ≤1×10 -3 Ω·cm, can load GOase and construct an electron conduction channel.

[0011] The mesoporous SiO2 protective shell is prepared by interfacial polymerization. The Fe-Zr@GOase immobilized enzyme powder is dispersed in PBS buffer solution, and a carboxyl or amino group is grafted on the surface of the immobilized enzyme by ultrasonic treatment for 30 min to form a uniform dispersion; 0.1-1 M tetraethyl orthosilicate and carboxypropyl triethoxysilane (volume ratio 1:1-1:5) are added dropwise as a silicon source, and the dispersion is stirred magnetically at room temperature at 500 rpm for 6 h; then, 3-10% poly(methacrylic acid) solution is added to continue stirring for 2 h for surface carboxylation treatment; after the reaction, the coated product is collected by centrifugation at 6,000 rpm for 20 min, washed with deionized water for 2 times, and dried at 50℃ with air blowing for 6 h to obtain a biosensor core. The biosensor core has a particle size of 5-120 μm, a mesoporous SiO2 shell layer with a pore size of 5-12 nm, and a carboxyl loading capacity of ≥0.8 mmol / g, which can isolate the interference of polysaccharides and polyphenols in traditional Chinese medicine matrix and protect the immobilized enzyme, and also provides a certain temperature resistance effect.

[0012] The composite coated film electrode assembly adopts a layered coating-curing process to construct a multilayer gradient structure of a conductive bottom layer-biosensor core fixed layer-protection surface layer-protection outer layer, and the specific process is as follows:

[0013] The conductive bottom layer is a graphene-carbon nanotube-polyimide composite film, and graphene, carbon nanotubes and polyimide are weighed according to a mass ratio of 10:(3-5):(2-3), added into N-methyl pyrrolidone, and ultrasonically treated for 0.5-5 h to form a uniform dispersion; the glassy carbon electrode is ultrasonically cleaned with anhydrous methanol and deionized water for 5 min each, dried with nitrogen, and then uniformly coated with the dispersion at a speed of 5 cm / s using a micro-syringe, and dried at 120℃ for 50 min to form a conductive bottom layer with a thickness of 3-10 μm, a sheet resistance of ≤5 Ω / sq, and a surface roughness Ra<3.2 μm, which is used to enhance the conductivity of the electrode and provide a bonding substrate.

[0014] The biosensor core fixing layer is a composite of the biosensor core and polyvinyl alcohol-polyethylene glycol adhesive. The polyvinyl alcohol and polyethylene glycol are dissolved in deionized water at a mass ratio of (1-5):1, heated and stirred at 80°C for 30 min until completely dissolved, and then cooled to room temperature to obtain the composite adhesive. The biosensor core is added to the composite adhesive at a mass ratio of (2-6):1, and stirred for 25 min to form a paste. The paste-like mixture is printed on the surface of the conductive bottom layer using a 300-mesh silk screen, and is allowed to stand at 4°C for 24-72 h to form a biosensor core fixing layer with a thickness of 5-11 μm, which is used to fix the biosensor core and ensure the efficiency of enzymatic reaction.

[0015] The protective surface layer comprises a chitosan-gelatin-nano ZnO inner layer and a polytetrafluoroethylene-TiO2 outer layer. The inner layer is prepared by mixing chitosan, gelatin and nano ZnO at a mass ratio of 1:(1-5):(0.001-0.01), dissolving them in deionized water to form a solution with a mass fraction of 5-25%, and spraying the solution on the surface of the biosensor core fixing layer at a pressure of 0.1-0.2 MPa and a speed of 1-5 cm / s, and then allowing it to stand and dry at room temperature for 5-24 h to form an inner layer with a thickness of 3-5 μm. The outer layer is prepared by mixing polytetrafluoroethylene and TiO2 at a mass ratio of (1-5):1, dispersing them in ethanol to form a dispersion liquid with a mass fraction of 5-35%, and then immersing the dispersion liquid on the surface of the protective inner layer, and allowing it to stand and dry at room temperature for 5-24 h to form an outer layer with a thickness of 3-5 μm, which is used to further trap interfering substances and resist high temperature and humidity.

[0016] The electrode assembly further comprises a saturated calomel reference electrode and a platinum wire counter electrode, which are fixed with the glassy carbon working electrode coated with the composite film through a polytetrafluoroethylene electrode holder to form a three-electrode detection unit.

[0017] The environment adaptation module comprises a frying scene adaptation unit and a cooking scene adaptation unit, and is internally provided with a high-temperature cooling and heat insulation device, which is prepared as follows:

[0018] The high-temperature cooling and heat insulation device comprises an aerogel heat insulation layer and a micro cooling system. The aerogel heat insulation layer has a thickness of 3-5 μm and a thermal conductivity of ≤0.02 W / (m·K), and is used to block the transmission of external high temperature. The micro cooling system is a water cooling circulation system, which comprises a micro water pump, a circulating water pipe and a cooling fin, and is adapted to the frying and cooking scenes.

[0019] The front end of the adaptation unit is provided with a PTFE dustproof and breathable film, and the electrode window is covered with a cellulose ester hydrophilic and breathable film. The shell is made of polytetrafluoroethylene material, and the inner wall is pasted with an aerogel heat insulation layer. A hollow heat insulation cavity is formed between the shell and the accommodation cavity accommodating the electrode assembly coated with the film. The outer surface of the accommodation cavity is densely wound with a copper pipe for condensate water. The condensate water pipe is connected with a condensate water tank with a micro pump. The water inlet and outlet mode is lower inlet and upper outlet, which ensures that the internal temperature of the sensor is controlled at 25-35°C.

[0020] The signal conversion module contains a signal amplifier, a filter, and an analog-to-digital converter. The signal amplifier is an Au-modified amplifier used to enhance the weak current signal generated by the biosensor core. The filter is a 50Hz low-pass filter used to remove electromagnetic interference in the processing environment. The analog-to-digital converter is a 16-bit high-precision converter used to convert the amplified analog current signal into a digital signal. The signal processing delay of the signal conversion module is less than or equal to 5ms, ensuring real-time detection.

[0021] The data transmission module contains an STM32F103 microcontroller and a Bluetooth 5.0 module. The microcontroller has a HMF calibration algorithm and a processing completion judgment program. Different HMF endpoint concentration thresholds for sugar-containing traditional Chinese medicines are preset. The module can receive the current value output by the signal conversion module and convert it to HMF concentration through the HMF calibration algorithm. When the HMF concentration reaches the corresponding threshold and remains stable, it is determined that the processing is complete. The microcontroller sends a processing completion reminder signal to the terminal device (mobile phone / computer) through the Bluetooth 5.0 module and stores the monitoring data.

[0022] Preferably, the sensor realizes regeneration by regulating the valence state of Fe element through a Fe-Zr-based framework. The specific mechanism is as follows: during the detection process, GOase catalyzes the HMF reaction to generate H2O2; Zr 4+ (O2 - ) reduces Fe 3+ to Fe 2+ ; Fe 2+ decomposes H2O2 to generate ·OH, while Fe 2+ is oxidized to Fe 3+ ; the generated ·OH can activate the active center of GOase, realizing the regeneration of the sensor, and the regeneration process does not require external oxygen supply, and the number of regenerations is greater than or equal to 30.

[0023] The application provides a preparation method and application of a regenerable biosensor for detecting the preparation of sugar-containing drugs.

[0024] 1. In the application, GOase is fixed on the biosensor core by a Fe-Zr bimetallic framework, and is protected by a mesoporous SiO2 shell and a multilayer gradient composite coating film. The enzyme activity retention rate of the immobilized enzyme is greater than or equal to 85% in an environment of 15-80°C, effectively resisting the high temperature and high humidity environment during processing, while ensuring the detection sensitivity and accuracy.

[0025] 2. The carboxylation treatment of the mesoporous SiO2 shell and the protective surface layer and protective outer layer of the composite coating film can effectively isolate the interfering substances such as polysaccharides and polyphenols in the traditional Chinese medicine matrix, reducing the detection errors caused by signal interference.

[0026] 3、The ROS-driven regeneration mechanism based on the Fe-Zr-based framework of the application does not require external oxygen supply, has short regeneration time, can be regenerated for more than 30 times, greatly reduces the use cost, and meets the needs of industrial continuous production.

[0027] 4、The application can switch the frying / steaming scene through the quick release buckle of the modular environment adaptation unit, and the high-temperature cooling insulation device ensures the stability of the internal detection environment;The signal conversion module processing delay is less than or equal to 5ms, the data transmission module real-time feedback HMF concentration and processing completion prompt, the processing completion degree judgment accuracy is greater than or equal to 98%. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 It is a schematic diagram of the overall structure of the biosensor of the application;

[0029] Figure 2 It is a schematic diagram of the structure of the biosensor core;

[0030] Figure 3 It is a schematic diagram of the cross section of the multilayer gradient composite coating film;

[0031] Figure 4 It is a schematic diagram of the renewable mechanism of the sensor.

[0032] 1, environmental adaptation module;101, PTFE dustproof and breathable film;102, circulating water pipe;103, micro water pump;2, multilayer gradient composite coating film;201, protective outer layer;202, protective surface layer;203, biosensor core fixing layer;204, conductive bottom layer;205, glassy carbon working electrode;206, saturated mercury reference electrode;207, platinum wire counter electrode;3, signal conversion module;301, signal amplifier;302, 50Hz low-pass filter;303, 16-bit analog-to-digital converter;4, data transmission module;401, STM32F103 microcontroller;402, Bluetooth 5.0 module. DETAILED DESCRIPTION

[0033] The technical solutions in the embodiments of the application will be described clearly and completely below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the application.

[0034] Example 1

[0035] As Figures 1-4As shown, the embodiment of the present application provides a renewable biosensor for detecting the preparation of sugar-containing drugs, which comprises a biosensor core, a composite coated film electrode assembly, an environment adaptation module 1, a signal conversion module 3 and a data transmission module 4.

[0036] The biosensor core comprises a mesoporous SiO2 protective shell and a Fe-Zr bimetallic@GOase immobilized enzyme; the composite coated film electrode assembly comprises a multilayer gradient composite coated film 2, a glassy carbon working electrode 205, a saturated calomel reference electrode 206 and a platinum wire counter electrode 207; the environment adaptation module 1 comprises a frying scene adaptation unit and a cooking scene adaptation unit, and is internally provided with a high-temperature cooling and heat insulation device; the signal conversion module 3 comprises a signal amplifier 301, a filter and an analog-to-digital converter 303; the data transmission module 4 comprises an STM32F103 microcontroller 401 and a Bluetooth 5.0 module 402, and the microcontroller 401 is internally provided with an HMF calibration algorithm and a processing degree judgment program; the sensor realizes regeneration through the Fe-Zr-based framework to regulate the valence state of Fe element, and further activates the active center of GOase through ·OH.

[0037] The Fe-Zr@GOase immobilized enzyme is constructed by one-pot method, wherein the molar ratio of Fe 3+ to Zr 4+ is 1:1, and the pore size is 2nm, which is used for loading GOase and constructing an electron conduction channel; the mass ratio of GOase in the immobilized enzyme is 1:1, the enzyme activity retention rate of the immobilized enzyme is ≥85% in the environment of 15-80℃, and the surface resistivity is ≤1×10 -3 Ω·cm;

[0038] The mesoporous SiO2 protective shell is introduced with carboxyl or amino on the surface of the Fe-Zr@GOase immobilized enzyme by interfacial polymerization, and silane such as carboxypropyl triethoxysilane, 3-glycidyl ether oxypropyl trimethoxysilane, etc. is used, the silicon source concentration is 0.1M, and after reaction at room temperature for 5-8 hours, it is air-dried, the pore size is in the range of 1-100nm, and hexanedioic acid, maleic anhydride, polymethacrylic acid, silane coupling agent, etc. are used for surface carboxylation, which is used for isolating the interference of polysaccharides and polyphenols in traditional Chinese medicine matrix and protecting the immobilized enzyme, and at the same time provides certain temperature resistance effect.

[0039] The multilayer gradient composite coated film 2 comprises, from bottom to top, a conductive bottom layer 204, a biosensor core fixing layer 203, a protective surface layer 202 and a protective outer layer 201. The conductive bottom layer 204 is a graphene-carbon nanotube-polyimide composite film, and the three are mixed in a mass ratio of 10:3:2, dispersed in N-methyl pyrrolidone, then ultrasonically treated for 0.5 hours, scraped on the surface of the glassy carbon working electrode 205, dried at 120℃ for 50min, the thickness is 3μm, the square resistance is ≤5Ω / sq, and the surface roughness Ra is <3.2μm, which is used for enhancing the conductivity of the electrode and providing a bonding substrate.

[0040] The biosensor core fixing layer 203 is a composite of the biosensor core and a polyvinyl alcohol-polyethylene glycol adhesive, the mass ratio of polyvinyl alcohol to polyethylene glycol in the adhesive is 1:1, the thickness is 5 μm, and the biosensor core fixing layer 203 is printed on the surface of the conductive bottom layer 204 by 300 mesh silk screen printing, and is placed at 4°C for 24 hours for curing, for fixing the biosensor core and ensuring the efficiency of enzymatic reaction, and the mass ratio of the biosensor core to the adhesive is 2:1;

[0041] The protective surface layer 202 includes a chitosan-gelatin-nano ZnO inner layer and a polytetrafluoroethylene-TiO2 outer layer, the inner layer is prepared by mixing chitosan, gelatin and nano ZnO according to a mass ratio of 1:1:0.001, dissolving in deionized water to form a solution with a mass fraction of 5%, and spraying on the surface of the biosensor core fixing layer 203 at a pressure of 0.1 MPa and a speed of 1 cm / s, and then being left to dry at room temperature for 5 hours, and the thickness is 3 μm. The protective outer layer 201 is prepared by mixing polytetrafluoroethylene and TiO2 according to a mass ratio of 1:1, dispersing in ethanol to form a dispersion liquid with a mass fraction of 5%, and then immersing and coating on the surface of the protective inner layer, and then being left to dry at room temperature for 5 hours, and the thickness is 3 μm, for further trapping interfering substances and resisting high temperature and high humidity.

[0042] The high-temperature cooling and heat insulation device includes an aerogel heat insulation layer with a thickness of 3 μm and a thermal conductivity of ≤0.02 W / (m·K), for blocking external high temperature transmission; the environmental adaptation module 1 further includes a miniature cooling system, which is suitable for frying and cooking scenes, and the frying scene adaptation unit and the cooking scene adaptation unit are water cooling circulation systems, which contain a miniature water pump 103, a circulating water pipe 102 and a heat sink, and are provided with a PTFE dustproof and breathable film 101 at the front end, and the electrode window is covered with a cellulose ester hydrophilic and breathable film.

[0043] The signal amplifier 301 is an Au-modified amplifier for enhancing the weak current signal generated by the biosensor core; the filter is a 50 Hz low-pass filter 302 for removing electromagnetic interference in the processing environment; the analog-to-digital converter 303 is a 16-bit high-precision converter for converting the amplified analog current signal into a digital signal; the signal processing delay of the signal conversion module 3 is ≤5 ms, ensuring the real-time detection.

[0044] The data transmission module 4 can output the current value of the signal conversion module 3; the processing completion degree judgment program presets the HMF end concentration threshold of different sugar-containing traditional Chinese medicines; the microcontroller 401 sends a “processing is completed” reminder signal to the terminal device through the Bluetooth 5.0 module 402, and stores the monitoring data at the same time.

[0045] Example two:

[0046] As Figures 1-4As shown, the embodiment of the present application provides a renewable biosensor for detecting the preparation of sugar-containing drugs, which comprises a biosensor core, a composite coated film electrode assembly, an environment adaptation module 1, a signal conversion module 3 and a data transmission module 4.

[0047] The biosensor core comprises a mesoporous SiO2 protective shell and a Fe-Zr bimetallic@GOase immobilized enzyme; the composite coated film electrode assembly comprises a multilayer gradient composite coated film 2, a glassy carbon working electrode 205, a saturated calomel reference electrode 206 and a platinum wire counter electrode 207; the environment adaptation module 1 comprises a frying scene adaptation unit and a cooking scene adaptation unit, and is internally provided with a high-temperature cooling and heat insulation device; the signal conversion module 3 comprises a signal amplifier 301, a filter and an analog-to-digital converter 303; the data transmission module 4 comprises an STM32F103 microcontroller 401 and a Bluetooth 5.0 module 402, and the microcontroller 401 is internally provided with an HMF calibration algorithm and a processing degree judgment program; the sensor realizes regeneration through the Fe-Zr-based framework to regulate the valence state of Fe element, and further activates the active center of GOase through ·OH.

[0048] The Fe-Zr@GOase immobilized enzyme is constructed by one-pot method, in which the molar ratio of Fe 3+ to Zr 4+ is 1:10, and the pore size is 800 nm, which is used for loading GOase and constructing an electron conduction channel; the mass ratio of GOase in the immobilized enzyme is 1:60, and the enzyme activity retention rate of the immobilized enzyme is ≥85% at 80°C environment, and the surface resistivity is ≤1×10 -3 Ω·cm;

[0049] The mesoporous SiO2 protective shell is introduced with carboxyl or amino on the surface of the Fe-Zr@GOase immobilized enzyme by interfacial polymerization, and silane such as carboxypropyl triethoxysilane, 3-glycidyl ether oxypropyl trimethoxysilane, etc. is used, the silicon source concentration is 1M, and after reaction at room temperature for 8 hours, it is air-dried, the pore size is in the range of 1-100 nm, and hexanedioic acid, maleic anhydride, polymethacrylic acid, silane coupling agent, etc. are used for surface carboxylation, which is used to isolate the interference of polysaccharides and polyphenols in traditional Chinese medicine matrix and protect the immobilized enzyme, and also provides certain temperature resistance effect.

[0050] The multilayer gradient composite coated film 2 comprises, from bottom to top, a conductive bottom layer 204, a biosensor core fixing layer 203, a protective surface layer 202 and a protective outer layer 201. The conductive bottom layer 204 is a graphene-carbon nanotube-polyimide composite film, and the three are mixed in a mass ratio of 10:5:3, dispersed in N-methyl pyrrolidone, then ultrasonically treated for 5 hours, scraped on the surface of the glassy carbon working electrode 205, dried at 120°C for 50 min, with a thickness of 10μm, a square resistance ≤5Ω / sq, and a surface roughness Ra<3.2μm, which is used to enhance the conductivity of the electrode and provide a bonding substrate.

[0051] The biosensor core fixing layer 203 is a composite of the biosensor core and a polyvinyl alcohol-polyethylene glycol adhesive, the mass ratio of polyvinyl alcohol to polyethylene glycol in the adhesive is 5:1, the thickness is 11 μm, and the biosensor core fixing layer 203 is printed on the surface of the conductive bottom layer 204 by 300 mesh screen printing, and is placed at 4°C for 72 hours for curing, for fixing the biosensor core and ensuring the efficiency of enzymatic reaction, and the mass ratio of the biosensor core to the adhesive is 6:1;

[0052] The protective surface layer 202 includes a chitosan-gelatin-nano ZnO inner layer and a polytetrafluoroethylene-TiO2 outer layer, the inner layer is prepared by mixing chitosan, gelatin and nano ZnO according to a mass ratio of 1:5:0.01, dissolving in deionized water to form a solution with a mass fraction of 25%, and spraying on the surface of the biosensor core fixing layer 203 at a pressure of 0.2 MPa and a speed of 5 cm / s, and then being left to dry at room temperature for 24 hours, and the thickness is 5 μm. The protective outer layer 201 is prepared by mixing polytetrafluoroethylene and TiO2 according to a mass ratio of 5:1, dispersing in ethanol to form a dispersion liquid with a mass fraction of 35%, and then immersing and coating on the surface of the protective inner layer, and then being left to dry at room temperature for 24 hours, and the thickness is 5 μm, for further trapping interfering substances and resisting high temperature and high humidity.

[0053] The high-temperature cooling and heat insulation device includes an aerogel heat insulation layer with a thickness of 5 μm and a thermal conductivity of ≤0.02 W / (m·K) for blocking external high temperature transmission; the environmental adaptation module 1 further includes a miniature cooling system suitable for frying and steaming scenes, and the frying scene adaptation unit and the steaming scene adaptation unit are water cooling circulation systems containing a miniature water pump 103, a circulating water pipe 102 and a heat sink, and the front end is provided with a PTFE dustproof and breathable film 101, and the electrode window is covered with a cellulose ester hydrophilic and breathable film.

[0054] The signal amplifier 301 is an Au-modified amplifier for enhancing the weak current signal generated by the biosensor core; the filter is a 50 Hz low-pass filter 302 for removing electromagnetic interference in the processing environment; the analog-to-digital converter 303 is a 16-bit high-precision converter for converting the amplified analog current signal into a digital signal; the signal processing delay of the signal conversion module 3 is ≤5 ms, ensuring the real-time detection.

[0055] The data transmission module 4 can output the current value of the signal conversion module 3; the processing completion judgment program presets the HMF end concentration threshold of different sugar-containing traditional Chinese medicines; the microcontroller 401 sends a “processing is completed” reminder signal to the terminal device through the Bluetooth 5.0 module 402, and stores the monitoring data at the same time.

[0056] Experiment 1: Application of the biosensor in the frying scene of Fructus Corni

[0057] Fresh Fructus Corni was selected as the experimental sample, a constant temperature electric frying pan with a frying frequency of 30 times / min was used as the processing equipment, a sensor was equipped with a frying scene adaptation unit, the assembly of the frying scene adaptation unit was completed through quick-release buckles, the power condensate pump was started to ensure that the internal temperature of the sensor was stable at 30°C. During the formal experiment, 500 g of cored Fructus Corni was mixed with 50 g of honey (mass ratio 10:1, which meets the requirements of honey processing) and then put into the frying pan. The frying temperature was set to 100°C and the frying frequency was set to 30 times / min. The sensor probe was attached to the inner wall of the frying pan (25 mm away from the surface of the material), and the change of HMF concentration was monitored in real time through the Bluetooth terminal. In the initial frying stage (0-30 min), the HMF concentration slowly increased from 0 to 0.58 μmol / L, corresponding to the initial dehydration process of sugar components; as the frying time was prolonged to 30-60 min, the concentration quickly increased to 1.1 μmol / L and stabilized for 5 min, reaching the preset HMF threshold value of Fructus Corni honey processing, and the terminal timely reminded the completion of processing, at which time the frying was stopped.

[0058] Experiment two: application of biosensor in the processing scene of Radix Rehmanniae Preparata

[0059] Radix Rehmanniae was used as the experimental object, and a high-pressure steaming tank with temperature control accuracy of ±1°C and pressure control range of 0-0.2 MPa was used for processing. The sensor was equipped with a steaming scene adaptation unit. Before the experiment, the steaming adaptation unit was assembled through quick-release buckles, the power condensate pump was started to ensure that the internal temperature of the sensor was stable at 30°C, and the detection requirements of the steaming scene were met. During the steaming experiment, 1000 g of sliced Radix Rehmanniae was mixed with 300 mL of yellow wine (mass ratio 10:3, which meets the yellow wine steaming process) and then loaded into the steaming tank. The temperature was set to 100°C and the pressure was set to 0.1 MPa. The sensor probe was inserted through the sampling port at the top of the steaming tank (25 mm away from the surface of the material). In the initial steaming stage (0-2 h), the HMF concentration slowly increased from 0 to 0.79 μmol / L, corresponding to the gradual hydrolysis and dehydration of polysaccharides in Radix Rehmanniae; within 2-4 h, the concentration quickly increased to 1.53 μmol / L; at 4-6 h, the concentration stabilized at 1.86 μmol / L, reaching the HMF threshold value of Radix Rehmanniae processing, and the sensor immediately reminded the completion of processing, stopping the steaming.

[0060] Experiment three: application of biosensor in the processing scene of Radix Rehmanniae

[0061] The licorice decoction pieces are processed using an electric heating constant temperature boiling pot with a temperature control precision of ±1℃ and a stirring speed of 50 rpm, a power condensate pump is started to ensure that the internal temperature of the sensor is stable at 30℃. During the boiling experiment, 800 g of licorice decoction pieces are weighed, 4000 mL of ultrapure water is added into the boiling pot, the temperature is set to 100℃, the stirring speed is set to 50 rpm, the sensor probe is fixed on the side wall of the boiling pot and is 25 mm away from the liquid surface, and the concentration change of HMF is monitored in real time. When boiling for 1.5-3 h, the concentration rises to 1.15 μmol / L and stabilizes, reaching the HMF threshold value of licorice boiling, the sensor sends a processing completion reminder, and boiling is stopped.

[0062] While embodiments of the present application have been shown and described with reference to particular embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the spirit and scope of the application. The scope of the application is defined by the appended claims and their equivalents.

Claims

1. A renewable biosensor for determining the degree of completion of the preparation of a drug containing saccharides by detecting HMF, characterized by: The biosensor core comprises a mesoporous SiO2 protective shell and a Fe-Zr bimetallic GOase immobilized enzyme; the composite coated film electrode assembly comprises a multilayer gradient composite coated film, a glassy carbon working electrode, a saturated mercury-mercury reference electrode and a platinum wire counter electrode; the environment adaptation module comprises a frying scene adaptation unit and a cooking scene adaptation unit, and is internally provided with a high-temperature cooling and heat insulation device; the signal conversion module comprises a signal amplifier, a filter and an analog-to-digital converter; the data transmission module comprises an STM32F103 microcontroller and a Bluetooth 5.0 module, and the microcontroller is internally provided with an HMF calibration algorithm and a processing degree judgment program; the sensor can be regenerated by regulating the valence state of Fe element through a Fe-Zr base frame, and the active center of GOase is further activated by ·OH. The mesoporous SiO2 protective shell is prepared by an interfacial polymerization method, carboxyl or amino groups are introduced on the surface of the Fe-Zr@GOase immobilized enzyme, and a silane source with a concentration of 0.1-1M, such as carboxypropyl triethoxysilane, 3-glycidyl ether propyl trimethoxysilane, etc., is used to react at room temperature for 5-8 hours and then air-dried, the pore size is in the range of 1-100 nm, and the surface is carboxylated by using adipic acid, maleic anhydride, polymethylacrylic acid, silane coupling agent, etc., to isolate the interference of polysaccharides and polyphenols in the traditional Chinese medicine matrix and protect the immobilized enzyme, and to provide certain temperature resistance.

2. A renewable biosensor for detecting the preparation of a drug containing saccharide according to claim 1, characterized in that: The sensor catalyzes HMF reaction to generate H2O2, Zr 4+ (O 2- ) changes Fe 3+ into Fe 2+ , Fe 2+ decomposes H2O2 into ·OH, and Fe 2+ changes into Fe 3+ , and the generated ·OH activates the active center of GOase.

3. The method for preparing a renewable biosensor for detecting the preparation of a drug containing saccharide according to claim 1, characterized in that: The Fe-Zr@GOase immobilized enzyme is constructed by one-pot method, wherein Fe 3+ has a molar ratio of 1:(1-10) with Zr 4+ , a pore size of 2-800 nm, is used for loading GOase and constructing an electron conduction channel; GOase has a mass ratio of 1:(1-60) in the immobilized enzyme, the immobilized enzyme has an enzyme activity retention rate of ≥85% under an environment of 15-80℃, and a surface resistivity of ≤1×10 -3 Ω·cm. The multilayer gradient composite coated film comprises, from bottom to top, a conductive bottom layer, a biosensor core fixing layer, a protective surface layer and a protective outer layer. The conductive bottom layer is a graphene-carbon nanotube-polyimide composite film, the three are mixed in a mass ratio of 10:(3-5):(2-3), dispersed in N-methylpyrrolidone, then ultrasonically treated for 0.5-5 hours, scraped and coated on the surface of the glassy carbon working electrode, dried at 120℃ for 50min, the thickness is 3-10μm, the square resistance is ≤5Ω / sq, and the surface roughness Ra is <3.2μm, for enhancing the conductivity of the electrode and providing a bonding substrate; 4. The method for preparing a renewable biosensor for detecting the preparation of a drug containing saccharide according to claim 1, characterized in that: The biosensor core fixing layer is a composite of the biosensor core and a polyvinyl alcohol-polyethylene glycol adhesive, the mass ratio of polyvinyl alcohol to polyethylene glycol in the adhesive is (1-5):1, the thickness is 5-11μm, and the adhesive is printed on the surface of the conductive bottom layer with a 300-mesh screen, and is cured at 4℃ for 24-72h, for fixing the biosensor core and ensuring the efficiency of enzymatic reaction, and the mass ratio of the biosensor core to the adhesive is (2-6):

1. ​ The protective surface layer comprises a chitosan-gelatin-nano ZnO inner layer and a polytetrafluoroethylene-TiO2 outer layer, the inner layer is prepared by mixing chitosan, gelatin and nano ZnO at a mass ratio of 1:(1-5):(0.001-0.01), dissolving in deionized water to form a solution with a mass fraction of 5-25%, spraying on the surface of the biosensor core fixation layer at a pressure of 0.1-0.2 MPa and a speed of 1-5 cm / s, and air-drying at room temperature for 5-24 h, with a thickness of 3-5 μm. The protective outer layer is prepared by mixing polytetrafluoroethylene and TiO2 at a mass ratio of (1-5):1, dispersing in ethanol to form a dispersion liquid with a mass fraction of 5-35%, and immersing and coating on the surface of the protective inner layer, and air-drying at room temperature for 5-24 h, with a thickness of 3-5 μm, for further trapping interfering substances and resisting high temperature and humidity.

5. The method for preparing a renewable biosensor for detecting the preparation of a drug containing saccharide according to claim 1, characterized in that: The high-temperature cooling and heat insulation device comprises an aerogel heat insulation layer with a thickness of 3-5 μm and a thermal conductivity of ≤0.02 W / (m·K) for blocking external high temperature transmission; the environmental adaptation module further comprises a miniature cooling system suitable for frying and steaming scenes, the frying scene adaptation unit and the steaming scene adaptation unit are water cooling circulation systems containing a miniature water pump, a circulating water pipe and a cooling fin, and the front end is provided with a PTFE dustproof and breathable membrane, and the electrode window is covered with a cellulose ester hydrophilic and breathable membrane.

6. The method for preparing a renewable biosensor for detecting the preparation of a drug containing saccharide according to claim 1, characterized in that: The signal amplifier is an Au-modified amplifier for enhancing the weak current signal generated by the biosensor core; the filter is a 50 Hz low-pass filter for removing electromagnetic interference in the processing environment; the analog-to-digital converter is a 16-bit high-precision converter for converting the amplified analog current signal into a digital signal; the signal processing delay of the signal conversion module is ≤5 ms, ensuring real-time detection.

7. The method for preparing a renewable biosensor for detecting the preparation of a drug containing saccharide according to claim 1, characterized in that: The data transmission module can output the current value of the signal conversion module; the processing completion degree judgment program presets the HMF end point concentration threshold of different sugar-containing traditional Chinese medicines; the microcontroller sends a "processing completion" reminder signal to the terminal device through the Bluetooth 5.0 module, and stores the monitoring data.

8. The application of the renewable biosensor prepared by the preparation method of any one of claims 3-7 in the HMF concentration detection of the processing completion degree judgment of the sugar-containing traditional Chinese medicine frying or steaming processing.

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