CN108018329A - Control and in-situ detection method of a kind of electron beam irradiation to enzymatic activity - Google Patents
Control and in-situ detection method of a kind of electron beam irradiation to enzymatic activity Download PDFInfo
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- 238000010894 electron beam technology Methods 0.000 title claims abstract description 48
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 25
- 238000001514 detection method Methods 0.000 title claims abstract description 16
- 230000002255 enzymatic effect Effects 0.000 title 1
- 230000000694 effects Effects 0.000 claims abstract description 37
- 108090000790 Enzymes Proteins 0.000 claims abstract description 35
- 102000004190 Enzymes Human genes 0.000 claims abstract description 35
- 230000005669 field effect Effects 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 14
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000010931 gold Substances 0.000 claims abstract description 13
- 229910052737 gold Inorganic materials 0.000 claims abstract description 13
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N Titan oxide Chemical compound O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000004065 semiconductor Substances 0.000 claims abstract description 9
- 239000010409 thin film Substances 0.000 claims abstract description 8
- WABPQHHGFIMREM-UHFFFAOYSA-N lead(0) Chemical compound [Pb] WABPQHHGFIMREM-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 5
- 239000010703 silicon Substances 0.000 claims abstract description 5
- 239000004408 titanium dioxide Substances 0.000 claims abstract description 5
- 239000000523 sample Substances 0.000 claims description 10
- 229920001661 Chitosan Polymers 0.000 claims description 4
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 4
- 239000003431 cross linking reagent Substances 0.000 claims description 4
- 238000005566 electron beam evaporation Methods 0.000 claims description 4
- 239000003292 glue Substances 0.000 claims description 4
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 4
- 239000012498 ultrapure water Substances 0.000 claims description 4
- 238000012625 in-situ measurement Methods 0.000 claims description 3
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 229910052751 metal Inorganic materials 0.000 claims description 2
- 239000002184 metal Substances 0.000 claims description 2
- 238000011056 performance test Methods 0.000 claims description 2
- 238000003466 welding Methods 0.000 claims description 2
- 238000012360 testing method Methods 0.000 abstract description 8
- 230000005855 radiation Effects 0.000 abstract description 5
- 238000012544 monitoring process Methods 0.000 abstract description 4
- 230000002285 radioactive effect Effects 0.000 abstract description 3
- 238000005259 measurement Methods 0.000 abstract description 2
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 8
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 8
- 108010046334 Urease Proteins 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 6
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 5
- 108090000623 proteins and genes Proteins 0.000 description 5
- 239000000126 substance Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 4
- 229910010413 TiO 2 Inorganic materials 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 235000014655 lactic acid Nutrition 0.000 description 3
- 239000004310 lactic acid Substances 0.000 description 3
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000011066 ex-situ storage Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000002925 chemical effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000005865 ionizing radiation Effects 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 238000006241 metabolic reaction Methods 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/001—Enzyme electrodes
- C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
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- G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
- G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
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Abstract
本发明公开了一种电子束辐照对酶活性的控制与原位检测方法。包括如下步骤：（1）制备二氧化钛薄膜场效应管；（2）酶的固定化；（3）将器件放入SEM样品台，以硅作为栅电极，金电极阵列中任意两个相邻电极作为源、漏电极，电极通过引线与腔体上航空接头连接；（4）将上述电极的器件放置于扫描电镜中，用电子束照射；（4）电导性能测试。基于二氧化钛薄膜场效应管，将酶固定于场效应管，以扫描电子显微镜的电子束作为辐照源，并将场效应管的电极通过引线及腔体接口引出至SEM腔体外的半导体特性分析仪进行原位的电导测量，通过电导的变化以监测酶的活性。电子束的产生不需要借助任何放射性元素，实现电子束辐照下酶活性的原位监测。
The invention discloses a method for controlling and in-situ detection of enzyme activity by electron beam irradiation. It includes the following steps: (1) Preparation of titanium dioxide thin film field effect transistor; (2) Immobilization of enzyme; (3) Putting the device into the SEM sample stage, using silicon as the gate electrode, and any two adjacent electrodes in the gold electrode array as Source and drain electrodes, the electrodes are connected to the aviation joints on the chamber through lead wires; (4) Place the devices of the above electrodes in the scanning electron microscope and irradiate them with electron beams; (4) Test the electrical conductivity. Based on titanium dioxide thin film field effect tube, the enzyme is immobilized on the field effect tube, the electron beam of the scanning electron microscope is used as the radiation source, and the electrode of the field effect tube is led out to the semiconductor characteristic analyzer outside the SEM cavity through the lead wire and the cavity interface Conduct in situ conductometric measurements to monitor enzyme activity through changes in conductance. The generation of electron beams does not require any radioactive elements, and the in-situ monitoring of enzyme activity under electron beam irradiation is realized.
Description
技术领域technical field
本发明涉及一种电子束辐照对酶活性的控制与原位检测的技术。The invention relates to a technique for controlling and in-situ detection of enzyme activity by electron beam irradiation.
背景技术Background technique
辐照技术是通过辐照源 （x射线、γ射线或电子束）对物质进行电离辐射，通过电离辐射与物质相互作用的物理效应、化学效应和生物效应，来提供消毒灭菌、改变物质性状等相关技术。电子束辐照是一种冷杀菌技术，与传统的x射线和γ射线相比，电子束的产生以电能为能源，不需要借助任何放射性元素，因此电子束辐照技术具有更大优势和安全性。近年来，随着化学、食品等领域对辐照技术研究的不断深入，辐照作用于蛋白质和多肽的研究也逐渐增多。不同强度的辐照作用于蛋白质上，可以改变蛋白质的结构，从而对蛋白质的性质进行控制。Irradiation technology is to ionize substances through irradiation sources (x-rays, γ-rays or electron beams), and to provide disinfection and change the properties of substances through the physical, chemical and biological effects of ionizing radiation interacting with substances. and other related technologies. Electron beam irradiation is a cold sterilization technology. Compared with traditional x-rays and γ-rays, electron beams are generated with electric energy and do not require any radioactive elements. Therefore, electron beam irradiation technology has greater advantages and safety sex. In recent years, with the continuous deepening of research on radiation technology in the fields of chemistry and food, the research on the effect of radiation on proteins and peptides has also gradually increased. Irradiation of different intensities acts on the protein, which can change the structure of the protein, thereby controlling the properties of the protein.
酶是作用于体内特定分子（也叫做酶的底物）的特殊蛋白质，人体内几乎所有的细胞活动进程都需要酶的参与，有增加代谢反应和变化等作用。酶的活性可以被温度、化学环境、电磁波以及底物浓度等因素影响。那么，电子束作为一种重要的辐射源，其在辐照酶后是否会使酶结构发生变化进而影响其活性，能否实现对酶活性进行原位监测，这一方面的技术手段尚未见报道。Enzymes are special proteins that act on specific molecules (also called enzyme substrates) in the body. Almost all cell activity processes in the human body require the participation of enzymes, which can increase metabolic reactions and changes. Enzyme activity can be affected by factors such as temperature, chemical environment, electromagnetic waves, and substrate concentration. Then, as an important radiation source, whether the electron beam will change the structure of the enzyme after irradiating the enzyme and affect its activity, and whether it can monitor the enzyme activity in situ has not been reported yet. .
发明内容Contents of the invention
本发明的目的在于克服现有技术的不足，提供一种基于电子束辐照对酶活性的控制与原位检测方法，从而实现电子束辐照下酶电导特性的原位测量。The purpose of the present invention is to overcome the deficiencies of the prior art, and provide a control and in-situ detection method for enzyme activity based on electron beam irradiation, so as to realize in-situ measurement of enzyme conductance characteristics under electron beam irradiation.
为了实现上述目的，本发明采用如下技术方案：In order to achieve the above object, the present invention adopts the following technical solutions:
一种电子束辐照对酶活性的控制与原位检测方法，包括如下步骤：A method for controlling and in-situ detection of enzyme activity by electron beam irradiation, comprising the following steps:
（1）制备二氧化钛薄膜场效应管，将条状阵列掩膜版置于场效应管表面，采用电子束蒸发镀金电极，将多条引线分别焊接至金电极，选用无机胶水保护电极；(1) To prepare a titanium dioxide thin film field effect tube, place a strip array mask on the surface of the field effect tube, use electron beam evaporation to plate gold electrodes, weld multiple leads to the gold electrodes, and use inorganic glue to protect the electrodes;
（2）酶的固定化： 在源-漏电极间依次滴加连接剂、交联剂和酶，每步滴加完溶液固定一个小时，最后用超纯水洗净；(2) Enzyme immobilization: Add linking agent, cross-linking agent and enzyme between the source-drain electrodes sequentially, fix the solution for one hour after each step, and finally wash with ultrapure water;
（3）将器件放入SEM样品台，以硅作为栅电极，金电极阵列中任意两个相邻电极作为源、漏电极，电极通过引线与腔体上航空接头连接，将引线另一悬空端连接BNC接头并与腔体上航空接头连接；(3) Put the device into the SEM sample stage, use silicon as the gate electrode, and any two adjacent electrodes in the gold electrode array as the source and drain electrodes. Connect the BNC connector and connect with the aviation connector on the cavity;
（4）将上述具有多个电极的器件放置于扫描电镜中，用30kV电子束照射15min，分别记录0 min、5 min、10 min和15 min时数据；该器件上另选四个D-S电极分别设置未辐照、10kV、 20kV和30kV电子束各照射15min；(4) Place the above-mentioned device with multiple electrodes in a scanning electron microscope, irradiate it with a 30kV electron beam for 15 minutes, and record the data at 0 min, 5 min, 10 min and 15 min respectively; select four D-S electrodes on the device respectively Set unirradiated, 10kV, 20kV and 30kV electron beams to irradiate for 15 minutes each;
（5）电导性能测试：将半导体特性分析仪接头接至腔体航空接头外侧，实现电子束辐照下酶电导特性的原位测量。(5) Conductivity performance test: Connect the connector of the semiconductor characteristic analyzer to the outside of the aviation connector of the cavity to realize the in-situ measurement of the conductivity characteristic of the enzyme under electron beam irradiation.
在上述的电子束辐照对酶活性的控制与原位检测方法中，步骤（3）所述电极通过引线与腔体上航空接头连接采用如下二种方法：用小型探针台把电极和BNC接头连接，或者用引线连接仪或者超声焊接仪将电极和BNC接头通过金属引线连接。In the above method of controlling and in situ detection of enzyme activity by electron beam irradiation, the electrode in step (3) is connected to the aviation connector on the cavity through the lead wire using the following two methods: use a small probe station to connect the electrode and the BNC Joint connection, or use a lead wire connection device or an ultrasonic welding device to connect the electrode and the BNC connector through metal leads.
在上述的电子束辐照对酶活性的控制与原位检测方法中，步骤（2）所述连接剂优选为壳聚糖。In the method for controlling and in situ detection of enzyme activity by electron beam irradiation, the linker in step (2) is preferably chitosan.
在上述的电子束辐照对酶活性的控制与原位检测方法中，步骤（2）所述交联剂优选为戊二醛。In the method for controlling and in situ detection of enzyme activity by electron beam irradiation, the cross-linking agent in step (2) is preferably glutaraldehyde.
本发明基于SEM的电子束以及半导体特性分析仪，建立电子束辐照下酶活性的控制方法，酶的活性能够简便地通过设置电子束的加速高压以及辐照时间来实现，并且其活性可进行实时的原位检测。The present invention is based on the SEM electron beam and semiconductor characteristic analyzer, and establishes the control method of the enzyme activity under electron beam irradiation. The activity of the enzyme can be easily realized by setting the accelerated high voltage of the electron beam and the irradiation time, and its activity can be carried out. Real-time in situ detection.
与现有技术相比，本发明具有如下有益效果：Compared with the prior art, the present invention has the following beneficial effects:
1.本发明基于二氧化钛薄膜场效应管，将酶固定于场效应管，以扫描电子显微镜（SEM）的电子束作为辐照源，并将场效应管的电极通过引线及腔体接口引出至SEM腔体外的半导体特性分析仪进行原位的电导测量，通过电导的变化以监测酶的活性。电子束的产生不需要借助任何放射性元素，因此电子束辐照技术具有更大优势和安全性。1. The present invention is based on a titanium dioxide thin film field effect tube, immobilizes the enzyme on the field effect tube, uses the electron beam of the scanning electron microscope (SEM) as the radiation source, and leads the electrode of the field effect tube to the SEM through the lead wire and the cavity interface The semiconductor characteristic analyzer outside the cavity performs in-situ conductometric measurement, and the enzyme activity is monitored through the change of conductance. The generation of electron beams does not require any radioactive elements, so electron beam irradiation technology has greater advantages and safety.
2.本发明实现了电子束辐照下酶活性的原位监测。2. The present invention realizes the in-situ monitoring of enzyme activity under electron beam irradiation.
3.本发明的测试系统搭建简单，测试方法简便，易于操作与监测。3. The test system of the present invention is simple to build, the test method is simple and convenient, and it is easy to operate and monitor.
附图说明Description of drawings
图1是电子束辐照对酶活性的控制与原位检测的技术示意图；Figure 1 is a technical schematic diagram of the control and in-situ detection of enzyme activity by electron beam irradiation;
图2是不同剂量和时间下，乳酸脱氢酶的IDS-VDS曲线图；其中（a）为电压30kV，分别辐照0 min、 5 min、 10 min和 15 min（b）为固定时间15 min，电压分别为未辐照 、10kV、 20kV和30kV；Figure 2 is the I DS -V DS curve of lactate dehydrogenase at different doses and times; where (a) is a voltage of 30kV, respectively irradiated for 0 min, 5 min, 10 min and 15 min (b) is a fixed time 15 min, the voltages were unirradiated, 10kV, 20kV and 30kV;
图3是向TiO2薄膜场效应管的乳酸脱氢酶中加入不同浓度乳酸后I-V曲线图，其中（a）无电子束照射，（b）30kV电子束照射15min。Figure 3 is the IV curve after adding different concentrations of lactic acid to the lactate dehydrogenase of the TiO 2 thin film field effect tube, in which (a) no electron beam irradiation, (b) 30kV electron beam irradiation for 15min.
具体实施方式Detailed ways
实施例1：以乳酸脱氢酶为研究对象。首先制备TiO2薄膜场效应管，将条状阵列掩膜版置于场效应管表面，采用电子束蒸发镀金电极，并使用引线连接仪将多条引线分别焊接至金电极，随后使用无机胶水保护电极，固化时间为6h以上。其次，在多个源-漏电极中间分别滴加2µL的0.5wt% 壳聚糖，均匀涂布；1h后，滴加2µL的2.5%戊二醛，均匀涂布；1h后，继续滴加2µL乳酸脱氢酶；固定1h后，最后用超纯水洗净。将器件放入SEM样品台，以硅作为栅电极，金电极阵列中任意两个相邻电极作为源、漏电极，将引线另一悬空端连接BNC接头并与腔体上航空接头连接。腔体外侧，将半导体特性分析仪的信号线连接至所需要测试器件的栅、源、漏电极上，装置示意图如图1。在第一个位置，设置30kV高压，电子束持续辐照15min，并记录多个辐照时间点下（0min、 5min、10min和15 min）的电流值；连接其它测试位置，将辐照条件分别设置为：不辐照、10kV、 20kV和 30kV电子束辐照各15min。同时，通过半导体特性分析仪原位测试乳酸脱氢酶的电学性能，从而对其活性进行监测。测试不同辐照强度和不同时间下乳酸脱氢酶的I-V曲线，如图2，可以得出，同一时间下，电子束辐照的强度越大，LDH的活性减小；同一辐照强度下，时间越长，LDH的活性减小。此外，还进一步通过非原位的测试手段对上述结果进行验证。将样品从SEM腔体中取出，记录加入不同浓度乳酸后的I-V曲线，结果如图3所示。可以看出，一方面，固定化乳酸脱氢酶后，在其上面滴加不同浓度的乳酸，其活性的变化引起场效应管中电流的改变，能通过电导的测试对其活性进行判断；另一方面，样品经过30kV电子束照射15min后，峰的强度降低，且峰的数目发生了改变，在3.0V-4.0V左右的峰消失。因此，通过本发明的检测方法可以原位监测电子束辐照下乳酸脱氢酶的活性，为其活性的控制与原位监测提供了一种可行的途径。Example 1: Taking lactate dehydrogenase as the research object. First prepare the TiO 2 thin film field effect tube, place the strip array mask on the surface of the field effect tube, use electron beam evaporation to plate gold electrodes, and use a lead wire connector to weld multiple leads to the gold electrodes respectively, and then use inorganic glue to protect For electrodes, the curing time is more than 6h. Secondly, 2µL of 0.5wt% chitosan was added dropwise between multiple source-drain electrodes to spread evenly; after 1h, 2µL of 2.5% glutaraldehyde was added dropwise to spread evenly; after 1h, continue to drop 2µL Lactate dehydrogenase; after fixing for 1h, wash with ultrapure water at last. Put the device into the SEM sample stage, use silicon as the gate electrode, any two adjacent electrodes in the gold electrode array as the source and drain electrodes, connect the other floating end of the lead to the BNC connector and connect it to the aviation connector on the cavity. Outside the cavity, connect the signal lines of the semiconductor characteristic analyzer to the gate, source, and drain electrodes of the device to be tested. The schematic diagram of the device is shown in Figure 1. In the first position, set 30kV high voltage, and continuously irradiate the electron beam for 15 minutes, and record the current value at multiple irradiation time points (0min, 5min, 10min and 15min); The settings are: no irradiation, 10kV, 20kV and 30kV electron beam irradiation for 15 minutes each. At the same time, the electrical performance of lactate dehydrogenase was tested in situ by a semiconductor characteristic analyzer, so as to monitor its activity. Test the IV curves of lactate dehydrogenase under different irradiation intensities and different times, as shown in Figure 2, it can be concluded that at the same time, the greater the intensity of electron beam irradiation, the activity of LDH decreases; The longer the time, the less active LDH. In addition, the above results were further verified by means of ex-situ testing. The sample was taken out of the SEM cavity, and the IV curves after adding different concentrations of lactic acid were recorded, and the results are shown in Figure 3. It can be seen that, on the one hand, after the lactate dehydrogenase is immobilized, and different concentrations of lactic acid are added dropwise on it, the change of its activity will cause the change of the current in the field effect tube, and its activity can be judged by the conductance test; on the other hand On the one hand, after the sample was irradiated with 30kV electron beam for 15min, the intensity of the peaks decreased, and the number of peaks changed, and the peaks around 3.0V-4.0V disappeared. Therefore, the detection method of the present invention can monitor the activity of lactate dehydrogenase in situ under electron beam irradiation, which provides a feasible way for the control and in situ monitoring of its activity.
实施例2：以脲酶为研究对象。首先制备TiO2薄膜场效应管，将条状阵列掩膜版置于场效应管表面，采用电子束蒸发镀金电极，并使用引线连接仪将多条引线分别焊接至金电极，随后使用无机胶水保护电极，固化时间为6h以上。其次，在多个源-漏电极中间分别滴加2µL的0.5wt% 壳聚糖，均匀涂布；1h后，滴加2µL的2.5%戊二醛，均匀涂布；1h后，继续滴加2µL脲酶；固定1h后，最后用超纯水洗净。将器件放入SEM样品台，以硅作为栅电极，金电极阵列中任意两个相邻电极作为源、漏电极，将引线另一悬空端连接BNC接头并与腔体上航空接头连接。腔体外侧，将半导体特性分析仪的信号线连接至所需要测试器件的栅、源、漏电极上，装置示意图如图1。在第一个位置，设置30kV高压，电子束持续辐照15min，并记录多个辐照时间点下（0min、 5min、10min和15 min）的电流值；连接其它测试位置，将辐照条件分别设置为：不辐照、10kV、 20kV和 30kV电子束辐照各15min。同时，通过半导体特性分析仪原位测试脲酶的电学性能，从而对其活性进行监测。测试不同辐照强度和不同时间下脲酶的I-V曲线，可以得出，同一时间下，电子束辐照的强度越大，脲酶的活性减小；同一辐照强度下，时间越长，脲酶的活性越小。此外，还进一步通过非原位的测试手段对上述结果进行验证。将样品从SEM腔体中取出，记录加入不同浓度尿素后的I-V曲线。可以得出，一方面，固定化脲酶后，在其上面滴加不同浓度的尿素，其活性的变化引起场效应管中电流的改变，能通过电导的测试对其活性进行判断；另一方面，样品经过30kV电子束照射15min后，峰的强度和数目均发生了改变。因此，通过本发明的检测方法可以原位监测电子束辐照下脲酶的活性，为其活性的控制与原位监测提供了一种可行的途径。Embodiment 2: Taking urease as the research object. First prepare the TiO 2 thin film field effect tube, place the strip array mask on the surface of the field effect tube, use electron beam evaporation to plate gold electrodes, and use a lead wire connector to weld multiple leads to the gold electrodes respectively, and then use inorganic glue to protect For electrodes, the curing time is more than 6h. Secondly, 2µL of 0.5wt% chitosan was added dropwise between multiple source-drain electrodes to spread evenly; after 1h, 2µL of 2.5% glutaraldehyde was added dropwise to spread evenly; after 1h, continue to drop 2µL Urease; after fixing for 1 h, wash with ultrapure water at last. Put the device into the SEM sample stage, use silicon as the gate electrode, any two adjacent electrodes in the gold electrode array as the source and drain electrodes, connect the other floating end of the lead to the BNC connector and connect it to the aviation connector on the cavity. Outside the cavity, connect the signal lines of the semiconductor characteristic analyzer to the gate, source, and drain electrodes of the device to be tested. The schematic diagram of the device is shown in Figure 1. In the first position, set 30kV high voltage, and continuously irradiate the electron beam for 15 minutes, and record the current value at multiple irradiation time points (0min, 5min, 10min and 15min); The settings are: no irradiation, 10kV, 20kV and 30kV electron beam irradiation for 15 minutes each. At the same time, the electrical properties of urease were tested in situ by a semiconductor characteristic analyzer, so as to monitor its activity. Test the IV curve of urease under different irradiation intensities and different times, it can be concluded that at the same time, the greater the intensity of electron beam irradiation, the activity of urease decreases; under the same irradiation intensity, the longer the time, the greater the activity of urease smaller. In addition, the above results were further verified by means of ex-situ testing. The sample was taken out from the SEM cavity, and the IV curve after adding different concentrations of urea was recorded. It can be concluded that, on the one hand, after the immobilized urease is added dropwise on it with different concentrations of urea, the change of its activity causes the change of the current in the field effect tube, and its activity can be judged by the test of the conductance; on the other hand, After the sample was irradiated with 30kV electron beam for 15min, both the intensity and the number of the peaks changed. Therefore, the detection method of the present invention can in situ monitor the activity of urease under electron beam irradiation, which provides a feasible way for its activity control and in situ monitoring.
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