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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- CN108018329A CN108018329A CN201711070808.7A CN201711070808A CN108018329A CN 108018329 A CN108018329 A CN 108018329A CN 201711070808 A CN201711070808 A CN 201711070808A CN 108018329 A CN108018329 A CN 108018329A
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- electron beam
- electrode
- beam irradiation
- enzymatic activity
- detection method
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- 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
Abstract
The invention discloses a kind of control of electron beam irradiation to enzymatic activity and in-situ detection method.Include the following steps：（1）Prepare titanium deoxid film field-effect tube；（2）Enzyme immobilizatio；（3）Device is put into SEM sample stages, using silicon as gate electrode, any two adjacent electrode is connected as source, drain electrode, electrode by lead with Aviation Connector on cavity in gold electrode array；（4）The device of above-mentioned electrode is positioned in scanning electron microscope, uses electron beam irradiation；（4）Conductivity is tested.Based on titanium deoxid film field-effect tube, enzyme is fixed on field-effect tube, irradiation bomb is used as using the electron beam of scanning electron microscope, and the electrode of field-effect tube is led into characteristic of semiconductor analyzer outside SEM cavitys by lead and cavity interface and carries out conductance measurement in situ, by the change of conductance to monitor the activity of enzyme.The generation of electron beam by any radioactive element, need not realize the in-situ monitoring of enzymatic activity under electron beam irradiation.
Description
Technical field
The present invention relates to a kind of control of electron beam irradiation to enzymatic activity and the technology of in situ detection.
Background technology
Irradiation technique is to pass through irradiation bomb（X-ray, gamma-rays or electron beam）Ionising radiation is carried out to material, passes through electricity
From the physical effect, chemical effect and biological effect of coupled radiation and convection heat transfer, to provide sterilization, change physical characters
Etc. correlation technique.Electron beam irradiation is a kind of cold sterilization technology, compared with traditional x-ray and gamma-rays, the generation of electron beam
Using electric energy as the energy, it is not necessary to which by any radioactive element, therefore electronic beam irradiation technology has greater advantages and security.
In recent years, as the fields such as chemistry, food deepen continuously irradiation technique research, effect of irradiation is ground in proteins and peptides
Study carefully and also gradually increase.The effect of irradiation of varying strength is on protein, thus it is possible to vary the structure of protein, so as to protein
Property is controlled.
Enzyme is to act on internal specific molecular（The also referred to as substrate of enzyme）Specific proteins, it is almost all of thin in human body
Intracellular activity process is required for the participation of enzyme, there is the effect such as increase metabolic response and change.The activity of enzyme can be by temperature, chemical ring
The factors such as border, electromagnetic wave and concentration of substrate influence.So, electron beam is as a kind of important radiation source, it is after enzyme is irradiated
Whether enzymatic structure can be made to change and then influence its activity, can realized and in-situ monitoring is carried out to enzymatic activity, this aspect
There is not been reported for technological means.
The content of the invention
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of based on control of the electron beam irradiation to enzymatic activity
System and in-situ detection method, so as to fulfill the in site measurement of enzyme conductance property under electron beam irradiation.
To achieve these goals, the present invention adopts the following technical scheme that：
Control and in-situ detection method of a kind of electron beam irradiation to enzymatic activity, include the following steps：
（1）Titanium deoxid film field-effect tube is prepared, strip array mask plate is placed in field-effect tube surface, is steamed using electron beam
Gold-plated electrode is sent out, a plurality of leads is respectively welded to gold electrode, selects inorganic glue water conservation electrode；
（2）Enzyme immobilizatio：Bridging agent, crosslinking agent and enzyme are added dropwise successively in source-electric leakage interpolar, often step drips solution and fixes
One hour, finally cleaned with ultra-pure water；
（3）Device is put into SEM sample stages, using silicon as gate electrode, in gold electrode array any two adjacent electrode as source,
Drain electrode, electrode are connected by lead with Aviation Connector on cavity, by another free end of lead connection BNC connector and with cavity
Aviation Connector connects；
（4）The above-mentioned device with multiple electrodes is positioned in scanning electron microscope, with 30kV electron beam irradiation 15min, is remembered respectively
Data when recording 0 min, 5 min, 10 min and 15 min；On the device select else four D-S electrodes set respectively it is non-irradiated,
10kV, 20kV and 30kV electron beam respectively irradiate 15min；
（5）Conductivity is tested：Characteristic of semiconductor analyzer connector is connected on the outside of cavity Aviation Connector, realizes electron beam irradiation
The in site measurement of lower enzyme conductance property.
In above-mentioned electron beam irradiation in the control and in-situ detection method of enzymatic activity, step（3）The electrode passes through
Lead is connected with Aviation Connector on cavity using following two kinds of methods：Electrode is connected with BNC connector with compact probe platform, or
Instrument is connected with lead or ultra-sonic welded instrument connects electrode with BNC connector by metal lead wire.
In above-mentioned electron beam irradiation in the control and in-situ detection method of enzymatic activity, step（2）The bridging agent is excellent
Elect chitosan as.
In above-mentioned electron beam irradiation in the control and in-situ detection method of enzymatic activity, step（2）The crosslinking agent is excellent
Elect glutaraldehyde as.
Electron beam and characteristic of semiconductor analyzer of the invention based on SEM, establish the control of enzymatic activity under electron beam irradiation
Method processed, the activity of enzyme can be realized easily by setting acceleration high pressure and the exposure time of electron beam, and it is lived
Property can carry out real-time in situ detection.
Compared with prior art, the present invention has the advantages that：
1. the present invention is based on titanium deoxid film field-effect tube, enzyme is fixed on field-effect tube, with scanning electron microscope（SEM）
Electron beam partly leading outside SEM cavitys is led to by lead and cavity interface as irradiation bomb, and by the electrode of field-effect tube
Bulk properties analyzer carries out conductance measurement in situ, by the change of conductance to monitor the activity of enzyme.The generation of electron beam is not required to
Will be by any radioactive element, therefore electronic beam irradiation technology has greater advantages and security.
2. the present invention realizes the in-situ monitoring of enzymatic activity under electron beam irradiation.
3. the test system building of the present invention is simple, test method is easy, easily operated with monitoring.
Brief description of the drawings
Fig. 1 is the technology schematic diagram of control and in situ detection of the electron beam irradiation to enzymatic activity；
Fig. 2 is the I of lactic dehydrogenase under various dose and timeDS-VDSCurve map；Wherein（a）For voltage 30kV, irradiate respectively
0 min, 5 min, 10 min and 15 min（b）For 15 min of set time, voltage is respectively non-irradiated, 10kV, 20kV
And 30kV；
Fig. 3 is to TiO2I-V curve figure after addition various concentrations lactic acid in the lactic dehydrogenase of thin film field-effect pipe, wherein（a）
Without electron beam irradiation,（b）30kV electron beam irradiations 15min.
Embodiment
Embodiment 1：Using lactic dehydrogenase as research object.TiO is prepared first2Thin film field-effect pipe, strip array is covered
Film version is placed in field-effect tube surface, welds a plurality of leads respectively using electron beam evaporation gold-plated electrode, and using lead connection instrument
Gold electrode is connected to, then uses inorganic glue water conservation electrode, hardening time is more than 6h.Secondly, among multiple source-drain electrodes
The 0.5wt% chitosans of 2 μ L, even spread are added dropwise respectively；After 1h, 2.5% glutaraldehyde of 2 μ L, even spread is added dropwise；After 1h, after
It is continuous that 2 μ L lactic dehydrogenases are added dropwise；After fixed 1h, finally cleaned with ultra-pure water.Device is put into SEM sample stages, grid are used as using silicon
Electrode, any two adjacent electrode is as source, drain electrode in gold electrode array, by another free end connection BNC connector of lead simultaneously
It is connected with Aviation Connector on cavity.On the outside of cavity, test device required for the signal wire of characteristic of semiconductor analyzer is connected to
Grid, source, on drain electrode, schematic device such as Fig. 1.First position, 30kV high pressures, electron-beam sustainer irradiation are set
15min, and record under multiple exposure time points（0min, 5min, 10min and 15 min）Current value；Connect other test positions
Put, radiation parameter is respectively set to：Do not irradiate, each 15min of 10kV, 20kV and 30kV electron beam irradiation.Meanwhile pass through half
The electric property of conductor characteristics analyzer in-situ test lactic dehydrogenase, so as to be monitored to its activity.Test different irradiation
The I-V curve of lactic dehydrogenase, such as Fig. 2 under intensity and different time, it can be deduced that, under the same time, electron beam irradiation it is strong
Degree is bigger, the activity reduction of LDH；Under same irradiation intensity, the time is longer, the activity reduction of LDH.In addition, also further pass through
The means of testing of ex situ verifies the above results.Sample is taken out from SEM cavitys, record adds various concentrations lactic acid
I-V curve afterwards, the results are shown in Figure 3.It can be seen that, on the one hand, after Immobilized lactate dehydrogenase, it is added dropwise in the above different
The lactic acid of concentration, the change of its activity cause the change of electric current in field-effect tube, its activity can be carried out by the test of conductance
Judge；On the other hand, sample is after 30kV electron beam irradiations 15min, the intensity decreases at peak, and the number at peak is changed
Become, disappear at the peak of 3.0V-4.0V or so.Therefore, can be with in-situ monitoring electron beam irradiation by the detection method of the present invention
The activity of lactic dehydrogenase, a kind of feasible approach is provided for its active control and in-situ monitoring.
Embodiment 2：Using urase as research object.TiO is prepared first2Thin film field-effect pipe, strip array mask plate is put
In field-effect tube surface, a plurality of leads is respectively welded to gold using electron beam evaporation gold-plated electrode, and using lead connection instrument
Electrode, then uses inorganic glue water conservation electrode, hardening time is more than 6h.Secondly, dripped respectively among multiple source-drain electrodes
Add the 0.5wt% chitosans of 2 μ L, even spread；After 1h, 2.5% glutaraldehyde of 2 μ L, even spread is added dropwise；After 1h, continue to be added dropwise
2 μ L urases；After fixed 1h, finally cleaned with ultra-pure water.Device is put into SEM sample stages, using silicon as gate electrode, gold electrode battle array
Any two adjacent electrode connects another free end connection BNC connector of lead and with aviation on cavity as source, drain electrode in row
Head connection.On the outside of cavity, grid, source, the drain electrode of test device required for the signal wire of characteristic of semiconductor analyzer is connected to
On, schematic device such as Fig. 1.First position, 30kV high pressures, electron-beam sustainer irradiation 15min are set, and record multiple spokes
According under time point（0min, 5min, 10min and 15 min）Current value；Other test positions are connected, radiation parameter is distinguished
It is arranged to：Do not irradiate, each 15min of 10kV, 20kV and 30kV electron beam irradiation.It is meanwhile former by characteristic of semiconductor analyzer
The electric property of bit test urase, so as to be monitored to its activity.Test urase under different irradiation intensities and different time
I-V curve, it can be deduced that, under the same time, the intensity of electron beam irradiation is bigger, the activity reduction of urase；Same irradiation intensity
Under, the time is longer, urase it is active smaller.In addition, also further the above results are tested by the means of testing of ex situ
Card.Sample is taken out from SEM cavitys, record adds the I-V curve after various concentrations urea.It can draw, on the one hand, fixed
After changing urase, the urea of various concentrations is added dropwise in the above, the change of its activity causes the change of electric current in field-effect tube, can lead to
The test for crossing conductance judges its activity；On the other hand, sample is after 30kV electron beam irradiations 15min, the intensity at peak
Changed with number.Therefore, can be with the work of urase under in-situ monitoring electron beam irradiation by the detection method of the present invention
Property, provide a kind of feasible approach for its active control and in-situ monitoring.
Claims (4)
1. control and in-situ detection method of a kind of electron beam irradiation to enzymatic activity, it is characterised in that include the following steps：
（1）Titanium deoxid film field-effect tube is prepared, strip array mask plate is placed in field-effect tube surface, is steamed using electron beam
Gold-plated electrode is sent out, a plurality of leads is respectively welded to gold electrode, selects inorganic glue water conservation electrode；
（2）Enzyme immobilizatio：Bridging agent, crosslinking agent and enzyme are added dropwise successively in source-electric leakage interpolar, often step drips solution and fixes
One hour, finally cleaned with ultra-pure water；
（3）Device is put into SEM sample stages, using silicon as gate electrode, in gold electrode array any two adjacent electrode as source,
Drain electrode, electrode are connected by lead with Aviation Connector on cavity, by another free end of lead connection BNC connector and with cavity
Aviation Connector connects；
（4）The above-mentioned device with multiple electrodes is positioned in scanning electron microscope, with 30kV electron beam irradiation 15min, is remembered respectively
Data when recording 0 min, 5 min, 10 min and 15 min；On the device select else four D-S electrodes set respectively it is non-irradiated,
10kV, 20kV and 30kV electron beam respectively irradiate 15min；
（5）Conductivity is tested：Characteristic of semiconductor analyzer connector is connected on the outside of cavity Aviation Connector, realizes electron beam irradiation
The in site measurement of lower enzyme conductance property.
2. control and in-situ detection method of the electron beam irradiation as claimed in claim 1 to enzymatic activity, it is characterised in that step
（3）The electrode is connected by lead with Aviation Connector on cavity using following two kinds of methods：With compact probe platform electrode and
BNC connector connects, and either connects instrument with lead or ultra-sonic welded instrument connects electrode with BNC connector by metal lead wire.
3. control and in-situ detection method of the electron beam irradiation as claimed in claim 1 to enzymatic activity, it is characterised in that step
（2）The bridging agent is chitosan.
4. control and in-situ detection method of the electron beam irradiation as claimed in claim 1 to enzymatic activity, it is characterised in that step
（2）The crosslinking agent is glutaraldehyde.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201711070808.7A CN108018329A (en) | 2017-11-03 | 2017-11-03 | Control and in-situ detection method of a kind of electron beam irradiation to enzymatic activity |
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| CN201711070808.7A CN108018329A (en) | 2017-11-03 | 2017-11-03 | Control and in-situ detection method of a kind of electron beam irradiation to enzymatic activity |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913725A (en) * | 2018-07-23 | 2018-11-30 | 东北农业大学 | A method of improving structured lipid OPO yield |
| CN112611775A (en) * | 2020-12-16 | 2021-04-06 | 聚束科技（北京）有限公司 | Method for observing biological tissue and electron microscope |
| KR20220013816A (en) * | 2020-07-27 | 2022-02-04 | 서강대학교산학협력단 | Apparatus and method for analysing device function |
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| CN101813660A (en) * | 2010-04-02 | 2010-08-25 | 北京工业大学 | Method for preparing fixed enzyme electrode by using TiO2 as carrier |
| CN102346164A (en) * | 2011-07-01 | 2012-02-08 | 北京科技大学 | Method for constructing uric acid sensor on the basis of super-long zinc oxide nano-wire |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108913725A (en) * | 2018-07-23 | 2018-11-30 | 东北农业大学 | A method of improving structured lipid OPO yield |
| KR20220013816A (en) * | 2020-07-27 | 2022-02-04 | 서강대학교산학협력단 | Apparatus and method for analysing device function |
| KR102649229B1 (en) * | 2020-07-27 | 2024-03-20 | 서강대학교 산학협력단 | Apparatus and method for analysing device function |
| CN112611775A (en) * | 2020-12-16 | 2021-04-06 | 聚束科技（北京）有限公司 | Method for observing biological tissue and electron microscope |
| CN112611775B (en) * | 2020-12-16 | 2023-08-11 | 聚束科技（北京）有限公司 | Method for observing biological tissue and electron microscope |
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Application publication date: 20180511 |