CN103149111A - Method for detecting odor substance butanedione based on olfactory receptor sensor - Google Patents
Method for detecting odor substance butanedione based on olfactory receptor sensor Download PDFInfo
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- CN103149111A CN103149111A CN201310060405XA CN201310060405A CN103149111A CN 103149111 A CN103149111 A CN 103149111A CN 201310060405X A CN201310060405X A CN 201310060405XA CN 201310060405 A CN201310060405 A CN 201310060405A CN 103149111 A CN103149111 A CN 103149111A
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Abstract
The invention discloses a method for detecting an odor substance butanedione based on an olfactory receptor sensor. The method comprises the following steps of: at first, expressing olfactory receptor protein ODR-10 with a His6-tag on an HEK-293 cell surface, extracting and effectively immobilizing the olfactory receptor protein ODR-10 on a quartz crystal microbalance surface through an aptamer so as to form the olfactory receptor sensor; testing a series of odor material butanedione with different set concentrations, thus obtaining a butanedione-resonance frequency variation standard curve; and then, testing a gas which has unknown concentration and needs to be tested, thereby obtaining the concentration of the odor substance-butanedione in the gas to be tested according to the quartz crystal microbalance resonant frequency and the butanedione-resonance frequency variation standard curve. The method is simple in required instrument and convenient to operate, a problem that the olfactory receptor protein ODR-10a of a sensitive element and a quartz crystal microbalance are stably coupled with each other is solved, and the odor substance butanedione is quickly and specially detected.
Description
Technical field
The present invention relates to a kind of detection technique, relate in particular to a kind of method that detects the odoring substance diacetyl based on the olfactory receptor sensor.
Background technology
Airborne odoring substance can be experienced and tell to the Biologic Olfaction system fast, delicately, and this has benefited from olfactory receptor can be combined specifically by scent molecule in air.In the design of tradition gas sensor, sensitive material adopts conducting polymer, macromolecular material etc. more, and shortcoming is poor specificity.Scent molecule is of a great variety, is mostly the little molecular weight volatile organic gas of configurations, while especially in the air in testing environment, containing multiple smell, detects specifically certain smell and becomes relatively difficult.Emerging biology sensor is intended addressing this problem from bionical angle, and the mimic biology olfactory system utilizes the specific recognition of olfactory receptor to scent molecule, for specific detection gas provides new technological approaches.Therefore, how to obtain the gas sensitive material of high special, and, by the effectively stable coupling of itself and diode sensor, the specific detection that realizes scent molecule is that current gas sensor further develops a difficult problem of needing solution badly.
Summary of the invention
The object of the invention is to for the deficiencies in the prior art, provide a kind of olfactory receptor that utilizes to build in conjunction with QCM (Quartz Crystal Microbalance) the method that the olfactory receptor sensor carries out the gas specific detection as sensitive material.
The objective of the invention is to be achieved through the following technical solutions: a kind of method that detects the odoring substance diacetyl based on the olfactory receptor sensor, the method comprises the following steps:
(1) prepare olfactory receptor ODR-10;
(2) fixedly olfactory receptor ODR-10 at QCM (Quartz Crystal Microbalance) surface construction olfactory receptor sensor;
(3) by pure air, the flow velocity with 1mL/s pumps in test chamber, after the resonance frequency of quartz crystal microbalance sensor reaches a stable state, the gas to be measured containing the odoriferous substance diacetyl of again concentration being determined pumps into test chamber, after the resonance frequency of quartz crystal microbalance sensor is stablized again, the change amount of resonance frequency is the response of olfactory receptor sensor to the gaseous matter diacetyl under this concentration; Finally again pure air is pumped in test chamber, clean test chamber, complete the one-time detection process; At least, after the 10min of interval, can pump into another concentration gas to be measured and detect to test chamber next time after the resonance frequency of quartz crystal microbalance sensor is stablized; Repeat above-mentioned steps, obtain the sensor resonant frequency change amount that the gaseous matter diacetyl of a series of concentration causes, obtain diacetyl concn-resonance frequency and change discharge curve;
(4) for the detection of the gaseous matter diacetyl of unknown concentration, at first by pure air, the flow velocity with 1mL/s pumps in test chamber, treats that the resonance frequency of quartz crystal microbalance sensor reaches a stable state F
0after, then the gas to be measured of concentration the unknown is pumped into to test chamber, treat that the resonance frequency of quartz crystal microbalance sensor is stabilized to F again
1after, obtain resonance frequency change amount F
1-F
0, the diacetyl concn obtained according to above-mentioned steps-resonance frequency changes the concentration that discharge curve is extrapolated gaseous matter diacetyl to be measured, realizes the concentration determination to diacetyl.
Further, described step (1) comprises following sub-step:
(1.1) construction of expression vector: at first adopt the plasmid of technique for gene engineering to containing olfactory receptor ODR-10 gene and rho-tag signal peptide sequence full-length cDNA
pEGFP-N1/rho-tag/odr-10(its gene order is as shown in SEQ ID NO.1) carries out appropriate reconstruction, builds the expression vector of the olfactory receptor that is applicable to the use of olfactory receptor sensor, makes the His of its N end with amalgamation and expression
6-tag label; At first, design corresponding primer, obtain and increase olfactory receptor ODR-10 gene and rho-tag signal peptide sequence full-length cDNA, and add the His of amalgamation and expression at the N end
6-tag label, upstream primer sequence SEQ ID NO.2; The downstream primer sequence is as shown in SEQ ID NO.3; Carry out in 50 μ L reaction systems PCR (PCR), comprises 1 μ L
pEGFP-N1/rho-tag/odr-10plasmid, 1 μ L upstream primer, 1 μ L downstream primer, the dNTP potpourri that 4 μ L concentration are 10mM, 0.5 μ L exo+ polymerase STAR
tM(Takara, Japan), 10 μ L PCR damping fluids are (containing Mg
2+) and 32.5 μ L deionized waters, at first temperature cycles is 95 ℃ of denaturations 3 minutes, then carries out 30 seconds, 60 ℃ 30 seconds, 72 ℃ extensions 70 seconds of annealing of 94 ℃ of sex change of 30 circulations, is finally 10 ℃ of coolings 10 seconds.The product obtained after amplification
his 6 -tag/rho-tag/odr-10double digestion reaction by restriction enzyme is subcloned into expression vector
pcDNA3.1 (+)multiple clone site
kpn Iwith
bamH Ibetween, complete the expression vector of olfactory receptor
pcDNA3.1 (+)/his 6 -tag/rho-tag/odr-10structure, its gene order is identified by order-checking as SEQ ID NO.4;
(1.2) transfected with human embryonic kidney cell HEK-293:HEK-293 cell culture fluid is the DMEM high glucose medium that has added hyclone, penicillin (100 U/mL) and streptomysin (100 μ g/mL) that percent by volume is 10%, and condition of culture is that 37 ℃, percent by volume are 5% CO
2incubator.The previous day of transfection, the HEK-293 cell is inoculated in six hole double dish, and it is 0.5～2 * 10 that 500 μ L concentration are inoculated in every hole
5the cell of cells/mL, containing in antibiotic DMEM high glucose medium, growing to degrees of fusion, not being about 80-90%, treat transfection; Transfection reagent is lipofectamin 2000, the expression vector that uses step 1.1 to build
pcDNA3.1 (+)/his 6 -tag/rho-tag/odr-10transfection HEK-293 cell; With serum-free DMEM nutrient solution, 4 μ g expression vectors are diluted to 50 μ L, mix; Dilute again lipofectamine lipfectamin 2000 to the 50 μ L of 10 μ L with serum-free DMEM nutrient solution, at room temperature hatch 5 minutes; Then the expression vector after dilution and lipfectamin 2000 being mixed to cumulative volume is 100 μ L, shakes up incubated at room 20 minutes; Adding the mixed liquor of 100 μ L expression vectors and lipfectamin 2000 in each hole of six orifice plates, after the front and back rocker mixes, is 5% CO at 37 ℃, percent by volume
2incubator hatch 24-48 hour after, olfactory receptor expresses the surface of cell membrane at HEK-293, in order to as the sensitive material of olfactory receptor sensor;
(1.3) extract olfactory receptor ODR-10: be the sensitive material of the olfactory receptor ODR-10 by preparation in step 1.2 as the olfactory receptor sensor, use in the HEK-293 cell of Membrane protein extraction kit from transfection and extract olfactory receptor ODR-10; First collect the transfection HEK-293 cell of 48 hours, wash 3 times each 5 minutes with the deionized water of 4 ℃ is centrifugal; Then add 1mL protein extraction damping fluid, 1 μ L protease inhibitors, 1 μ L DDT, ultrasonication 4 times, then 4 ℃ of 14000g centrifugal 10 minutes, abandon precipitation, supernatant goes in a new centrifuge tube water-bath 10 minutes, under room temperature 13000 centrifugal 5 minutes, sample was divided into two-layer up and down, and epicyte protein is contained in lower floor; Take off a layer sample, add 4 ℃ of aqua sterilisas of 500 μ L, place 5 minutes for 4 ℃, then 37 ℃ of water-baths are 10 minutes, and under room temperature 13000 centrifugal 5 minutes, sample was divided into two-layer up and down, repeats this step 5 time; The lower floor's sample finally obtained is the memebrane protein potpourri, wherein comprises with merging His
6the olfactory receptor ODR-10 of-tag label;-70 ℃ of preservations are stand-by.
Described step (2) is specially: olfactory receptor sensor preparation process of the present invention is as follows: QCM (Quartz Crystal Microbalance) is placed in to ethanol and soaks 10 minutes, nitrogen drying; Then soak 20 minutes washed with de-ionized water in 1M NaOH aqueous solution; In 1M HCl solution, soak 5 minutes; The dense H of new configuration
2sO
4with H
2o
2(volume ratio 2:3) mixed liquid dipping 1 minute, nitrogen drying, to complete the thoroughly clean of QCM (Quartz Crystal Microbalance); Utilize golden mercapto key self-assembling reaction covalency His that fixedly one deck can the specific recognition amalgamation and expression on the gold electrode of QCM (Quartz Crystal Microbalance)
6the sulfhydrylation single stranded DNA aptamers of-tag label, this aptamers by 5 ' end covalent modification HS-(CH
2)
6gene order shown in the SEQ ID NO.5 of group forms, and under room temperature, reaction is 21 hours; Then seal the unreacted site of gold electrode surfaces of molecule (11-MUA) sealing quartz crystal microbalance sensor, sealing condition is lower 1 hour of room temperature; The epicyte protein mixed liquor that contains olfactory receptor of step 1.5 preparation evenly is incubated in to the gold electrode surfaces of quartz crystal microbalance sensor, and is placed on incubated at room and spends the night, make olfactory receptor by the His of amalgamation and expression
6specific association reaction occurs in-tag label and aptamers, realizes that olfactory receptor fixes and purifying in the gold electrode surfaces specificity of quartz crystal microbalance sensor; The phospholipid bilayer of cell membrane is conducive to keep the native conformation of olfactory receptor ODR-10; N
2the QCM (Quartz Crystal Microbalance) that after drying up, surface is fixed with to olfactory receptor is arranged in detection system, completes the structure of olfactory receptor sensor, with the detection for odoring substance.
Beneficial effect of the present invention, the present invention adopts modern molecule clone technology, prepare can specific binding the olfactory receptor ODR-10 of scent molecule diacetyl, as the sensitive material of gas sensor, overcome traditional gas detect in special poor, the shortcoming that sensitivity is low of sensitive material.Adopt aptamers self assembly technique for fixing to realize that olfactory receptor fixes at the efficient coupling on diode sensor surface, not only can improve the stability of sensor, and the special effective ability in conjunction with olfactory receptor of aptamers realizes the single step purification of olfactory receptor on sensor chip, other irrelevant memebrane protein is because the affinity lacked with aptamers can not be fixed on sensor surface, and then improved fixed efficiency and the distribution density of olfactory receptor at sensor surface, having overcome traditional sensitive material can not be effectively and the problem of diode sensor coupling, finally contribute to improve the sensitivity of sensor, specificity and stability.This detection method adopts and has effectively fixed the QCM (Quartz Crystal Microbalance) of olfactory receptor as the olfactory receptor sensor, can realize real-time, quick, specific detection to the scent molecule diacetyl, overcome that the traditional detection techniqueflow is complicated, specificity is low, susceptibility is poor, can't realize the shortcoming of Site Detection, be applicable to the real-time specificity monitoring of gas in complicated gaseous environment.The specificity that this olfactory receptor can meet scent molecule in complicated gaseous environment as these advantages and the function of sensitive material specific detection gas detects real-time, can be widely used in the association areas such as biomedicine, environmental protection and food service industry.
The accompanying drawing explanation
Fig. 1 is the gene structure figure that the present invention prepares olfactory receptor ODR-10;
Fig. 2 is the fixedly schematic diagram of olfactory receptor ODR-10 on QCM (Quartz Crystal Microbalance) of the present invention;
Fig. 3 is olfactory receptor sensor detecting system structural drawing of the present invention;
Fig. 4 is the RT-PCR figure as a result that the present invention prepares olfactory receptor ODR-10;
Fig. 5 is the Western blot figure as a result that the present invention prepares olfactory receptor ODR-10;
Fig. 6 is the figure as a result that olfactory receptor sensor of the present invention detects several odoring substances;
Fig. 7 is the figure as a result that olfactory receptor sensor of the present invention detects the variable concentrations diacetyl;
In figure, the His of amalgamation and expression
6the His of-tag gene 1, rho-tag signal peptide gene 2, olfactory receptor ODR-10 gene 3, lipid bilayer 4, olfactory receptor 5, amalgamation and expression
6-tag label 6, sealing molecule 7, aptamers 8, gold electrode 9, quartz crystal 10, the first air valve 11, syringe pump 12, waste gas airbag 13, gas piping 14, the lid 15 of test chamber, test chamber 16, rubber seal 17, QCM (Quartz Crystal Microbalance) 18, signal output electrode 19, QCM200 detection system 20, pure air airbag 21, gas airbag to be measured 22, the second air valve 23.
Embodiment
Below introduce in detail and using olfactory receptor ODR-10 and prepare in conjunction with quartz crystal microbalance sensor ultimate principle and the concrete steps that the olfactory receptor sensor detects gas as sensitive element.
The present invention is based on the method for olfactory receptor sensor detection odoring substance diacetyl, comprise the following steps:
1, prepare olfactory receptor ODR-10
1.1 the structure of expression vector
At first adopt the plasmid of technique for gene engineering to containing olfactory receptor ODR-10 gene 3 and rho-tag signal peptide 2 sequence full-length cDNAs
pEGFP-N1/rho-tag/odr-10(its gene order is as shown in SEQ ID NO.1) carries out appropriate reconstruction, builds the expression vector of the olfactory receptor 5 that is applicable to the use of olfactory receptor sensor, makes the His of its N end with amalgamation and expression
6-tag label 6; At first, design corresponding primer, obtain and increase olfactory receptor ODR-10 gene 3 and rho-tag signal peptide 2 sequence full-length cDNAs, and add the His of amalgamation and expression at the N end
6-tag label, upstream primer sequence SEQ ID NO.2:5 '-AGA
gGTACCgATG
cACCACCACCACCACCACaTGAACGGGACCGAGGGCCCAA-3 ', wherein underscore is partly
kpnthe I restriction enzyme site, the His that dotted portion is amalgamation and expression
6-tag gene 1 sequence, so that the His that makes the N end of the olfactory receptor 5 of follow-up expression contain amalgamation and expression
6-tag label 6, contribute to olfactory receptor 5 effectively fixing at sensor surface; The downstream primer sequence is as shown in SEQ ID NO.3: 5 '-cgc
ggatcctcacgtcggaacttgagacaaat-3 ', wherein underscore is partly
bamHthe I restriction enzyme site; Carry out in 50 μ L reaction systems PCR (PCR), comprises 1 μ L
pEGFP-N1/rho-tag/odr-10plasmid, 1 μ L upstream primer, 1 μ L downstream primer, the dNTP potpourri that 4 μ L concentration are 10mM, 0.5 μ L exo+ polymerase STAR
tM, the PCR damping fluid of 10 μ L is (containing Mg
2+) and 32.5 μ L deionized waters, at first temperature cycles is 95 ℃ of denaturations 3 minutes, then carries out 30 seconds, 60 ℃ 30 seconds, 72 ℃ extensions 70 seconds of annealing of 94 ℃ of sex change of 30 circulations, is finally 10 ℃ of coolings 10 seconds.The product obtained after amplification
his 6 -tag/rho-tag/odr-10double digestion reaction by restriction enzyme is subcloned into expression vector
pcDNA3.1 (+)multiple clone site
kpn Iwith
bamH Ibetween, complete the expression vector of olfactory receptor 5
pcDNA3.1 (+)/his 6 -tag/rho-tag/odr-10structure, its gene order is identified by order-checking as SEQ ID NO.4.
1.2 transfected with human embryonic kidney cell HEK-293
The HEK-293 cell culture fluid is the DMEM high glucose medium that has added hyclone, penicillin (100 U/mL) and streptomysin (100 μ g/mL) that percent by volume is 10%, and condition of culture is that 37 ℃, percent by volume are 5% CO
2incubator.The previous day of transfection, the HEK-293 cell is inoculated in six hole double dish, and it is 0.5～2 * 10 that 500 μ L concentration are inoculated in every hole
5the cell of cells/mL, containing in antibiotic DMEM high glucose medium, growing to degrees of fusion, not being about 80-90%, treat transfection; Transfection reagent is lipofectamin 2000, the expression vector that uses step 1.1 to build
pcDNA3.1 (+)/his 6 -tag/rho-tag/odr-10transfection HEK-293 cell; With serum-free DMEM nutrient solution, 4 μ g expression vectors are diluted to 50 μ L, mix; Dilute again lipofectamine lipfectamin 2000 to the 50 μ L of 10 μ L with serum-free DMEM nutrient solution, at room temperature hatch 5 minutes; Then the expression vector after dilution and lipfectamin 2000 being mixed to cumulative volume is 100 μ L, shakes up incubated at room 20 minutes; Adding the mixed liquor of 100 μ L expression vectors and lipfectamin 2000 in each hole of six orifice plates, after the front and back rocker mixes, is 5% CO at 37 ℃, percent by volume
2incubator hatch 24-48 hour after, olfactory receptor 5 expresses the surface of cell membrane at HEK-293, with the standby sensitive material of making the olfactory receptor sensor.
Reverse transcription-PCR 1.3 (RT-PCR)
Adopt the method for RT-PCR to detect the expression of olfactory receptor 5 mRNA level in cell to verify its successful expression preparation; The HEK-293 cell after 48 hours, is pressed total RNA that the Trizol single stage method is extracted cell in transfection; First take out the superclean bench of six orifice plate cells to the bacterium of having gone out, inhale and abandon nutrient culture media, wash lightly one time with phosphate buffer (PBS, pH7.4); Then add 0.5 mL Trizol reagent to every hole, with pipettor piping and druming cell make it to come off, cracking; Again cell liquid after above-mentioned Trizol cracking is proceeded in 1.5 mL EP pipes, the lower placement of room temperature (15 ℃～30 ℃) 5 minutes; Add chloroform to the ratio that adds 0.2 mL chloroform according to every 1 mL Trizol in above-mentioned EP pipe, cover lid firmly shakes 15 seconds in hand, after under room temperature, (15 ℃～30 ℃) place 2～3 minutes, and 12000 g(2 ℃～8 ℃) centrifugal 18 minutes; Take out the EP pipe from hydro-extractor after, sample is divided into three layers, the water on yellow organic phase, middle layer and the upper strata of bottom; The upper strata water is taken out in new 1.5 mL EP pipes, the ratio that adds 0.5 mL isopropyl alcohol according to every 1 mL Trizol adds isopropyl alcohol, at room temperature (15 ℃～30 ℃) are placed 10 minutes, centrifugal 10 minutes of 12000 g (2 ℃～8 ℃), then abandoning supernatant; The ratio that adds 1 mL 75% ethanol according to every 1 mL Trizol is again carried out the vortex mixed washing, 7500 g(2 ℃～8 ℃ afterwards) centrifugal 5 minutes, abandoning supernatant; Sediment is total RNA of cell, makes it natural drying under room temperature; With not containing the deionized water dissolving RNA precipitation of RNA enzyme, be placed in-70 ℃ of Refrigerator stores stand-by; Then get 5 μ L RNA solution, by detecting ultraviolet absorptivity under 260 nm and 280 nm wavelength, estimate its purity, and calculate nucleic acid content (RNA absorbance under 260 nm wavelength is, concentration is equivalent to 40 μ g/mL) at 1 o'clock.
By RNA solution dilution to concentration, be 0.2 μ g/ μ L, then therefrom get 5 μ L to PCR tubules, the dNTP potpourri, the 3 μ L that add the poly-primer of 1 μ L random six, 1 μ L concentration to be 10 mM do not mix containing the deionized water of RNA enzyme again, the of short duration centrifugal several seconds, on the PCR instrument, sex change is carried out in 70 ℃ of reactions in 5 minutes; Cooling annealing under ice bath; Add successively again the RNA enzyme inhibitor that 5 times of concentration PrimerScript damping fluids of 4 μ L, 0.5 μ L concentration are 40 U/ μ L, the PrimeScript that 1 μ L concentration is 200 U/ μ L
rTase and 4.5 μ L are not containing the deionized water of RNA enzyme, and mixing is the reverse transcription reaction liquid of 20 μ L; Carry out reverse transcription reaction on the PCR instrument, reaction conditions is: 45 minutes, 95 ℃ reactions of 10 minutes, 42 ° C reactions of 30 ℃ of reactions 5 minutes; After stopping reaction, take out the reverse transcription reaction product and be placed in immediately on ice; Product is placed on-20 ℃ of preservations.
The reverse transcription product of above-mentioned acquisition of usining afterwards carries out pcr amplification as template, and the upstream primer gene order is as SEQ ID NO.2:5 '-AGAGGTACCGATGCACCACCACCACCACCACATGAACGGGACCGAGGGCCCAA-3 '; The downstream primer gene order is as shown in SEQ ID NO.3: 5 '-cgc
ggatcctcacgtcggaacttgagacaaat-3 '; Under ice bath, dNTP potpourri, 0.5 μ L exo+ polymerase STAR that synthetic cDNA template, 1 μ L upstream primer, 1 μ L downstream primer, the 4 μ L concentration of 1 μ L reverse transcription are 10mM
tM, 10 μ L PCR damping fluids are (containing Mg
2+) and 32.5 μ L deionized waters be mixed into the reaction system that total amount is 50 μ L, 95 ℃ of denaturations 3 minutes, then carry out 30 seconds, 72 ℃ of 30 seconds, 60 ℃ annealing of 94 ℃ of sex change of 30 circulations and extend 70 seconds, is finally 10 ℃ of coolings 10 seconds; Observe by the ultraviolet imagery system expression that electrophoretic band is determined olfactory receptor ODR-10 gene 3 mRNA level in cell after PCR product electrophoresis.
1.4 Western blot identifies
Adopt Western blot technology to identify that from protein level olfactory receptor 5 is at the intracellular expression of HEK-293; At first add PMSF at RIPA fine melt liquid, making the PMSF final concentration is that 1mM is stand-by; After the taking-up transfection, the HEK-293 cell of 48 hours, to superclean bench, is inhaled and is abandoned nutrient culture media, and phosphate buffer is washed once, discards waste liquid, is placed on ice; Add 200 μ L lysates in each hole of six orifice plates, repeatedly blow and beat cell, make lysate fully contact 30 minutes with cell; After the abundant cracking of cell, centrifugal 5 minutes of 14000g, get supernatant and obtain the olfactory receptor sample to centrifuge tube; Get 50 μ L samples to the EP pipe of 1.5mL, add 5 times of loading damping fluids of 12.5 μ L to mix, 100 ℃ are boiled 10 minutes, centrifugal 1 minute of 12000g room temperature, and supernatant adds the protein SDS-PAGE gel, carries out protein electrophoresis under 120V voltage; Then go to pvdf membrane, in the bovine serum albumin solution that to be dipped in mass percent be 2.5%, the room temperature shaking table is hatched 1 hour, completes sealing; Then wash 3 times with TBST solution, each 10 minutes, add primary antibodie (anti-His
6-tag mouse IgG), 4 ℃ of overnight incubation; Take out pvdf membrane and wash 3 times with TBST solution, each 10 minutes, add two anti-(DyLight680 mark goat anti-mouse IgG) room temperature lucifuge shaking tables of 1:500 to hatch 1 hour; Be placed in scanning analysis under Odyssey Dual band IR laser imaging system; Scheduled target albumen size is 45kD; Whether successful expression in the HEK-293 cell of olfactory receptor 5 is identified in position by Western blot protein electrophoresis band.
1.5 extract olfactory receptor ODR-10
For the sensitive material of the olfactory receptor 5 by preparation in step 1.2 as the olfactory receptor sensor, use in the HEK-293 cell of Membrane protein extraction kit from transfection and extract olfactory receptor ODR-10; First collect the transfection HEK-293 cell of 48 hours, wash 3 times each 5 minutes with the deionized water of 4 ℃ is centrifugal; Then add 1mL protein extraction damping fluid, 1 μ L protease inhibitors, 1 μ L DDT, ultrasonication 4 times, then 4 ℃ of 14000g centrifugal 10 minutes, abandon precipitation, supernatant goes in a new centrifuge tube water-bath 10 minutes, under room temperature 13000 centrifugal 5 minutes, sample was divided into two-layer up and down, and epicyte protein is contained in lower floor; Take off a layer sample, add 4 ℃ of aqua sterilisas of 500 μ L, place 5 minutes for 4 ℃, then 37 ℃ of water-baths are 10 minutes, and under room temperature 13000 centrifugal 5 minutes, sample was divided into two-layer up and down, repeats this step 5 time; The lower floor's sample finally obtained is the memebrane protein potpourri, wherein comprises with merging His
6the olfactory receptor ODR-10 of-tag label;-70 ℃ of preservations are stand-by.
In this step 1, sub-step 1.3 and 1.4 for verify olfactory receptor successful expression at surface of cell membrane, the necessary sub-step of this step 1 not.
2, fixedly olfactory receptor ODR-10 at QCM (Quartz Crystal Microbalance) surface construction olfactory receptor sensor
In the present invention, the olfactory receptor sensor be take olfactory receptor ODR-10 as sensitive material, is fixed on the QCM (Quartz Crystal Microbalance) surface, and concrete building process is as follows; QCM (Quartz Crystal Microbalance) 18 is placed in to ethanol and soaks 10 minutes, nitrogen drying; Then soak 20 minutes washed with de-ionized water in 1M NaOH aqueous solution; In 1M HCl solution, soak 5 minutes; The dense H of new configuration
2sO
4with H
2o
2(volume ratio 2:3) mixed liquid dipping 1 minute, nitrogen drying, to complete the thoroughly clean of QCM (Quartz Crystal Microbalance) 18; Utilize golden mercapto key self-assembling reaction covalency His that fixedly one deck can the specific recognition amalgamation and expression on the gold electrode 9 of QCM (Quartz Crystal Microbalance) 18
6the sulfhydrylation single stranded DNA aptamers 8 of-tag label 6, this aptamers 8 by 5 ' end covalent modification HS-(CH
2)
6gene order shown in the SEQ ID NO.5 of group forms (5 '-HS-(CH
2)
6-GCTATGGGTGGTCTGGTTGGGATTGGCCCCGGGAGCTGGC-3 '), under room temperature, react 21 hours; Then seal molecule 7(11-MUA) sealing quartz crystal microbalance sensor the surperficial unreacted site of gold electrode 17, sealing condition is lower 1 hour of room temperature; The epicyte protein mixed liquor that contains olfactory receptor 5 of step 1.5 preparation evenly is incubated in to gold electrode 9 surfaces of quartz crystal microbalance sensor, and is placed on incubated at room and spends the night, make olfactory receptor 5 by the His of amalgamation and expression
6specific association reaction occurs with aptamers 8 in-tag label 6, realizes that olfactory receptor 5 fixes and purifying at gold electrode 9 surface specifics of quartz crystal microbalance sensor; The phospholipid bilayer 4 of cell membrane is conducive to keep the native conformation of olfactory receptor ODR-10; N
2the QCM (Quartz Crystal Microbalance) 18 that after drying up, surface is fixed with to olfactory receptor 5 is arranged in detection system, completes the structure of olfactory receptor sensor, with the detection for odoring substance.
3, the detection of odoring substance diacetyl
3.1 quartz crystal microbalance sensor principle
Use the diode sensor of QCM (Quartz Crystal Microbalance) as the olfactory receptor sensor in the present invention, QCM (Quartz Crystal Microbalance) is a kind of mass-sensitive type sensor, ultimate principle is that the resonance frequency of the quartz crystal pressure change small to the surface on its surface is relevant, by the small pressure of change monitoring strand DNA on Surface of Quartz crystal that records quartz-crystal resonance frequency, changes.Based on this principle, also can detect the mass change that strand DNA on Surface of Quartz crystal is small by the variation of monitoring quartz-crystal resonance frequency.Utilize this principle that QCM (Quartz Crystal Microbalance) is combined with olfactory receptor and build the olfactory receptor sensor, olfactory receptor is fixed on QCM (Quartz Crystal Microbalance) sensitive electrode surface as sensitive element, after the effect of test substance generation specific adsorption, QCM (Quartz Crystal Microbalance) can arrive this process that detects.When the olfactory receptor that is fixed in strand DNA on Surface of Quartz crystal when odoring substance adsorbs, the quality on QCM (Quartz Crystal Microbalance) surface also changes, cause the change of quartz-crystal resonance frequency, and the change of frequency and strand DNA on Surface of Quartz crystal quality change over direct ratio, meet formula:
Δ F=K Δ m, wherein
Δ Ffor the variation (Hz) of quartz-crystal resonance frequency,
Δ mfor the mass change (g) of strand DNA on Surface of Quartz crystal,
kfor related coefficient,
k=-F/ (Art),
ffor the original frequency (MHz) of quartz crystal,
afor the total surface area (cm of electrode
2),
rdensity (g/cm for crystal
3),
tthickness (cm) for crystal.Therefore, variation by the monitoring quartz-crystal resonance frequency can calculate its surperficial mass change, obtain odoring substance and surperficial olfactory receptor interaction information, indirect calculation goes out the concentration of odoring substance, the final specific detection realized odoring substance.
3.2 the olfactory receptor sensor detects the process of gas
The signal of quartz crystal microbalance sensor 18 resonance frequencies is sent to QCM200 detection system 20(Stanford Research Systems by sensor electrode interface 19, Inc., the change amount of quartz crystal microbalance sensor 18 resonance frequencies that USA), cause with recording odor material and olfactory receptor 5 interactions, at first open the first air valve 11 and close the second air valve 23, by syringe pump 12, by gas piping 14, by the air in pure air airbag 21, the flow velocity with 1mL/s pumps in test chamber, after the resonance frequency of quartz crystal microbalance sensor 18 reaches a stable state, open again the second air valve 23 and close the first air valve 11 gas to be measured containing the odoriferous substance diacetyl that concentration is definite and pump into test chamber from gas airbag 22 to be measured, after the resonance frequency of quartz crystal microbalance sensor 18 is stablized again, the change amount of resonance frequency is the response of sensor to the gaseous matter diacetyl under this concentration, finally open the first air valve 11 again and close the second air valve 23, pure air is pumped in test chamber, the purge gas test chamber, complete the one-time detection process, at least, after the 10min of interval, can pump into another gas to be measured and detect to test chamber next time after the resonance frequency of quartz crystal microbalance sensor 18 is stablized, repeat above-mentioned steps, obtain the sensor resonant frequency change amount that the gaseous matter diacetyl of a series of concentration causes, obtain diacetyl concn-resonance frequency and change discharge curve.
Detection for the gaseous matter diacetyl of unknown concentration, at first open the first air valve 11 and close the second air valve 23, by syringe pump 12, by gas piping 14, by the air in pure air airbag 21, the flow velocity with 1mL/s pumps in test chamber, treats that the resonance frequency of quartz crystal microbalance sensor 18 reaches a stable state F
0after, then open the second air valve 23 and close the first air valve 11 gas to be measured of concentration the unknown is pumped into to test chamber from gas airbag 22 to be measured, treat that the resonance frequency of quartz crystal microbalance sensor 18 is stabilized to F again
1after, obtain resonance frequency change amount F
1-F
0, the diacetyl concn obtained according to above-mentioned steps-resonance frequency changes the concentration that discharge curve is extrapolated gaseous matter diacetyl to be measured, realizes the concentration determination to diacetyl.
Further illustrate the present invention below in conjunction with drawings and Examples, it is more obvious that purpose of the present invention and effect will become.
As shown in Figure 1, the gene structure figure of olfactory receptor ODR-10 prepared by the present invention, in figure, the His of amalgamation and expression
6-tag gene 1, rho-tag signal peptide gene 2, olfactory receptor ODR-10 gene 3, the olfactory receptor ODR-10 that this gene structure design is expressed is the His with amalgamation and expression at the N end
6-tag label and rho-tag signal peptide, the His of amalgamation and expression
6-tag label contributes to olfactory receptor ODR-10 is fixed on effectively the gold electrode of QCM (Quartz Crystal Microbalance), and the rho-tag signal peptide is conducive to the correct location of olfactory receptor ODR-10 at cell membrane, Figure 2 shows that in the present invention, olfactory receptor ODR-10 is fixed on the lip-deep schematic diagram of QCM (Quartz Crystal Microbalance), N end amalgamation and expression has His
6the olfactory receptor 5 of-tag label 6, pass through His
6specificity between-tag label 6 and aptamers 8 mutually combines and is fixed on the gold electrode 9 on quartz crystal 10 surfaces, the lipid bilayer 4 of cell membrane component is conducive to keep the native conformation of olfactory receptor 5, and sealing molecule 7 is for sealing the exposed site of gold electrode surfaces, Figure 3 shows that olfactory receptor sensor detecting system structural drawing, wherein the surface of QCM (Quartz Crystal Microbalance) 18 is shown in Fig. 2, surface has successfully fixed olfactory receptor ODR-10, by controlling the first air valve 11 and the second air valve 23, pure air airbag 21 and gas airbag 22 to be measured are pumped in test chamber 16 by syringe pump 12, the lid 15 of test chamber is spun on test chamber 16 tops by screw thread, waste gas enters waste gas airbag 13 by gas piping 14, rubber seal 17 is for being fixed on test chamber 16 by QCM (Quartz Crystal Microbalance) 18, signal output electrode 19 is connected to the resonant frequency signal of QCM (Quartz Crystal Microbalance) 18 real time record of carrying out signal in QCM200 detection system 20, Fig. 4 is the RT-PCR figure as a result that the present invention prepares olfactory receptor ODR-10, and target stripe is greatly about 1.1kb, and prompting olfactory receptor 5 is significantly expressed on the mRNA level in cell, the Western blot that Fig. 5 the present invention prepares olfactory receptor ODR-10 is figure as a result, and target stripe is greatly about 45KD, shows at protein level olfactory receptor 5 successful expression in the HEK-293 cell, Fig. 6 is the figure as a result that olfactory receptor sensor of the present invention detects several odoring substances, this presentation of results adopts olfactory receptor ODR-10 the most responsive to the odoring substance diacetyl as the olfactory receptor sensor of sensitive material, the response less that other several gases cause, so this olfactory receptor sensor has good specificity, Fig. 7 is the figure as a result that the olfactory receptor sensor for preparing of the present invention detects the variable concentrations diacetyl, the lowest detection lower limit is approximately 1.5ppm, detection sensitivity is approximately 0.55 ± 0.024 Hz/ppm, and this olfactory receptor sensor can detect the odoring substance diacetyl sensitive, specifically in sum.
<110 > Zhejiang University
<120 > a kind of method that detects the odoring substance diacetyl based on the olfactory receptor sensor
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 5778
<212> DNA
<213> Caenorhabditis elegans
<400> 1
tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120
gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180
atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240
aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300
catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360
catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420
atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480
ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540
acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600
ccggactcag atctcgagat gaacgggacc gagggcccaa acttctacgt gcctttctcc 660
aacaagacgg gcgtggtgga attcatgtcg ggagaattgt ggattaccct agttgacaca 720
gcggacattg tcggcgtcac cctcaccttc tgtgtcaaca ttgttcttct cggacttctg 780
aaaacacgtg gaaaaaactt gggcacttat aaatatctca tggcgttttt ctcagtattc 840
tcgatttttt acgccatcat cgagttcata ttacgaccta taatgcatat tgagaacacc 900
actttctttt tgatctcaag gaaaagattc aactactcca ccaaacttgg aaaaatcaac 960
tctgcgtttt actgtgcttg ttttgccacc agttttgttg tctcaggagt tcactttgtt 1020
tatcgatatt ttgcaacttg caaaccgaat ctacttcgtt tgttcaactt gccaactctt 1080
ctactttggc cacttggttg cagtgtaccc gtgacaatgt gggctagtgt ctcatatttt 1140
ttgtatccag ataccgagta cacggaagcg gctgtcacca atgtactaaa taaccactat 1200
aactggatca aaaaggagaa tgtatcgtac attgcatacg tctattacca atacgaaaac 1260
ggagtaaggc atatctacct caaaaacttg cttggatgct ttgttcatta ctttgtcatg 1320
tcgatgacgt ttgttgtgat gttctactgc ggatatgcca cgtggaaaac tatgaatgaa 1380
cacaaggatg tatctgatag aactcgagcg ctacagaaac aacttttcaa agctttagtt 1440
cttcagacac tcatcccaac tatcttcatg tacgccccaa ctggagtcat gttcatcgca 1500
ccgttttttg acgtgaattt gaatgcaaac gccaatttca ttgtgttttg ctcatttctg 1560
tacccgggac tcgatccact cattctgatt ttgatcattc gtgatttccg aagaacaata 1620
ttcaatttct tgtgtggaaa gaaaaacagt gttgatgaat cccgctcgac aacaagagcc 1680
aatttgtctc aagttccgac gtgaggatcc accggtcgcc accatggtga gcaagggcga 1740
ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca 1800
caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa 1860
gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga ccaccctgac 1920
ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg acttcttcaa 1980
gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa 2040
ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct 2100
gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta 2160
caacagccac aacgtctata tcatggccga caagcagaag aacggcatca aggtgaactt 2220
caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact accagcagaa 2280
cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc 2340
cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac 2400
cgccgccggg atcactctcg gcatggacga gctgtacaag taaagcggcc gcgactctag 2460
atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac 2520
ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca 2580
gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt 2640
tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaagg cgtaaattgt 2700
aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 2760
ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg agatagggtt 2820
gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact ccaacgtcaa 2880
agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac cctaatcaag 2940
ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga gcccccgatt 3000
tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg 3060
agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc 3120
cgcgcttaat gcgccgctac agggcgcgtc aggtggcact tttcggggaa atgtgcgcgg 3180
aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata 3240
accctgataa atgcttcaat aatattgaaa aaggaagagt cctgaggcgg aaagaaccag 3300
ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagc aggcagaagt 3360
atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 3420
gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 3480
actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 3540
ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag 3600
tagtgaggag gcttttttgg aggcctaggc ttttgcaaag atcgatcaag agacaggatg 3660
aggatcgttt cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt 3720
ggagaggcta ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt 3780
gttccggctg tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc 3840
cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc 3900
ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga 3960
agtgccgggg caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat 4020
ggctgatgca atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca 4080
agcgaaacat cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga 4140
tgatctggac gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc 4200
gagcatgccc gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat 4260
catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga 4320
ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg 4380
ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt 4440
ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa 4500
gcgacgccca acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg 4560
ggcttcggaa tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg 4620
ctggagttct tcgcccaccc tagggggagg ctaactgaaa cacggaagga gacaataccg 4680
gaaggaaccc gcgctatgac ggcaataaaa agacagaata aaacgcacgg tgttgggtcg 4740
tttgttcata aacgcggggt tcggtcccag ggctggcact ctgtcgatac cccaccgaga 4800
ccccattggg gccaatacgc ccgcgtttct tccttttccc caccccaccc cccaagttcg 4860
ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag gccctgccat agcctcaggt 4920
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 4980
aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 5040
gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 5100
atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 5160
gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 5220
gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 5280
tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 5340
accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 5400
ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 5460
cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 5520
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 5580
ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 5640
tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 5700
ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 5760
cgtattaccg ccatgcat 5778
<210> 2
<211> 53
<212> DNA
<213 > artificial design
<400> 2
agaggtaccg atgcaccacc accaccacca catgaacggg accgagggcc caa 53
<210> 3
<211> 32
<212> DNA
<213 > artificial design
<400> 3
cgcggatcct cacgtcggaa cttgagacaa at 32
<210> 4
<211> 6322
<212> DNA
<213> Caenorhabditis elegans
<400> 4
gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60
ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120
cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180
ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240
gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300
tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360
cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420
attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480
atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540
atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600
tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660
actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720
aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780
gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840
ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900
gtttaaactt aagcttggta ccgatgcacc accaccacca ccacatgaac gggaccgagg 960
gcccaaactt ctacgtgcct ttctccaaca agacgggcgt ggtggaattc atgtcgggag 1020
aattgtggat taccctagtt gacacagcgg acattgtcgg cgtcaccctc accttctgtg 1080
tcaacattgt tcttctcgga cttctgaaaa cacgtggaaa aaacttgggc acttataaat 1140
atctcatggc gtttttctca gtattctcga ttttttacgc catcatcgag ttcatattac 1200
gacctataat gcatattgag aacaccactt tctttttgat ctcaaggaaa agattcaact 1260
actccaccaa acttggaaaa atcaactctg cgttttactg tgcttgtttt gccaccagtt 1320
ttgttgtctc aggagttcac tttgtttatc gatattttgc aacttgcaaa ccgaatctac 1380
ttcgtttgtt caacttgcca actcttctac tttggccact tggttgcagt gtacccgtga 1440
caatgtgggc tagtgtctca tattttttgt atccagatac cgagtacacg gaagcggctg 1500
tcaccaatgt actaaataac cactataact ggatcaaaaa ggagaatgta tcgtacattg 1560
catacgtcta ttaccaatac gaaaacggag taaggcatat ctacctcaaa aacttgcttg 1620
gatgctttgt tcattacttt gtcatgtcga tgacgtttgt tgtgatgttc tactgcggat 1680
atgccacgtg gaaaactatg aatgaacaca aggatgtatc tgatagaact cgagcgctac 1740
agaaacaact tttcaaagct ttagttcttc agacactcat cccaactatc ttcatgtacg 1800
ccccaactgg agtcatgttc atcgcaccgt tttttgacgt gaatttgaat gcaaacgcca 1860
atttcattgt gttttgctca tttctgtacc cgggactcga tccactcatt ctgattttga 1920
tcattcgtga tttccgaaga acaatattca atttcttgtg tggaaagaaa aacagtgttg 1980
atgaatcccg ctcgacaaca agagccaatt tgtctcaagt tccgacgtga ggatccacta 2040
gtccagtgtg gtggaattct gcagatatcc agcacagtgg cggccgctcg agtctagagg 2100
gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt 2160
ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta 2220
ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg 2280
ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc 2340
ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca 2400
cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc 2460
tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac 2520
gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag 2580
tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc 2640
atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg 2700
actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata 2760
agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa 2820
cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca 2880
gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc 2940
ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata 3000
gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg 3060
ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag 3120
ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg 3180
ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg 3240
attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc 3300
tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg 3360
caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag 3420
gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc 3480
gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat 3540
ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg 3600
cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc 3660
gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag 3720
catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc 3780
gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc 3840
cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata 3900
gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc 3960
gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac 4020
gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc 4080
catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt 4140
tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc 4200
accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt 4260
tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg 4320
tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat 4380
agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa 4440
gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc 4500
gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc 4560
aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact 4620
cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac 4680
ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa 4740
aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg 4800
acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa 4860
gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc 4920
ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac 4980
gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac 5040
cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg 5100
taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt 5160
atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa 5220
cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct 5280
cttgatccgg caaacaaacc accgctggta gcggtttttt tgtttgcaag cagcagatta 5340
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 5400
agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 5460
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 5520
cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 5580
ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 5640
taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt 5700
tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 5760
ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 5820
atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 5880
gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 5940
tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 6000
cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 6060
taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 6120
ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 6180
ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 6240
cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 6300
ttactttcac cagcgtttct gg 6322
<210> 5
<211> 40
<212> DNA
<213 > artificial design
<400> 5
gctatgggtg gtctggttgg gattggcccc gggagctggc 40
Claims (3)
1. a method that detects the odoring substance diacetyl based on the olfactory receptor sensor, is characterized in that, the method comprises the following steps:
(1) prepare olfactory receptor ODR-10;
(2) fixedly olfactory receptor ODR-10 at QCM (Quartz Crystal Microbalance) surface construction olfactory receptor sensor;
(3) by pure air, the flow velocity with 1mL/s pumps in test chamber, after the resonance frequency of quartz crystal microbalance sensor reaches a stable state, the gas to be measured containing the odoriferous substance diacetyl of again concentration being determined pumps into test chamber, after the resonance frequency of quartz crystal microbalance sensor is stablized again, the change amount of resonance frequency is the response of olfactory receptor sensor to the gaseous matter diacetyl under this concentration; Finally pure air is pumped in test chamber, the purge gas test chamber, complete the one-time detection process again; At least, after the 10min of interval, can pump into another concentration gas to be measured and detect to test chamber next time after the resonance frequency of quartz crystal microbalance sensor is stablized; Repeat above-mentioned steps, obtain the sensor resonant frequency change amount that the gaseous matter diacetyl of a series of concentration causes, obtain diacetyl concn-resonance frequency and change discharge curve;
(4) for the detection of the gaseous matter diacetyl of unknown concentration, at first by pure air, the flow velocity with 1mL/s pumps in test chamber, treats that the resonance frequency of quartz crystal microbalance sensor reaches a stable state F
0after, then the gas to be measured of concentration the unknown is pumped into to test chamber, treat that the resonance frequency of quartz crystal microbalance sensor is stabilized to F again
1after, obtain resonance frequency change amount F
1-F
0, the diacetyl concn obtained according to above-mentioned steps 3-resonance frequency changes the concentration that discharge curve obtains gaseous matter diacetyl to be measured, realizes the concentration determination to diacetyl.
2. a kind of method that detects the odoring substance diacetyl based on the olfactory receptor sensor according to claim 1, is characterized in that, described step 1 comprises following sub-step:
(1.1) construction of expression vector: at first adopt the plasmid of technique for gene engineering to containing olfactory receptor ODR-10 gene and rho-tag signal peptide sequence full-length cDNA
pEGFP-N1/rho-tag/odr-10(its gene order is as shown in SEQ ID NO.1) carries out appropriate reconstruction, builds the expression vector of the olfactory receptor that is applicable to the use of olfactory receptor sensor, makes the His of its N end with amalgamation and expression
6-tag label; At first, design corresponding primer, obtain and increase olfactory receptor ODR-10 gene and rho-tag signal peptide sequence full-length cDNA, and add the His of amalgamation and expression at the N end
6-tag label, upstream primer sequence SEQ ID NO.2; The downstream primer sequence is as shown in SEQ ID NO.3; Carry out in 50 μ L reaction systems PCR, and at first temperature cycles is 95 ℃ of denaturations 3 minutes, then carries out 30 seconds, 60 ℃ 30 seconds, 72 ℃ extensions 70 seconds of annealing of 94 ℃ of sex change of 30 circulations, is finally 10 ℃ of coolings 10 seconds; The product obtained after amplification
his 6 -tag/rho-tag/odr-10double digestion reaction by restriction enzyme is subcloned into expression vector
pcDNA3.1 (+)multiple clone site
kpn Iwith
bamH Ibetween, complete the expression vector of olfactory receptor
pcDNA3.1 (+)/his 6 -tag/rho-tag/odr-10structure, its gene order is identified by order-checking as SEQ ID NO.4;
(1.2) transfected with human embryonic kidney cell HEK-293:HEK-293 cell culture fluid is the DMEM high glucose medium that has added hyclone, penicillin (100 U/mL) and streptomysin (100 μ g/mL) that percent by volume is 10%, and condition of culture is that 37 ℃, percent by volume are 5% CO
2incubator; The previous day of transfection, the HEK-293 cell is inoculated in six hole double dish, and it is 0.5～2 * 10 that 500 μ L concentration are inoculated in every hole
5the cell of cells/mL, containing in antibiotic DMEM high glucose medium, growing to degrees of fusion, not being about 80-90%, treat transfection; Transfection reagent is lipofectamin 2000, the expression vector that uses step 1.1 to build
pcDNA3.1 (+)/his 6 -tag/rho-tag/odr-10transfection HEK-293 cell; With serum-free DMEM nutrient solution, 4 μ g expression vectors are diluted to 50 μ L, mix; Dilute again lipofectamine lipfectamin 2000 to the 50 μ L of 10 μ L with serum-free DMEM nutrient solution, at room temperature hatch 5 minutes; Then the expression vector after dilution and lipfectamin 2000 being mixed to cumulative volume is 100 μ L, shakes up incubated at room 20 minutes; Adding the mixed liquor of 100 μ L expression vectors and lipfectamin 2000 in each hole of six orifice plates, after the front and back rocker mixes, is 5% CO at 37 ℃, percent by volume
2incubator hatch 24-48 hour after, olfactory receptor expresses the surface of cell membrane at HEK-293, in order to as the sensitive material of olfactory receptor sensor;
(1.3) extract olfactory receptor ODR-10: be the sensitive material of the olfactory receptor ODR-10 by preparation in step 1.2 as the olfactory receptor sensor, use in the HEK-293 cell of Membrane protein extraction kit from transfection and extract olfactory receptor ODR-10; First collect the transfection HEK-293 cell of 48 hours, wash 3 times each 5 minutes with the deionized water of 4 ℃ is centrifugal; Then add 1mL protein extraction damping fluid, 1 μ L protease inhibitors, 1 μ L DDT, ultrasonication 4 times, then 4 ℃ of 14000g centrifugal 10 minutes, abandon precipitation, supernatant goes in a new centrifuge tube water-bath 10 minutes, under room temperature 13000 centrifugal 5 minutes, sample was divided into two-layer up and down, and epicyte protein is contained in lower floor; Take off a layer sample, add 4 ℃ of aqua sterilisas of 500 μ L, place 5 minutes for 4 ℃, then 37 ℃ of water-baths are 10 minutes, and under room temperature 13000 centrifugal 5 minutes, sample was divided into two-layer up and down, repeats this step 5 time; The lower floor's sample finally obtained is the memebrane protein potpourri, wherein comprises with merging His
6the olfactory receptor ODR-10 of-tag label;-70 ℃ of preservations are stand-by.
3. a kind of method that detects the odoring substance diacetyl based on the olfactory receptor sensor according to claim 1, is characterized in that, described step 2 is specially: QCM (Quartz Crystal Microbalance) is placed in to ethanol and soaks 10 minutes, nitrogen drying; Then soak 20 minutes washed with de-ionized water in 1M NaOH aqueous solution; In 1M HCl solution, soak 5 minutes; Dense H
2sO
4with H
2o
2in the mixed liquor that 2:3 forms by volume, soak 1 minute, nitrogen drying, to complete the thoroughly clean of QCM (Quartz Crystal Microbalance); Utilize golden mercapto key self-assembling reaction covalency His that fixedly one deck can the specific recognition amalgamation and expression on the gold electrode of QCM (Quartz Crystal Microbalance)
6the sulfhydrylation single stranded DNA aptamers of-tag label, this aptamers by 5 ' end covalent modification HS-(CH
2)
6gene order shown in the SEQ ID NO.5 of group forms, and under room temperature, reaction is 21 hours; Then seal the unreacted site of gold electrode surfaces of molecule 1 1-MUA sealing quartz crystal microbalance sensor, sealing condition is lower 1 hour of room temperature; The epicyte protein mixed liquor that contains olfactory receptor of step 1.5 preparation evenly is incubated in to the gold electrode surfaces of quartz crystal microbalance sensor, and is placed on incubated at room and spends the night, make olfactory receptor by the His of amalgamation and expression
6specific association reaction occurs in-tag label and aptamers, realizes that olfactory receptor fixes and purifying in the gold electrode surfaces specificity of quartz crystal microbalance sensor.
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| CN104388570A (en) * | 2014-12-04 | 2015-03-04 | 中国科学院苏州生物医学工程技术研究所 | Piezoelectric thin film technology-based nucleic acid single-gene mutation detection method |
| CN107144610A (en) * | 2017-03-31 | 2017-09-08 | 国家纳米科学中心 | The method that gold nano grain adsorption albumen interacts with cell membrane associated receptor is detected based on quartz crystal chip in situ quantitation |
| CN107475349A (en) * | 2017-07-05 | 2017-12-15 | 成都奇璞生物科技有限公司 | A kind of quick detection bacterial drug resistance method based on QCM D |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104388570A (en) * | 2014-12-04 | 2015-03-04 | 中国科学院苏州生物医学工程技术研究所 | Piezoelectric thin film technology-based nucleic acid single-gene mutation detection method |
| CN108139344A (en) * | 2015-11-17 | 2018-06-08 | 株式会社而摩比特 | Smell sensor and measurement of odor system |
| CN108139344B (en) * | 2015-11-17 | 2021-10-15 | 株式会社而摩比特 | Odor sensor and odor measurement system |
| CN107144610A (en) * | 2017-03-31 | 2017-09-08 | 国家纳米科学中心 | The method that gold nano grain adsorption albumen interacts with cell membrane associated receptor is detected based on quartz crystal chip in situ quantitation |
| CN107144610B (en) * | 2017-03-31 | 2019-07-26 | 国家纳米科学中心 | Method based on quartz crystal chip in situ quantitation detection gold nano grain adsorption albumen and the interaction of cell membrane associated receptor |
| CN107475349A (en) * | 2017-07-05 | 2017-12-15 | 成都奇璞生物科技有限公司 | A kind of quick detection bacterial drug resistance method based on QCM D |
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