CN107976476A - A kind of assay method of the lactic acid dehydrogenase activity based on titanium deoxid film field-effect tube - Google Patents
A kind of assay method of the lactic acid dehydrogenase activity based on titanium deoxid film field-effect tube Download PDFInfo
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- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 101710088194 Dehydrogenase Proteins 0.000 title claims abstract description 26
- 239000004310 lactic acid Substances 0.000 title claims abstract description 24
- 235000014655 lactic acid Nutrition 0.000 title claims abstract description 24
- 230000005669 field effect Effects 0.000 title claims abstract description 18
- 230000000694 effects Effects 0.000 title claims abstract description 16
- RTAQQCXQSZGOHL-UHFFFAOYSA-N Titanium Chemical compound [Ti] RTAQQCXQSZGOHL-UHFFFAOYSA-N 0.000 title claims abstract description 12
- 229910052719 titanium Inorganic materials 0.000 title claims abstract description 12
- 239000010936 titanium Substances 0.000 title claims abstract description 12
- 238000003556 assay Methods 0.000 title claims abstract description 4
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 claims abstract description 25
- 229910052710 silicon Inorganic materials 0.000 claims abstract description 25
- 239000010703 silicon Substances 0.000 claims abstract description 25
- 238000012360 testing method Methods 0.000 claims abstract description 19
- 108090000790 Enzymes Proteins 0.000 claims abstract description 18
- 102000004190 Enzymes Human genes 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 17
- 238000001514 detection method Methods 0.000 claims abstract description 15
- 230000002255 enzymatic effect Effects 0.000 claims abstract description 12
- 230000008569 process Effects 0.000 claims abstract description 8
- 238000000137 annealing Methods 0.000 claims abstract description 5
- 230000035484 reaction time Effects 0.000 claims abstract description 5
- 238000004140 cleaning Methods 0.000 claims abstract description 3
- 238000001952 enzyme assay Methods 0.000 claims abstract description 3
- 238000004528 spin coating Methods 0.000 claims abstract description 3
- GWEVSGVZZGPLCZ-UHFFFAOYSA-N titanium dioxide Inorganic materials O=[Ti]=O GWEVSGVZZGPLCZ-UHFFFAOYSA-N 0.000 claims description 15
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
- 230000008859 change Effects 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 8
- 229910052737 gold Inorganic materials 0.000 claims description 7
- 239000010931 gold Substances 0.000 claims description 7
- 239000000523 sample Substances 0.000 claims description 7
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 229920001661 Chitosan Polymers 0.000 claims description 5
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 5
- 239000003153 chemical reaction reagent Substances 0.000 claims description 5
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 5
- 239000012498 ultrapure water Substances 0.000 claims description 5
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 4
- 238000001704 evaporation Methods 0.000 claims description 4
- 230000008020 evaporation Effects 0.000 claims description 4
- 239000000706 filtrate Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 4
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 238000007747 plating Methods 0.000 claims description 3
- CBENFWSGALASAD-UHFFFAOYSA-N Ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 claims description 2
- 238000002425 crystallisation Methods 0.000 claims description 2
- 230000008025 crystallization Effects 0.000 claims description 2
- 238000001035 drying Methods 0.000 claims description 2
- 238000010894 electron beam technology Methods 0.000 claims description 2
- 229910052709 silver Inorganic materials 0.000 claims description 2
- 239000004332 silver Substances 0.000 claims description 2
- 238000007740 vapor deposition Methods 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- VXUYXOFXAQZZMF-UHFFFAOYSA-N titanium(IV) isopropoxide Chemical compound CC(C)O[Ti](OC(C)C)(OC(C)C)OC(C)C VXUYXOFXAQZZMF-UHFFFAOYSA-N 0.000 claims 1
- 238000005259 measurement Methods 0.000 abstract description 5
- 210000002966 serum Anatomy 0.000 abstract description 5
- 230000004044 response Effects 0.000 abstract description 3
- 238000010998 test method Methods 0.000 abstract description 3
- 230000033001 locomotion Effects 0.000 abstract 1
- 239000010408 film Substances 0.000 description 22
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008279 sol Substances 0.000 description 4
- 210000001519 tissue Anatomy 0.000 description 3
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- 239000006059 cover glass Substances 0.000 description 2
- 230000005611 electricity Effects 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 239000004408 titanium dioxide Substances 0.000 description 2
- 238000012876 topography Methods 0.000 description 2
- 206010001557 Albinism Diseases 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 1
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 208000031845 Pernicious anaemia Diseases 0.000 description 1
- 108010069013 Phenylalanine Hydroxylase Proteins 0.000 description 1
- 102100038223 Phenylalanine-4-hydroxylase Human genes 0.000 description 1
- 201000011252 Phenylketonuria Diseases 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 208000034189 Sclerosis Diseases 0.000 description 1
- 102000003425 Tyrosinase Human genes 0.000 description 1
- 108060008724 Tyrosinase Proteins 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005566 electron beam evaporation Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000002414 glycolytic effect Effects 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000032839 leukemia Diseases 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 1
- BOPGDPNILDQYTO-NNYOXOHSSA-N nicotinamide-adenine dinucleotide Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 BOPGDPNILDQYTO-NNYOXOHSSA-N 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- OGIDPMRJRNCKJF-UHFFFAOYSA-N titanium oxide Inorganic materials [Ti]=O OGIDPMRJRNCKJF-UHFFFAOYSA-N 0.000 description 1
- 238000002211 ultraviolet spectrum Methods 0.000 description 1
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/403—Cells and electrode assemblies
- G01N27/414—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS
- G01N27/4145—Ion-sensitive or chemical field-effect transistors, i.e. ISFETS or CHEMFETS specially adapted for biomolecules, e.g. gate electrode with immobilised receptors
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microelectronics & Electronic Packaging (AREA)
- Engineering & Computer Science (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Electrochemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of assay method of the lactic acid dehydrogenase activity based on titanium deoxid film field-effect tube.Include the following steps：（1）Sol-gal process prepares titanium deoxid film：Cleaning silicon chip, prepares solution, spin coating, annealing；（2）Plated electrode and the connection of three electrodes；（3）Enzyme immobilizatio；（4）The detection of enzymatic activity：The electrology characteristic of the field-effect tube of Enzyme assay is tested using KEITHLEY 4200, first test the I V curves of lactic dehydrogenase between D S under different grid voltages, then test is detected immediately after various concentrations lactic acid is added dropwise, then it is detected in multiple reaction time points, observes the I V curvilinear motions between differential responses time D S.The present invention is without test paper, test period is short, response is fast, it is possible to achieve the quick detection of enzymatic activity.The enzyme concentration that the present invention needs is low, and volume is few, and can be immobilised enzymes.Test method is simple, and a new method is provided for enzymatic activity measurement in serum.
Description
Technical field
The present invention relates to a kind of determination techniques of the lactic acid dehydrogenase activity based on titanium deoxid film field-effect tube.
Background technology
The generation of many diseases and the exception of enzyme are closely related in human body, will trigger albinism as tyrosinase lacks,
Phenylalanine hydroxylase missing causes phenylketonuria, and Dehydrogenase Content is higher, it may be possible to because leukaemia, lung block
And caused by the Pleural effusions caused by metastases.The disease of many histoorgans often shows as the different of some enzymatic activitys in blood
Often, the measure of enzymatic activity contributes to the diagnose and treat of many diseases.Under normal circumstances, the enzyme to play a role in tissue is in serum
Middle content is little, only works as tissue damaged, and special marker enzyme can largely be released into blood, causes enzyme content in serum to increase.
The assay method of enzyme content mainly has performance rate method in serum（Continuous monitoring method）With timing colorimetric method.Both approaches are both needed to
The enzyme amount for being detected and being consumed with large-scale special instrument is more（It is generally necessary to gather 3-4 ml venous blood）.Meanwhile clinically
Need to undergo multiple processes such as collection, separation and the storage of blood preparation, any one process before the measure of enzymatic activity
Dealing with improperly can make it that measured value is inaccurate.Based on factors above, it is necessary to develop a kind of new test method avoiding or
Reduce produced problem in the above method.It is more sensitive to electric charge in view of the electrology characteristic of field-effect tube, and different enzymes leads to
Often carry different electric charge and polarity.Therefore, we are based on titanium dioxide field-effect tube, for its electrology characteristic with enzyme class and
Activity change and the characteristics of change, it is proposed that a kind of detection method of enzymatic activity.Involved detection method in the patent of invention
The detection process of enzymatic activity is greatly simplify, the diagnose and treat of relevant disease can be greatly facilitated.
Meanwhile lactic dehydrogenase is a kind of glycolytic ferment, it is present in all histiocytic kytoplasms of body.Lactic dehydrogenase
Enzyme content it is higher or relatively low, it is meant that the exception of some tissues in body, higher cardiac muscle is common in as lactic dehydrogenase checks
The illnesss such as inflammation, hepatic sclerosis, malignant tumour and pernicious anaemia, its detection have health particularly important meaning.Base of the present invention
In titanium dioxide field-effect tube, it is established that the detection method of lactic acid dehydrogenase activity, so that by electricity means fast and accurately
Examination goes out the root of disease, so as to preferably be treated.
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 titanium deoxid film field-effect tube
The technology of the detection method of lactic acid dehydrogenase activity.
To achieve these goals, the present invention adopts the following technical scheme that：
A kind of detection method of the lactic acid dehydrogenase activity based on titanium deoxid film field-effect tube, includes the following steps：
1st, the field-effect tube structure based on Enzyme assay is prepared
（1）Sol-gal process prepares titanium dioxide（TiO2）Film
Cleaning silicon chip：N-type silicon chip first（Resistivity< 0.1 Ω·cm）The back side a small amount of BOE solution is added dropwise to remove Si surfaces
The SiO of self-assembling formation2, cleaned with ultra-pure water after 30s, then silicon chip be positioned in beaker, successively with ethanol, acetone, ultrapure
Each ultrasonic 15 minutes of water, uses N2Drying, the back side of silicon chip is uniformly coated with conductive silver paste solution to prevent the Si surfaces appeared again
Secondary oxidation, after completely dry etc. silver paste, then silicon chip is positioned on ozone clean machine and is cleaned half an hour, clean silicon wafer is stand-by.
Prepare solution：In N2In glove box, take 5mL purity not less than 99.9% isopropanol in 10mL band cover glass reagent bottles
In, stirring magneton is put into, glass reagent bottle cap is covered tightly, is positioned on magnetic force heating stirrer, is added dropwise while stirring
350 μ L, 99.995% titanium tetraisopropylates, after mixing, add 10 μ L, 36.5% hydrochloric acid, and closeing the lid prevents hydrochloric acid from volatilizing,
Finally 5h is stirred at 60 DEG C.
Spin coating：Clean silicon wafer is placed on sol evenning machine, sol evenning machine rotates under the low speed, and measuring a small amount of filtrate with pipettor drips
It is added on silicon chip, then forms a film at a high speed, setting rotating speed is 6000r/min, rejection film 30s, and silicon chip is placed on warm table after rejection film
Upper 110 DEG C of heating 5min.
Annealing：The wet film that rejection film is obtained is placed in tube furnace, 350 DEG C of pre-burning 5min under 5kPa oxygen atmospheres, then
The 1h that anneals at 550 DEG C completes crystallization process, obtains TiO2Film.
（2）Plated electrode and the connection of three electrodes：Strip mask plate is placed in silicon chip surface, using the gold-plated electricity of electron-beam vapor deposition method
Pole, setting evaporation rate are 0.5/s, and Au thickness is 60nm.Using n-Si as gate electrode（gate）, source electrode（source）And leakage
Electrode（drain）For the gold electrode of surface plating.
（3）Enzyme immobilizatio：In source-drain electrode（S-D）Between chitosan, glutaraldehyde and lactic dehydrogenase are added dropwise successively, often
Step drips solution and fixes a hour, is finally cleaned with ultra-pure water, and it is stand-by to place 4 DEG C of refrigerator.
2nd, the detection of enzymatic activity
Conductivity is tested：Ready sample in step 1 is placed in four probe stations, enzyme activity is tested using KEITHLEY 4200
Property detection field-effect tube electrology characteristic, first test the I-V curve of lactic dehydrogenase between D-S under different grid voltages, so
Test is detected immediately after various concentrations lactic acid is added dropwise afterwards, is then detected in multiple reaction time points, is observed different anti-
I-V curve change between seasonable between D-S, test result and the results change trend that ultraviolet spectra detects are very consistent.
The present invention is based on titanium deoxid film field-effect tube, using chitosan and glutaraldehyde as crosslinking agent, by testing not
With the I-V curve between D-S after the I-V curve between the D-S of lactic dehydrogenase under grid voltage and dropwise addition various concentrations lactic acid
Change with time, realize the measure of lactic acid dehydrogenase activity.Test result shows, under 0 to 5V bias, TiO2On film
After lactic dehydrogenase, current value increase, while plus after grid voltage, the results showed that, gate voltage, which has this electric current, to be adjusted
Control acts on.Further, after various concentrations lactic acid being added dropwise, record the I-V curve between D-S and electric current changes with time.As a result
Show, during same concentration, electric current is very consistent with time downward trend and amplitude and result that spectral measurement obtains.Based on this
Patent of invention, the activity of enzyme can be directly obtained by the measurement of conductance, woth no need to excessive processing step and dedicated large-scale
Instrument, measurement result can provide certain reference value for the diagnosis of some diseases.
Compared with prior art, the present invention has the advantages that：
1. the present invention is without test paper, test period is short, response is fast, it is possible to achieve the quick detection of enzymatic activity.
2. the enzyme concentration that the present invention needs is low, volume is few, and can be immobilised enzymes.
3. test method of the present invention is simple, a new method is provided for enzymatic activity measurement in serum.
Brief description of the drawings
Fig. 1 is TiO2The schematic diagram of the lactic acid dehydrogenase activity detection method of thin film field-effect pipe, wherein chitosan are referred to as
CHIT, glutaraldehyde abbreviation GA, lactic dehydrogenase abbreviation LDH；
Fig. 2 is TiO2The XRD test results of film；
Fig. 3 is TiO2Film（a）AFM surface topography maps and（b）SEM surface topography maps；
Fig. 4 is to be based on TiO2The detection of the lactic acid dehydrogenase activity of thin film field-effect pipe：Wherein（a）VgDuring=0.1V, to
TiO2After film lactic acid, VDS-IDSCurve map；（b）After the lactic acid of various concentrations is added dropwise on to LDH, VDS-IDSCurve map；
（c）After LDH 1h are added dropwise, it is 10 to test lactic acid concn with KEITHLEY 4200-3During M, maximum current with the reaction time change
Figure；（d）After LDH 1h are added dropwise, uv-vis spectra measures same concentrations lactic acid is added dropwise after, absorbance is at any time at 340nm by NADH
Between change.
Embodiment
Embodiment 1：In N2In glove box, first, 5mL purity is taken to be tried not less than 99.9% isopropanol in 10mL band cover glasses
In agent bottle, stirring magneton is put into, glass reagent bottle cap is covered tightly, is positioned on magnetic force heating stirrer, while stirring dropwise
350 μ L, 99.995% titanium tetraisopropylates are added, after mixing, add 10 μ L, 36.5% hydrochloric acid, closeing the lid prevents hydrochloric acid
Volatilization, finally stirs 5h at 60 DEG C.After, it is stand-by with 0.22 μm of PTFE filter element filtering, filtrate.Clean silicon wafer is placed in
On sol evenning machine, sol evenning machine rotates under the low speed, measures a small amount of filtrate added drop-wise to silicon chip with pipettor, then forms a film at a high speed, if
It is 6000r/min to put rotating speed, rejection film 30s, and silicon chip is placed on 110 DEG C of heating 5min on warm table after rejection film.By sample from hand
Take out, be positioned in tube furnace in casing, 350 DEG C of pre-burnings 5min, 550 DEG C of annealing 1h under 5kPa oxygen atmosphere.Treat that sample is cold
But to taking out after room temperature, XRD is used（Such as Fig. 2）, SEM and AFM（Such as Fig. 3）To TiO2Film carries out component, structure and morphology characterization,
The result shows that TiO2Film is uniform, smooth, fine and close.Strip mask plate is placed in sample surfaces, using electron beam evaporation gold evaporation
Electrode, setting evaporation rate are 0.5/s, and Au thickness is 60nm.Using n-type silicon as gate electrode, the two neighboring bar shaped of surface plating
Gold electrode is source electrode and drain electrode.TiO is tested by KEITHLEY 4200 first2The electric property that film has, Ran Hou
It is added dropwise the 0.5wt% chitosans of 2 μ L among source-drain electrode, even spread, after 1h, is added dropwise 2.5% glutaraldehyde of 2 μ L, uniformly applies
Cloth, after 1h, continues that 2 μ L lactic dehydrogenases are added dropwise, the electric property of lactic dehydrogenase is tested after fixed 1h, as a result such as Fig. 4（a）,
Then to the lactic acid of dropwise addition various concentrations on LDH, test result such as Fig. 4（b）, draw in VgDuring=0.1V, lactic acid concn is bigger,
Electric current is bigger.When lactic acid concn is added to maximum 10-3During M, every the I-V curve of 1min in record 5min, as a result such as Fig. 4（c）.
It can be seen that, on the one hand, the addition of lactic dehydrogenase so that conductance increases, and special indicatrix is presented, and gate voltage is to this
Electric current has regulating and controlling effect.On the other hand, after Immobilized lactate dehydrogenase, the lactic acid of various concentrations is added dropwise in the above, it is lived
The change of property causes the change of electric current in field-effect tube, its activity can be judged by the test of conductance.Experimental result table
Bright, the activity of lactic dehydrogenase is gradually reduced as the time increases, and conductance is gradually reduced, and electrical performance testing result is surveyed with spectrum
Test result is very consistent（Such as Fig. 4（c）With 4（d））.
Claims (1)
1. a kind of assay method of the lactic acid dehydrogenase activity based on titanium deoxid film field-effect tube, it is characterised in that including such as
Lower step：
（1）Sol-gal process prepares titanium deoxid film：
Cleaning silicon chip：BOE solution is added dropwise at the back side of n-type silicon chip to remove the SiO of Si surfaces self-assembling formation2, it is clear with ultra-pure water
Wash, then silicon chip is positioned in beaker, be ultrasonically treated successively with ethanol, acetone, ultra-pure water, use N2Drying, the back side of silicon chip
Conductive silver paste solution is uniformly coated with, after completely dry etc. silver paste, then silicon chip is positioned on ozone clean machine and is cleaned, clean silicon wafer is treated
With；
Prepare solution：In N2In glove box, isopropanol is taken to be put into stirring magneton in glass reagent bottle, by glass reagent bottle cap
Cover tightly, be positioned on magnetic force heating stirrer, titanium tetraisopropylate is added dropwise while stirring, after mixing, add salt
Acid, closeing the lid prevents hydrochloric acid from volatilizing, and finally stirs 5h at 60 DEG C；
Spin coating：Clean silicon wafer is placed on sol evenning machine, sol evenning machine rotates under the low speed, and measuring a small amount of filtrate added drop-wise with pipettor arrives
On silicon chip, then form a film at a high speed, setting rotating speed is 6000r/min, rejection film 30s, and silicon chip is placed on warm table after rejection film
110 DEG C of heating 5min；
Annealing：The wet film that rejection film is obtained is placed in tube furnace, 350 DEG C of pre-burning 5min under 5kPa oxygen atmospheres, then at 550 DEG C
Lower annealing 1h completes crystallization process, obtains TiO2Film；
（2）Plated electrode and the connection of three electrodes：Strip mask plate is placed in silicon chip surface, using electron-beam vapor deposition method gold-plated electrode,
Setting evaporation rate is 0.5/s, and Au thickness is 60nm；For n-Si as gate electrode, source electrode and drain electrode is the gold of surface plating
Electrode；
（3）Enzyme immobilizatio：Chitosan, glutaraldehyde and lactic dehydrogenase are added dropwise successively in source-electric leakage interpolar, often step drips
Solution fixes a hour, is finally cleaned with ultra-pure water；
（4）The detection of enzymatic activity：By step（1）In ready sample be placed in four probe stations, tested using KEITHLEY 4200
The electrology characteristic of the field-effect tube of Enzyme assay, the I-V for first testing lactic dehydrogenase between D-S under different grid voltages are bent
Line, then test are detected, are then detected in multiple reaction time points immediately after various concentrations lactic acid is added dropwise, and observation is not
With the I-V curve change between reaction time D-S.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373407A (en) * | 2019-07-31 | 2019-10-25 | 西南交通大学 | Utilize method, Biofunctional materials and the application of the fixed catalase of photocatalysis |
| WO2020043026A1 (en) * | 2018-08-28 | 2020-03-05 | 长沙理工大学 | Method and sensor for detecting l-cystine |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103472116A (en) * | 2013-08-29 | 2013-12-25 | 中国科学院化学研究所 | Ultrathin film field effect transistor sensor and application thereof |
| CN106098790A (en) * | 2016-08-30 | 2016-11-09 | 上海大学 | There is nitrogenous FET device of sull and preparation method thereof |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103472116A (en) * | 2013-08-29 | 2013-12-25 | 中国科学院化学研究所 | Ultrathin film field effect transistor sensor and application thereof |
| CN106098790A (en) * | 2016-08-30 | 2016-11-09 | 上海大学 | There is nitrogenous FET device of sull and preparation method thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020043026A1 (en) * | 2018-08-28 | 2020-03-05 | 长沙理工大学 | Method and sensor for detecting l-cystine |
| US11939625B2 (en) | 2018-08-28 | 2024-03-26 | Changsha University Of Science And Technology | Method and sensor for detecting L-cystine |
| CN110373407A (en) * | 2019-07-31 | 2019-10-25 | 西南交通大学 | Utilize method, Biofunctional materials and the application of the fixed catalase of photocatalysis |
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