CN103808773B - The preparation method of the citrus fruit fly OBP sensor that polyglycol is fixed - Google Patents
The preparation method of the citrus fruit fly OBP sensor that polyglycol is fixed Download PDFInfo
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- CN103808773B CN103808773B CN201410028821.6A CN201410028821A CN103808773B CN 103808773 B CN103808773 B CN 103808773B CN 201410028821 A CN201410028821 A CN 201410028821A CN 103808773 B CN103808773 B CN 103808773B
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- 241000255588 Tephritidae Species 0.000 title claims abstract description 62
- 235000020971 citrus fruits Nutrition 0.000 title claims abstract description 62
- 229920000151 polyglycol Polymers 0.000 title claims abstract description 57
- 239000010695 polyglycol Substances 0.000 title claims abstract description 57
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- 239000010931 gold Substances 0.000 claims abstract description 53
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 52
- 229910052737 gold Inorganic materials 0.000 claims abstract description 52
- 235000013399 edible fruits Nutrition 0.000 claims abstract description 38
- 239000000796 flavoring agent Substances 0.000 claims abstract description 38
- 235000019634 flavors Nutrition 0.000 claims abstract description 38
- 239000000463 material Substances 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 11
- 238000001514 detection method Methods 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 84
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 claims description 46
- 238000013016 damping Methods 0.000 claims description 39
- 239000012530 fluid Substances 0.000 claims description 39
- 229940117955 isoamyl acetate Drugs 0.000 claims description 23
- 230000005477 standard model Effects 0.000 claims description 21
- 238000005259 measurement Methods 0.000 claims description 18
- 239000012488 sample solution Substances 0.000 claims description 18
- UZFLPKAIBPNNCA-BQYQJAHWSA-N alpha-ionone Chemical compound CC(=O)\C=C\C1C(C)=CCCC1(C)C UZFLPKAIBPNNCA-BQYQJAHWSA-N 0.000 claims description 14
- 239000002904 solvent Substances 0.000 claims description 12
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 12
- 239000012498 ultrapure water Substances 0.000 claims description 12
- 230000005540 biological transmission Effects 0.000 claims description 11
- 239000011521 glass Substances 0.000 claims description 10
- 239000000126 substance Substances 0.000 claims description 9
- 239000000758 substrate Substances 0.000 claims description 9
- 238000004140 cleaning Methods 0.000 claims description 8
- 239000000902 placebo Substances 0.000 claims description 7
- 229940068196 placebo Drugs 0.000 claims description 7
- 208000011580 syndromic disease Diseases 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 125000003368 amide group Chemical group 0.000 claims description 6
- 239000012895 dilution Substances 0.000 claims description 6
- 238000010790 dilution Methods 0.000 claims description 6
- 238000001755 magnetron sputter deposition Methods 0.000 claims description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 5
- -1 potassium ferricyanide Chemical compound 0.000 claims description 5
- 239000000276 potassium ferrocyanide Substances 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- XOGGUFAVLNCTRS-UHFFFAOYSA-N tetrapotassium;iron(2+);hexacyanide Chemical compound [K+].[K+].[K+].[K+].[Fe+2].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-].N#[C-] XOGGUFAVLNCTRS-UHFFFAOYSA-N 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 4
- 239000010408 film Substances 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 239000011259 mixed solution Substances 0.000 claims description 4
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 3
- UCKMPCXJQFINFW-UHFFFAOYSA-N Sulphide Chemical compound [S-2] UCKMPCXJQFINFW-UHFFFAOYSA-N 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000001312 dry etching Methods 0.000 claims description 3
- 239000003822 epoxy resin Substances 0.000 claims description 3
- 239000003292 glue Substances 0.000 claims description 3
- 238000001459 lithography Methods 0.000 claims description 3
- 238000002156 mixing Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 229920002120 photoresistant polymer Polymers 0.000 claims description 3
- 229920000647 polyepoxide Polymers 0.000 claims description 3
- 239000011550 stock solution Substances 0.000 claims description 3
- 239000001117 sulphuric acid Substances 0.000 claims description 3
- 235000011149 sulphuric acid Nutrition 0.000 claims description 3
- 230000015572 biosynthetic process Effects 0.000 claims description 2
- 239000010409 thin film Substances 0.000 claims description 2
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 claims 3
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims 1
- 238000012544 monitoring process Methods 0.000 abstract description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 6
- 239000007987 MES buffer Substances 0.000 description 5
- 241000238631 Hexapoda Species 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000001103 potassium chloride Substances 0.000 description 3
- 235000011164 potassium chloride Nutrition 0.000 description 3
- 238000000840 electrochemical analysis Methods 0.000 description 2
- 235000012055 fruits and vegetables Nutrition 0.000 description 2
- 230000002209 hydrophobic effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000005518 electrochemistry Effects 0.000 description 1
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- 235000013305 food Nutrition 0.000 description 1
- 238000001453 impedance spectrum Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000011895 specific detection Methods 0.000 description 1
- WFKWXMTUELFFGS-UHFFFAOYSA-N tungsten Chemical compound [W] WFKWXMTUELFFGS-UHFFFAOYSA-N 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 239000010937 tungsten Substances 0.000 description 1
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- Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
Abstract
The invention discloses the preparation method of the citrus fruit fly OBP biology sensor that a kind of polyglycol is fixed, utilize the interdigital gold electrode of round line style of the microelectromechanical systems processes of standard, use the interdigital gold electrode surfaces of polyglycol process circle line style of modifying, for fixing citrus fruit fly OBP, obtain the citrus fruit fly OBP sensor that described polyglycol is fixing.Citrus fruit fly OBP is fixed on the interdigital gold electrode of round line style by the present invention, for the detection of fruit volatile flavor material.Citrus fruit fly OBP can be stablized by the citrus fruit fly OBP sensor fixed of polyglycol that the method builds, easy be fixed on the interdigital gold electrode surfaces of round line style, and then detect fruit volatile flavor material, the citrus fruit fly OBP biology sensor that polyglycol prepared by the present invention is fixed is highly sensitive, Monitoring lower-cut is low, high specificity.
Description
Technical field
The present invention relates to a kind of technology of preparing of senser element, particularly relate to a kind of method utilizing polyglycol to fix citrus fruit fly OBP, and utilize citrus fruit fly OBP sensor to detect the method for fruit volatile flavor material (alpha, beta-lonone and isoamyl acetate).
Background technology
Citrus fruit fly is a kind of insect multiple fruits and vegetables to very large threat, and its host range is extremely wide, relates to more than 250 kind of fruits and vegetables.So be agricultural, forestry quarantine departments vital task for the control of citrus fruit fly.For the apperceive characteristic that citrus fruit fly is sensitive to fruity odours, the control of sensor for citrus fruit fly of special fruity odours can be set up.OBP is that one is present in the extracellular low-molecular-weight hydrophobic proteins of citrus fruit fly antenna factory.It reversibly can be combined with targeted odorous molecule, plays the effect of transport hydrophobic small molecules.Citrus fruit fly OBP easily obtains, Stability Analysis of Structures, can build biology sensor as biosensor.Utilize the advantage that citrus fruit fly OBP and impedance transducer combine, develop a kind of citrus fruit fly OBP biology sensor based on electrochemical analysis, can specific detection fruit volatile flavor material (alpha, beta-lonone and isoamyl acetate), may be used for that food composition detects fast, the fields such as insect control such as cultured insect and citrus fruit fly.
Summary of the invention
The object of the present invention is to provide a kind of preparations and applicatio of the citrus fruit fly OBP electrochemical impedance sensor fixed based on polyglycol.
The object of the invention is to be achieved through the following technical solutions: it is characterized in that, comprise the following steps:
(1) the interdigital gold electrode impedance transducer of processing circle line style: take glass as substrate, magnetron sputtering titanizing W film is as adhesion layer on the glass substrate, and then magnetron sputtering gold thin film is as electrode layer; The interdigital figure of round line style is made by lithography with AZ photoresist, dry etching is adopted to be etched away in non-electrode region again, obtain the circle interdigital gold electrode of line style and external pin, finally chip chamber epoxide-resin glue is sealed on the glass substrate, the interdigital gold electrode impedance transducer of obtained circle line style;
(2) fixing citrus fruit fly OBP, this step is realized by following sub-step:
(2.1) first, by circle line style interdigital gold electrode by mass concentration be more than 98% the concentrated sulphuric acid and volumetric concentration be soak 3-5min in the mixed solution that is mixed into for 7:3 by volume of hydrogen peroxide solution of 30%, then ultrapure water is used more than 3 times, last absolute ethyl alcohol and the ultrapure water used successively cleans the above-mentioned interdigital gold electrode of round line style, dries up with nitrogen;
(2.2) compound concentration is the polyglycol solution of 1.5mg/ml, and solvent is made up of absolute ethyl alcohol and ultrapure water 3:7 mixing by volume; Polyglycol solution is dripped on the interdigital gold electrode of circle line style to covering the interdigital gold electrode of circle line style completely, at room temperature leave standstill 24h, sulfydryl in polyglycol reacts with the interdigital gold electrode of circle line style and generates golden sulfide linkage, and then polyglycol is fixed on the interdigital gold electrode of round line style, the residual polyglycol solution in sucking-off chip chamber more afterwards;
(2.3) EDC(1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride that concentration that volume is polyglycol solution half is 8mg/ml is added respectively) solution and concentration is the NHS(N-N-Hydroxysuccinimide of 12mg/ml) solution, the solvent of EDC solution and NHS solution is 0.1MMES damping fluid, after 15min, then add NaHCO
3solution regulates pH to 7.2-7.4;
(2.4) the citrus fruit fly OBP solution that isopyknic concentration with polyglycol solution is 67 μ g/ml is then added, the solvent of citrus fruit fly OBP solution is 0.1MPBS damping fluid, standing at room temperature 2h, the carboxyl effect of the polyglycol in citrus fruit fly OBP and polyglycol solution is made to form stable amido link, and then formation amido link structure, then residual in sucking-off chip chamber liquid, 0.1MPBS damping fluid is filled it up with again in chip chamber, sucking-off PBS damping fluid after standing 5min, for cleaning loose citrus fruit fly OBP, the citrus fruit fly OBP sensor that polyglycol is fixing can be obtained after repeating 3 times, for subsequent use under being placed in 4 DEG C of conditions.
Apply the method for the citrus fruit fly OBP sensor detection fruit volatile flavor material that above-mentioned polyglycol is fixed, comprise the following steps:
(1) fruit volatile flavor standard of physical sample solution to be measured is prepared: adopt substance withdrawl syndrome to be the standard model solution of standard stock solution dilution preparation 5 kinds of respective concentration gradients of 0.01M fruit volatile flavor material to be measured; Dilution is 0.1MPBS damping fluid;
(2) fruit volatile flavor standard of physical sample solution is detected, set up fruit volatile flavor standard of physical concentration-normalized impedance curve: the measurement of placebo solution electrochemical impedance collection of illustrative plates: two input ends first two external pins of the interdigital gold electrode of circle line style being connected electrochemical workstation respectively by wire, then in chip chamber, isopyknic redox couple solution with polyglycol solution is added, containing the 10mM potassium ferricyanide in redox couple solution, the KCl of 10mM potassium ferrocyanide and 0.1M, solvent is ultrapure water, isopyknic PBS damping fluid is added with redox couple solution again in chip chamber, the scanning of electrochemical impedance collection of illustrative plates is carried out as the citrus fruit fly OBP sensor that the polyglycol that instrument platform is right is fixing using electrochemical workstation, obtain electrochemical impedance collection of illustrative plates and electron transmission resistance, measure and terminate the complete soln that in rear sucking-off chip chamber, measurement last time is residual, then 0.1MPBS damping fluid is filled it up with, slow sucking-off PBS damping fluid after leaving standstill 5min, for cleaning the interdigital gold electrode of round line style, carry out the measurement of fruit volatile flavor standard of physical sample solution electrochemical impedance collection of illustrative plates again, in chip chamber, add equivalent with the isopyknic redox couple solution of polyglycol solution and a certain substance withdrawl syndrome fruit volatile flavor substance solution, carry out the measurement of electrochemical impedance collection of illustrative plates, obtain electrochemical impedance collection of illustrative plates corresponding to fruit volatile flavor standard of physical sample solution under this substance withdrawl syndrome and electron transmission resistance, measure and terminate the complete soln that in rear sucking-off chip chamber, measurement last time is residual, then 0.1MPBS damping fluid is filled it up with, slow sucking-off PBS damping fluid after leaving standstill 15min, for cleaning the interdigital gold electrode of round line style,
(3) fruit volatile flavor substance solution normal concentration-normalized impedance curve is set up: the measuring process repeating the electrochemical impedance collection of illustrative plates of above-mentioned steps 2 empty contrast solution and fruit volatile flavor standard of physical sample solution, until complete the measurement of the fruit volatile flavor standard of physical sample solution of 5 kinds of respective concentration gradients, obtain the electrochemical impedance spectrogram under variable concentrations and electron transmission resistance, calculate normalized impedance (standard model solution and placebo solution difference are relative to the changing value of placebo solution), obtain the relation curve y=a between fruit volatile flavor standard of physical sample solution concentration and normalized impedance
-bx, wherein, x is fruit volatile flavor standard of physical sample solution concentration, and y is normalized impedance, a and b is constant, realizes the detection to fruit volatile flavor material.
The invention has the beneficial effects as follows, the present invention utilizes polyglycol to be fixed on by citrus fruit fly OBP on the interdigital gold electrode of round line style, construct the citrus fruit fly OBP sensor that polyglycol is fixing, may be used for the detection of fruit volatile flavor material (isoamyl acetate and alpha, beta-lonone).Experiment shows that citrus fruit fly OBP sensor that the method builds can by easy for citrus fruit fly OBP and be stably fixed on the interdigital gold electrode surfaces of round line style, and the citrus fruit fly OBP sensor detection sensitivity that polyglycol is fixed is high, Monitoring lower-cut is low.
Accompanying drawing explanation
Fig. 1 is the interdigital gold electrode layout viewing of the present invention's circle line style;
Fig. 2 is citrus fruit fly OBP sensor test figure of the present invention;
Fig. 3 is citrus fruit fly OBP sensor preparation figure of the present invention;
Fig. 4 is in citrus fruit fly OBP sensor preparation process of the present invention, and the interdigital gold electrode of circle line style, the interdigital gold electrode of circle line style are fixed polyglycol (SH-PEG-COOH) and justified the electrochemical impedance collection of illustrative plates that the interdigital gold electrode of line style fixes polyglycol (SH-PEG-COOH) and citrus fruit fly OBP;
Fig. 5 is the electrochemical impedance collection of illustrative plates that citrus fruit fly OBP sensor of the present invention detects isoamyl acetate scent molecule solution;
Fig. 6 is the electrochemical impedance collection of illustrative plates that citrus fruit fly OBP sensor of the present invention detects alpha, beta-lonone scent molecule solution;
Fig. 7 be OBP biology sensor of the present invention respectively and the fruit volatile flavor material (isoamyl acetate and alpha, beta-lonone) of variable concentrations react after graph of a relation between electron transmission resistance and fruit volatile flavor material (isoamyl acetate and alpha, beta-lonone) log concentration;
In figure: substrate of glass 1, the interdigital gold electrode 2 of circle line style, external pin 3, cylindrical 4 inner circle 5, interdigital roundlet 6, chip chamber 7, wire 8, electrochemical workstation 9.
Embodiment
Below in conjunction with drawings and the specific embodiments, the present invention is described in detail, but be not restriction the present invention.
The preparation method of the citrus fruit fly OBP sensor that polyglycol of the present invention is fixed, comprises the following steps:
1, the processing of the interdigital gold electrode impedance transducer of line style is justified.
The processing of the interdigital gold electrode 2 of circle line style adopts the processing technology of standard.Take thickness as 0.5mm, diameter is 1.6cm glass is substrate, titanizing tungsten (TiW) film that magnetron sputtering 20nm is thick on glass is as adhesion layer, and gold (Au) film that then magnetron sputtering 300nm is thick is as electrode layer; Make the interdigital figure of round line style by lithography with AZ photoresist, wherein in the round interdigital gold electrode 2 of line style, the diameter of cylindrical 4 is 6000 μm, and the diameter of inner circle 5 is 5400 μm, and the diameter of each interdigital roundlet 6 of circle line style interdigital gold electrode 2 is 50 μm.Finally adopt dry etching to be etched away in non-electrode region, obtain the interdigital gold electrode of circle line style 2 and external pin 3, as shown in Figure 1.Chip chamber 7 epoxide-resin glue be finally made by pmma material is enclosed in substrate of glass 1, as shown in Figure 2, and the interdigital gold electrode impedance transducer of obtained circle line style.
2, citrus fruit fly OBP is fixing.
First, the mass concentration adding 100 μ l in chip chamber 7 be more than 98% the concentrated sulphuric acid and concentration of volume percent be 30% hydrogen peroxide solution be mixed solution 3-5min that 7:3 is mixed into by volume, for soaking the interdigital gold electrode 2 of round line style, then with the round interdigital gold electrode 2 of line style of ultrapure water more than 3 times.Finally again clean the interdigital gold electrode 2 of above-mentioned round line style with absolute ethyl alcohol and ultrapure water, dry up with nitrogen.Compound concentration is that (polyglycol (SH-PEG-COOH) used in the application all refers to that mean molecular weight is 2100 for polyglycol (SH-PEG-COOH) solution of 1.5mg/ml, the polyglycol of sulfydryl and carboxyl is modified at two ends respectively), solvent is made up of absolute ethyl alcohol and ultrapure water 3:7 mixing by volume.100 μ l polyglycol (SH-PEG-COOH) solution are dripped on the interdigital gold electrode of round line style 2 in chip chamber 7 to covering the interdigital gold electrode 2 of circle line style completely.At room temperature leave standstill 24h, the sulfydryl (-SH) in polyglycol (SH-PEG-COOH) reacts with the interdigital gold electrode of circle line style 2 and generates golden sulfide linkage, and then is fixed on the interdigital gold electrode 2 of round line style by polyglycol (SH-PEG-COOH).Residual polyglycol (SH-PEG-COOH) solution in slow sucking-off chip chamber 7 afterwards, add EDC(1-(3-the dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride that concentration is 8mg/ml more respectively) solution and concentration is the NHS(N-N-Hydroxysuccinimide of 12mg/ml) each 40 μ l(solvents of solution are 0.1MMES damping fluid, containing 0.1MKCl, pH=6,0.1MMES refers to the volumetric molar concentration of MES in MES damping fluid, and 0.1MKCl refers to the volumetric molar concentration of MES buffer solution potassium chloride.The MES damping fluid used in the application all refers to 0.1M, the MES damping fluid of pH=6).After 15 minutes, then add NaHCO
3solution regulates pH to 7.2-7.4.Then add 100 μ l, (solvent is 0.1MPBS damping fluid for citrus fruit fly OBP solution that concentration is 67 μ g/ml, pH=7.2,0.1M refers to phosphatic volumetric molar concentration in PBS damping fluid, the PBS damping fluid used in the application all refers to 0.1M, the PBS damping fluid of pH=7.2), standing at room temperature 2h, make the carboxyl effect of the polyglycol (SH-PEG-COOH) in citrus fruit fly OBP and polyglycol (SH-PEG-COOH) solution form stable amido link, and then form amido link structure as shown in Figure 3.After liquid residual in slow sucking-off chip chamber 7, the 0.1MPBS damping fluid of 400 μ l is added again in chip chamber 7, PBS damping fluid after standing 5min in sucking-off chip chamber 7, for cleaning loose citrus fruit fly OBP, the citrus fruit fly OBP sensor that polyglycol is fixing can be obtained after repeating 3 times, for subsequent use under being placed in 4 DEG C of conditions.Citrus fruit fly OBP has the amino acid sequence shown in SEQIDNO.1.
The present invention is based on the fixing citrus fruit fly OBP sensor of polyglycol to can be used for detecting fruit volatile flavor material (isoamyl acetate and alpha, beta-lonone).This application is specific as follows:
(1) the standard model solution of fruit volatile flavor material (isoamyl acetate and alpha, beta-lonone) to be measured is prepared: adopt substance withdrawl syndrome to be the standard model solution of standard stock solution dilution preparation 5 kinds of respective concentration gradients of 0.01M fruit volatile flavor material (isoamyl acetate and alpha, beta-lonone) to be measured; Dilution end liquid is 0.1MPBS damping fluid;
(2) isoamyl acetate scent molecule standard model solution is detected, set up isoamyl acetate scent molecule solution normal concentration-normalized impedance curve: the measurement first carrying out blank electrochemical impedance collection of illustrative plates, first two external pins 3 of the interdigital gold electrode of round line style 2 in Fig. 1 are connected two input ends of electrochemical workstation 9 respectively by wire 8, electrochemical workstation 9 can adopt the product of Shanghai Chen Hua Instrument Ltd. CHI660 model.Then in chip chamber 7, add 200 μ l redox couple solution, the KCl containing the 10mM potassium ferricyanide, 10mM potassium ferrocyanide and 0.1M in redox couple solution, solvent is ultrapure water.In chip chamber 7, add the PBS damping fluid of 200 μ l again as blank, using electrochemical workstation 9 as instrument platform, the scanning of electrochemical impedance collection of illustrative plates is carried out to the citrus fruit fly OBP sensor that the polyglycol that the present invention is based on electrochemical analysis is fixing.Concrete test parameter is initial voltage is 0.2V, and AC voltage magnitudes is 5mV, and swept frequency range is 0.1Hz ~ 100KHz, finally obtains electrochemical impedance collection of illustrative plates, utilizes the matching of typical equivalent impedance circuit to obtain the electron transmission resistance (R of blank
ct).After measurement terminates, first slowly sucking-off measured residual solution to the last time in chip chamber 7, then the PBS damping fluid of 400 μ l is added, slow sucking-off PBS damping fluid after leaving standstill 5min, for cleaning the mixed solution impact of measuring the residual potassium ferricyanide/potassium ferrocyanide and PBS damping fluid last time.Carry out isoamyl acetate scent molecule standard model solution afterwards again, add 10 of 200 μ l redox couple solution and 200 μ l respectively
-7the isoamyl acetate scent molecule standard model solution of M, in chip chamber 7, carries out the measurement of electrochemical impedance collection of illustrative plates, obtains 10
-7the electrochemical impedance collection of illustrative plates that M isoamyl acetate scent molecule standard model solution is corresponding.After one-shot measurement terminates, in first sucking-off chip chamber 7, last time measures residual solution, in chip chamber 7, then add the PBS damping fluid of 400 μ l, slow sucking-off PBS damping fluid after leaving standstill 15min, measures the residual potassium ferricyanide/potassium ferrocyanide and 10 to eliminate last time
-7the impact of M isoamyl acetate scent molecule standard model solution.Repeat the measurement of above-mentioned blank measure and isoamyl acetate scent molecule standard model solution, until complete 10
-6m, 10
-5m, 10
-4m and 10
-3the measurement of M isoamyl acetate scent molecule standard model solution.Finally obtain the fixing citrus fruit fly OBP sensor of polyglycol and 5 variable concentrations (10
-7m, 10
-6m, 10
-5m, 10
-4m and 10
-3m) the interactional electrochemical impedance collection of illustrative plates of isoamyl acetate scent molecule standard model solution under, as shown in Figure 5.Utilize 5 variable concentrations (10
-7m, 10
-6m, 10
-5m, 10
-4m and 10
-3m) the electron transmission resistance of isoamyl acetate scent molecule standard model solution, calculate normalized impedance (standard model solution and placebo solution difference relative to the changing value of placebo solution, i.e. (R
ct-PBS-R
ct-odor)/R
ct-PBS, wherein R
ct-PBSfor the electron transmission resistance of blank electrochemical impedance collection of illustrative plates, R
ct-odorthe electron transmission resistance of isoamyl acetate scent molecule standard model solution electrochemistry impedance spectrum under respective concentration), obtain the relation curve y=a between isoamyl acetate scent molecule standard model solution concentration and normalized impedance
-bx, wherein, x is isoamyl acetate scent molecule standard model solution concentration, and y is normalized impedance, a and b is constant; As shown in Figure 7;
(3) alpha, beta-lonone scent molecule standard model solution is detected, set up alpha, beta-lonone scent molecule solution normal concentration-normalized impedance curve: method, with above-mentioned steps 2, finally obtains the fixing citrus fruit fly OBP sensor of polyglycol and 5 variable concentrations (10
-7m, 10
-6m, 10
-5m, 10
-4m and 10
-3m) the interactional electrochemical impedance collection of illustrative plates of alpha, beta-lonone scent molecule standard model solution under, as shown in Figure 6, and obtains the relation curve between alpha, beta-lonone scent molecule standard model solution and normalized impedance, as shown in Figure 7.
SEQUENCELISTING
<110> Zhejiang University
The preparation method of the citrus fruit fly OBP sensor that <120> polyglycol is fixed
<160>1
<170>PatentInversion3.3
<210>1
<211>148
<212>PRT
<213> citrus fruit fly
<400>1
MetHisSerArgLysThrLeuLeuGlyThrLeuLeuTrpIleGlyPhe
151015
LeuLeuAsnPheIleTrpAlaGlnLysGluLeuArgArgAspGluThr
202530
TyrProProProGluLeuLeuLysGluLeuGlnProValHisAspSer
354045
CysValAlaLysThrGlyValThrGluGluAlaIleLysGluPheSer
505560
AspGlyAspValHisGluAspGluLeuLeuLysCysTyrMetTyrCys
65707580
ValPheGluGluThrAspValLeuHisGluAspGlyGluValHisLeu
859095
GluLysIleLeuAspLysLeuProGluSerMetHisValIleAlaLeu
100105110
HisMetGlyLysLysCysLeuTyrProLysGlyAspAsnLysCysGlu
115120125
ArgAlaPheTrpLeuHisArgCysTrpLysGluAlaAspProLysHis
130135140
TyrPheLeuIle
145
Claims (2)
1. the preparation method of citrus fruit fly OBP sensor that fixes of polyglycol, is characterized in that, comprise the following steps:
(1) the interdigital gold electrode impedance transducer of processing circle line style: take glass as substrate, at the upper magnetron sputtering titanizing W film of substrate of glass (1) as adhesion layer, then magnetron sputtering gold thin film is as electrode layer; The interdigital figure of round line style is made by lithography with AZ photoresist, dry etching is adopted to be etched away in non-electrode region again, obtain the circle interdigital gold electrode of line style (2) and external pin (3), finally chip chamber (7) are enclosed in substrate of glass (1) with epoxide-resin glue, the interdigital gold electrode impedance transducer of obtained circle line style;
(2) fixing citrus fruit fly OBP, this step is realized by following sub-step:
(2.1) first, by justify the interdigital gold electrode of line style (2) by mass concentration be more than 98% the concentrated sulphuric acid and volumetric concentration be soak 3-5min in the mixed solution that is mixed into for 7:3 by volume of hydrogen peroxide solution of 30%, then ultrapure water is used more than 3 times, last absolute ethyl alcohol and the ultrapure water used successively cleans the above-mentioned interdigital gold electrode of round line style (2), dries up with nitrogen;
(2.2) compound concentration is the polyglycol solution of 1.5mg/ml, and described polyglycol is mean molecular weight is 2100, and the polyglycol of sulfydryl and carboxyl is modified at two ends respectively, and solvent is made up of absolute ethyl alcohol and ultrapure water 3:7 mixing by volume; 100 μ l polyglycol solutions are dripped at the circle interdigital gold electrode of line style (2) upper to covering the circle interdigital gold electrode of line style (2) completely, at room temperature leave standstill 24h, sulfydryl in polyglycol reacts with the circle interdigital gold electrode of line style (2) and generates golden sulfide linkage, and then polyglycol is fixed on the interdigital gold electrode of round line style (2), polyglycol solution residual in sucking-off chip chamber (7) more afterwards;
(2.3) NHS (N-hydroxy-succinamide) solution that EDC (1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride) solution that concentration that volume is 40 μ l is 8mg/ml and concentration are 12mg/ml is added respectively, the solvent of EDC solution and NHS solution is 0.1MMES damping fluid, in described MES damping fluid, the volumetric molar concentration of MES is 0.1M, and contain the pH=6 that concentration is the KCl of 0.1M, damping fluid; After 15min, then add NaHCO
3solution regulates pH to 7.2-7.4;
(2.4) 100 μ l are then added, concentration is the citrus fruit fly OBP solution of 67 μ g/ml, the solvent of citrus fruit fly OBP solution is 0.1MPBS damping fluid, standing at room temperature 2h, the carboxyl effect of the polyglycol in citrus fruit fly OBP and polyglycol solution is made to form stable amido link, and then formation amido link structure, then residual in sucking-off chip chamber (7) liquid, 0.1MPBS damping fluid is filled it up with again in chip chamber (7), sucking-off PBS damping fluid after standing 5min, for cleaning loose citrus fruit fly OBP, the citrus fruit fly OBP sensor that polyglycol is fixing can be obtained after repeating 3 times, for subsequent use under being placed in 4 DEG C of conditions.
2. the citrus fruit fly OBP biology sensor that the polyglycol prepared by preparation method described in claim 1 is fixed detects the method for fruit volatile flavor material, described fruit volatile flavor material is isoamyl acetate or alpha, beta-lonone, it is characterized in that, comprise the following steps:
(1) fruit volatile flavor standard of physical sample solution to be measured is prepared:
Adopt substance withdrawl syndrome to be the standard model solution of standard stock solution dilution preparation 5 kinds of respective concentration gradients of 0.01M fruit volatile flavor material to be measured, dilution is 0.1MPBS damping fluid;
(2) detect fruit volatile flavor standard of physical sample solution, set up fruit volatile flavor standard of physical sample solution concentration-normalized impedance curve:
First the measurement of placebo solution electrochemical impedance collection of illustrative plates is carried out, two the external pins (3) first justifying the interdigital gold electrode of line style (2) connect two input ends of electrochemical workstation (9) respectively by wire (8), then in chip chamber (7), 200 μ l redox couple solution are added, containing the 10mM potassium ferricyanide in redox couple solution, the KCl of 10mM potassium ferrocyanide and 0.1M, solvent is ultrapure water, isopyknic PBS damping fluid with redox couple solution is added again in chip chamber (7), using electrochemical workstation (9) as instrument platform, the scanning of electrochemical impedance collection of illustrative plates is carried out to the citrus fruit fly OBP sensor that polyglycol is fixing, obtain electrochemical impedance collection of illustrative plates and electron transmission resistance, measurement terminates last time in rear sucking-off chip chamber (7) and measures residual complete soln, then 0.1MPBS damping fluid is filled it up with, slow sucking-off PBS damping fluid after leaving standstill 5min, for cleaning the interdigital gold electrode of round line style (2),
Carry out the measurement of fruit volatile flavor standard of physical sample solution electrochemical impedance collection of illustrative plates again, the fruit volatile flavor standard of physical sample solution of 200 μ l redox couple solution and a certain substance withdrawl syndrome of 200 μ l is added in chip chamber (7), carry out the measurement of electrochemical impedance collection of illustrative plates, obtain electrochemical impedance collection of illustrative plates corresponding to fruit volatile flavor standard of physical sample solution under this substance withdrawl syndrome and electron transmission resistance, measurement terminates last time in rear sucking-off chip chamber (7) and measures residual complete soln, then 0.1MPBS damping fluid is filled it up with, slow sucking-off PBS damping fluid after leaving standstill 15min, for cleaning the interdigital gold electrode of round line style (2),
Repeat the measuring process of the electrochemical impedance collection of illustrative plates of placebo solution and fruit volatile flavor standard of physical sample solution, until complete the measurement of the fruit volatile flavor standard of physical sample solution of 5 kinds of respective concentration gradients, obtain the electrochemical impedance spectrogram under variable concentrations and electron transmission resistance, calculate normalized impedance, obtain the relation curve y=a between fruit volatile flavor standard of physical sample solution concentration and normalized impedance
-bx, wherein, x is fruit volatile flavor standard of physical sample solution concentration, and y is normalized impedance, a and b is constant, realizes the detection to fruit volatile flavor material.
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