CN104034761B - A kind of device and method detecting OBP and pheromone cohesive process - Google Patents
A kind of device and method detecting OBP and pheromone cohesive process Download PDFInfo
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- CN104034761B CN104034761B CN201410202760.0A CN201410202760A CN104034761B CN 104034761 B CN104034761 B CN 104034761B CN 201410202760 A CN201410202760 A CN 201410202760A CN 104034761 B CN104034761 B CN 104034761B
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- 239000003016 pheromone Substances 0.000 title claims abstract description 75
- 238000000034 method Methods 0.000 title claims abstract description 46
- MLFHJEHSLIIPHL-UHFFFAOYSA-N isoamyl acetate Chemical compound CC(C)CCOC(C)=O MLFHJEHSLIIPHL-UHFFFAOYSA-N 0.000 claims abstract description 114
- 229940117955 isoamyl acetate Drugs 0.000 claims abstract description 57
- 241000256844 Apis mellifera Species 0.000 claims abstract description 56
- 241000256846 Apis cerana Species 0.000 claims abstract description 47
- 230000002572 peristaltic effect Effects 0.000 claims abstract description 47
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Abstract
The invention discloses a kind of device and method detecting OBP and pheromone cohesive process, device include sampling system, measure converting system, to electrode, reference electrode, working electrode, measuring flume, agitator, go out liquid peristaltic pump, waste liquid tank, computer；Sampling system is made up of the first peristaltic pump, the second peristaltic pump, the 3rd peristaltic pump, the first syringe, the second syringe, the 3rd syringe, controller, measures converting system and is made up of measuring circuit, high resistant electrometer, measuring circuit.The present invention obtains a kind of method detecting OBP and pheromone cohesive process by the detection Studies of Honeybee Pheromones isoamyl acetate standard solution of variable concentrations, apis cerana OBP (Acer ASP2) standard solution, apis cerana OBP (Acer ASP2) with the electrochemical impedance spectroscopy changed over of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of variable concentrations.
Description
Technical field
The present invention relates to a kind of based on electrochemical impedance analysis of spectrum method, particularly relate to a kind of device and method detecting OBP and pheromone cohesive process.
Background technology
In bio-sensing field, electrochemical impedance detection technique is owing to its cost is relatively low, signal is easily handled and analyzes, and is capable of detecting in real time and being widely used.OBP is the low-molecular-weight hydrophobic proteins outside a kind of born of the same parents, it is easy to purification, and can be specific binding with target molecule.In previous studies, the research to OBP mainly uses fluorescent marker method, mass spectrography etc., and fluorescent marker method needs to be marked object, and mass spectrography equipment needed thereby instrument is sufficiently expensive.OBP is utilized to be combined with the advantage of electrochemical impedance detection technique, development is a kind of based on electrochemical impedance analysis of spectrum, and detection apis cerana OBP (Acer-ASP2) has important practical value and Research Significance with the sensing device of the cohesive process of variable concentrations Studies of Honeybee Pheromones isoamyl acetate with method.
Summary of the invention
Present invention aims to the deficiencies in the prior art, it is provided that a kind of device and method detecting OBP and pheromone cohesive process.
It is an object of the invention to be achieved through the following technical solutions: a kind of device detecting OBP and pheromone cohesive process of the present invention, this device includes with lower part: sampling system, measure converting system, to electrode, reference electrode, working electrode, measuring flume, agitator, go out liquid peristaltic pump, waste liquid tank, computer；Sampling system is made up of the first peristaltic pump, the second peristaltic pump, the 3rd peristaltic pump, the first syringe, the second syringe, the 3rd syringe, controller, measures converting system and is made up of measuring circuit, high resistant electrometer, measuring circuit.The outfan of sampling system accesses the input of measuring flume；Electrode, reference electrode, working electrode are measured end and immersed respectively at measuring flume build-in test liquid 2/3rds；Measuring flume outfan connects liquid peristaltic pump and accesses in waste liquid tank；Measure converting system to be made up of measuring circuit, high resistant electrometer, measuring circuit；Measuring circuit is connected by wire with to electrode；High resistant electrometer one end is connected by wire with reference electrode；The high resistant electrometer other end is connected by wire with working electrode after being connected with measuring circuit；Record signal by being connected with computer by USB serial ports after measuring converting system conversion.
A kind of method detecting OBP and pheromone cohesive process, it is characterised in that comprise the following steps:
(1) the preparation Studies of Honeybee Pheromones isoamyl acetate standard solution of variable concentrations, apis cerana OBP (Acer-ASP2) standard solution, apis cerana OBP (Acer-ASP2) and the Studies of Honeybee Pheromones isoamyl acetate mixed solution of variable concentrations；
(2) 3 kinds of solution electrochemical impedance collection of illustrative plates under different time in detecting step (1)；
(3) relation of 3 kinds of solution electrochemistry resistance value concentration in analytical procedure (1)；
(4) relation of 3 kinds of solution electrochemistry resistance value times in analytical procedure (1), obtains OBP and pheromone cohesive process.
Further, step (4) is particularly as follows: utilize the electrochemical impedance collection of illustrative plates of the different time that Randles equivalent impedance circuit fit procedure (2) obtains, obtain each solution electron transfer resistance of different time under variable concentrations, and with the electron transfer resistance that records for respective 1st time as radix, the electron transfer resistance recorded below all deducts the 1st the electron transfer resistance recorded, and i.e. obtains R3、R6、R9、R12、R15、R18、R21、R24、R27、R30；Wherein RiRepresent i-th minute and measure the electron transfer resistance obtained, i=3,6,9,12,15,18,21,24,27,30;The electron transfer resistance that 1st time records is=R3-R3；The electron transfer resistance that 2nd time records is R6-R3；The like, make solution electron transfer resistance to be measured and change over relation curve, obtain OBP and pheromone cohesive process.
The invention has the beneficial effects as follows: the present invention provides a kind of without labelling, without ankyrin, the method for simple and lower-cost detection OBP and pheromone cohesive process.The method detection sensitivity is high, and Monitoring lower-cut is low.
Accompanying drawing explanation
Fig. 1 is test experience device schematic diagram of the present invention；
Fig. 2 be substance withdrawl syndrome of the present invention be 10-9M、10-8M、10-7M and 10-6The electrochemical impedance spectroscopy curve chart of the Studies of Honeybee Pheromones isoamyl acetate standard solution of M；
Fig. 3 is apis cerana OBP (Acer-ASP2) of the present invention and substance withdrawl syndrome is 10-9M、10-8M、10-7M and 10-6The electrochemical impedance spectroscopy curve chart of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M；
Fig. 4 be substance withdrawl syndrome of the present invention be 10-9M、10-8M、10-7M and 10-6The Studies of Honeybee Pheromones isoamyl acetate standard solution of M and apis cerana OBP (Acer-ASP2) are 10 with substance withdrawl syndrome-9M、10-8M、10-7M and 10-6The graph of a relation of the electrochemical impedance spectral curve of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M；
Fig. 5 be substance withdrawl syndrome of the present invention be 10-9M、10-8M、10-7M and 10-6The electrochemical impedance spectroscopy Curve Electron transfer resistance of the Studies of Honeybee Pheromones isoamyl acetate standard solution of M changes over curve chart；
Fig. 6 be apis cerana OBP (Acer-ASP2) standard solution of the present invention, apis cerana OBP (Acer-ASP2) and substance withdrawl syndrome be 10-9M、10-8M、10-7M and 10-6The electrochemical impedance spectroscopy Curve Electron transfer resistance of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M changes over curve chart.
Detailed description of the invention
Below in conjunction with drawings and the specific embodiments, the present invention is described in detail, but is not to limit the present invention.
A kind of device detecting OBP and pheromone cohesive process of the present invention, for detecting the cohesive process of apis cerana OBP (Acer-ASP2) and Studies of Honeybee Pheromones isoamyl acetate, whole system is formed by with lower part: sampling system 1, measure converting system 2, to electrode 3(using platinum filament as to electrode 3), reference electrode 4(is using Ag/AgCl electrode as reference electrode 4), working electrode 5(is using gold electrode as working electrode 5), measuring flume 6, agitator 7, go out liquid peristaltic pump 8, waste liquid tank 9, computer 10 form；Sampling system 1 is made up of first peristaltic pump the 11, second peristaltic pump the 12, the 3rd peristaltic pump the 13, first syringe the 14, second syringe the 15, the 3rd syringe 16, controller 17, measures converting system 2 and is made up of measuring circuit 21, high resistant electrometer 22, measuring circuit 23.The outfan of sampling system 1 accesses the input of measuring flume 6；Electrode 3, reference electrode 4, working electrode 5 are measured end and immersed respectively at measuring flume 6 build-in test liquid 2/3rds；Measuring flume 6 outfan connects liquid peristaltic pump 8 and accesses in waste liquid tank 9；Measure converting system 2 to be made up of measuring circuit 21, high resistant electrometer 22, measuring circuit 23；Measuring circuit 21 is connected by wire with to electrode 3；High resistant electrometer 22 one end is connected by wire with reference electrode 4；High resistant electrometer 22 other end is connected by wire with working electrode 5 after being connected with measuring circuit 23；Record signal by being connected with computer 10 by USB serial ports after measuring converting system 2 conversion.
The sequence such as SEQ of described apis cerana OBP (Acer-ASP2)
Shown in ID NO.1.
A kind of method detecting OBP and pheromone cohesive process of the present invention, based on electrochemical impedance analysis of spectrum, detection apis cerana OBP (Acer-ASP2) and the cohesive process of variable concentrations Studies of Honeybee Pheromones isoamyl acetate, comprise the following steps:
(1) solution preparation
For ensureing the accuracy of experimental result, all solution are the most now with the current.
(1.1) standard solution of Studies of Honeybee Pheromones isoamyl acetate is prepared: the standard stock solution dilution preparation substance withdrawl syndrome using substance withdrawl syndrome to be Studies of Honeybee Pheromones isoamyl acetate to be measured for 0.01M is 10-9M、10-8M、10-7M and 10-6The standard solution of 4 kinds of respective concentration gradients of M；Solvent is 0.1M phosphate buffer, pH=7.2, (0.1M refers to phosphatic molar concentration in phosphate buffer, and the phosphate buffer used in this specification each means 0.1 M, the phosphate buffer of pH=7.2).
(1.2) preparation apis cerana OBP (Acer-ASP2) standard solution: add apis cerana OBP (Acer-ASP2) solution that 30 μ l concentration are 500 μ g/ml in every 540 μ l phosphate buffers.
(1.3) preparation apis cerana OBP (Acer-ASP2) and Studies of Honeybee Pheromones isoamyl acetate mixed solution: be 10 at substance withdrawl syndrome-9M、10-8M、10-7M and 10-6In the standard solution of the Studies of Honeybee Pheromones isoamyl acetate of M, every 540 μ l add apis cerana OBP (Acer-ASP2) solution that 30 μ l concentration are 500 μ g/ml, apis cerana OBP (Acer-ASP2) can be specific binding with Studies of Honeybee Pheromones isoamyl acetate, for guaranteeing both detections cohesive process, before measuring apis cerana OBP (Acer-ASP2) is added the Studies of Honeybee Pheromones isoamyl acetate standard solution of corresponding concentration.
(2) the electrochemical impedance collection of illustrative plates of 3 kinds of solution in detecting step (1).
(2.1) the electrochemical impedance collection of illustrative plates of the Studies of Honeybee Pheromones isoamyl acetate standard solution of detection variable concentrations:
Redox couple solution is injected (containing the 5mM potassium ferricyanide, 5mM potassium ferrocyanide and the KCl of 0.1M in redox couple solution to measuring flume 6 by the first syringe 14 in Fig. 1, solvent is deionized water), open the first peristaltic pump 11, perfusion rate 10 μ l/s, closing the first peristaltic pump 11 after Hemoperfusion time 40s, then the amount concentration by the second syringe 15 injected material is 10-9The Studies of Honeybee Pheromones isoamyl acetate standard solution of M, opens the second peristaltic pump 12, perfusion rate 10 μ l/s, closes the second peristaltic pump 12 after Hemoperfusion time 40s.Then electrode 3, reference electrode 4, working electrode 5 will be fixed in measuring flume 6, access computer 10 by measuring circuit 21, high resistant electrometer 22, the measurement converting system of measuring circuit 23 composition.Concrete test parameter be initial voltage be 0.23V, AC voltage magnitudes is 5mV, and swept frequency range is 1Hz ~ 100KHz.Measuring once every 3 minutes, measure 30 minutes altogether, the substance withdrawl syndrome obtaining continuous 10 times measuring is 10-9The electrochemical impedance collection of illustrative plates of the Studies of Honeybee Pheromones isoamyl acetate standard solution of M.
After measurement terminates, opening out liquid peristaltic pump 8, discharge waste liquid, to waste liquid tank 9, closes out liquid peristaltic pump 8 after waste liquid drains.Then inject phosphate buffer by the 3rd syringe 16, open the 3rd peristaltic pump 13, perfusion rate 10 μ l/s, close the 3rd peristaltic pump 13 after Hemoperfusion time 100s, be 10 for cleaning the redox couple measuring residual last time and substance withdrawl syndrome-9The impact of M Studies of Honeybee Pheromones isoamyl acetate standard solution.Repeat the measurement of above-mentioned Studies of Honeybee Pheromones isoamyl acetate standard solution afterwards, until completing substance withdrawl syndrome is 10-8M、107M and 10-6The measurement of M Studies of Honeybee Pheromones isoamyl acetate standard solution.Finally give the amount concentration (10 of different material-9M、10-8M、10-7M and 10-6The electrochemical impedance collection of illustrative plates of the Studies of Honeybee Pheromones isoamyl acetate standard solution under M), the curve that electrochemical impedance collection of illustrative plates is made up of impedance real part and imaginary impedance, take the last electrochemical impedance collection of illustrative plates measured under each concentration and make curve, as shown in Figure 2.
(2.2) the electrochemical impedance collection of illustrative plates of detection apis cerana OBP (Acer-ASP2) standard solution:
After step (2.1), opening out liquid peristaltic pump 8, discharge waste liquid, to waste liquid tank 9, closes out liquid peristaltic pump 8 after waste liquid drains.Then phosphate buffer is injected by the 3rd syringe 16, open the 3rd peristaltic pump 13, perfusion rate 10 μ l/s, closes the 3rd peristaltic pump 13 after Hemoperfusion time 100s, for cleaning the redox couple and the impact of Studies of Honeybee Pheromones isoamyl acetate standard solution measuring residual last time.Electrode 3, reference electrode 4, working electrode 5 will be fixed in measuring flume 6, inject redox couple solution by the first syringe 14 in Fig. 1 to measuring flume 6, and open the first peristaltic pump 11, perfusion rate 10 μ l/s, after Hemoperfusion time 40s, close the first peristaltic pump 11.Then inject apis cerana OBP (Acer-ASP2) standard solution by the second syringe 15, open the second peristaltic pump 12, perfusion rate 10 μ l/s, after Hemoperfusion time 40s, close the second peristaltic pump 12.Then electrode 3, reference electrode 4, working electrode 5 are fixed, and insert measuring flume 6, access computer 10 by measuring circuit 21, high resistant electrometer 22, the measurement converting system of measuring circuit 23 composition.Concrete test parameter be initial voltage be 0.23V, AC voltage magnitudes is 5mV, and swept frequency range is 1Hz ~ 100KHz.Measured once every 3 minutes, measure 30 minutes altogether, finally give the electrochemical impedance collection of illustrative plates of apis cerana OBP (Acer-ASP2) standard solution measured continuous 10 times.
(2.3) the electrochemical impedance collection of illustrative plates of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of detection apis cerana OBP (Acer-ASP2) and variable concentrations:
After step (2.2), opening out liquid peristaltic pump 8, discharge waste liquid, to waste liquid tank 9, closes out liquid peristaltic pump 8 after waste liquid drains.Then phosphate buffer is injected by the 3rd syringe 16, open the 3rd peristaltic pump 13, perfusion rate 10 μ l/s, the 3rd peristaltic pump 13 is closed, for cleaning the redox couple and the impact of apis cerana OBP (Acer-ASP2) standard solution measuring residual last time after Hemoperfusion time 100s.
Electrode 3, reference electrode 4, working electrode 5 will be fixed in measuring flume 6, inject redox couple solution by the first syringe 14 in Fig. 1 to measuring flume 6, and open the first peristaltic pump 11, perfusion rate 10 μ l/s, after Hemoperfusion time 40s, close the first peristaltic pump 11.Then injecting apis cerana OBP (Acer-ASP2) with substance withdrawl syndrome by the second syringe 15 is 10-9The Studies of Honeybee Pheromones isoamyl acetate mixed solution of M, opens the second peristaltic pump 12, perfusion rate 10 μ l/s, closes the second peristaltic pump 12 after Hemoperfusion time 40s.Then electrode 3, reference electrode 4, working electrode 5 are fixed, and insert measuring flume 6, access computer 10 by measuring circuit 21, high resistant electrometer 22, the measurement converting system of measuring circuit 23 composition.Concrete test parameter be initial voltage be 0.23V, AC voltage magnitudes is 5mV, and swept frequency range is 1Hz ~ 100KHz.Measuring once every 3 minutes, measure 30 minutes altogether, finally giving the apis cerana OBP (Acer-ASP2) measured continuous 10 times is 10 with substance withdrawl syndrome-9The electrochemical impedance collection of illustrative plates of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M.
After measurement terminates, opening out liquid peristaltic pump 8, discharge waste liquid, to waste liquid tank 9, closes out liquid peristaltic pump 8 after waste liquid drains.Then PBS is injected by the 3rd syringe 16, open the 3rd peristaltic pump 13, perfusion rate 10 μ l/s, close the 3rd peristaltic pump 13 after Hemoperfusion time 100s, be 10 for cleaning the redox couple measuring residual last time and apis cerana OBP (Acer-ASP2) with substance withdrawl syndrome-9The impact of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M.Repeat the measurement of above-mentioned apis cerana OBP (Acer-ASP2) and the isoamyl acetate abnormal smells from the patient mixed solution of variable concentrations afterwards, until completing apis cerana OBP (Acer-ASP2) with substance withdrawl syndrome is 10-8M、107M and 10-6The measurement of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M.Finally give the amount concentration (10 of apis cerana OBP (Acer-ASP2) different material-9M、10-8M、10-7M and 10-6The electrochemical impedance collection of illustrative plates of the Studies of Honeybee Pheromones isoamyl acetate mixed solution under M), the curve that electrochemical impedance collection of illustrative plates is made up of impedance real part and imaginary impedance, take the last electrochemical impedance collection of illustrative plates measured under each concentration and make curve, as shown in Figure 3.
(3) the Studies of Honeybee Pheromones isoamyl acetate mixed solution electrochemical impedance analysis of spectrum of Studies of Honeybee Pheromones isoamyl acetate standard solution electrochemical impedance spectroscopy, apis cerana OBP (Acer-ASP2) and variable concentrations.
Utilize the electrochemical impedance collection of illustrative plates that the Studies of Honeybee Pheromones isoamyl acetate standard solution of the variable concentrations of Randles equivalent impedance circuit fit procedure (2.1) is measured for the last time, and the electrochemical impedance collection of illustrative plates that the Studies of Honeybee Pheromones isoamyl acetate mixed solution of the apis cerana OBP (Acer-ASP2) of step (2.3) and variable concentrations is measured for the last time, obtaining substance withdrawl syndrome is 10-9M、10-8M、10-7M and 10-6The Studies of Honeybee Pheromones isoamyl acetate standard solution of M and apis cerana OBP (Acer-ASP2) are 10 with substance withdrawl syndrome-9M、10-8M、10-7M and 10-6The electron transmission resistance of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M.
For ease of showing the linear trend of variable concentrations under two kinds of solution the most intuitively, according to point each in figure with the principle not changing its slope divided by identical number, in figure, substance withdrawl syndrome is 10-9M、10-8M、10-7M and 10-6The Studies of Honeybee Pheromones isoamyl acetate standard solution of M and apis cerana OBP (Acer-ASP2) are 10 with substance withdrawl syndrome-9M、10-8M、10-7M and 10-6The electron transmission resistance of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M is all 10 divided by each maximum substance withdrawl syndrome-6The electron transmission resistance of M, respectively obtaining the concentration of Studies of Honeybee Pheromones isoamyl acetate standard solution and the relation curve of normalized impedance value and apis cerana OBP (Acer-ASP2) with substance withdrawl syndrome is 10-9M、10-8M、10-7M and 10-6The concentration of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M and the relation curve of normalized impedance value, as shown in Figure 4.
(4) electrochemical impedance spectroscopy of Studies of Honeybee Pheromones isoamyl acetate standard solution, the electrochemical impedance spectroscopy of apis cerana OBP (Acer-ASP2) standard solution, apis cerana OBP (Acer-ASP2) change over analysis with the electrochemical impedance spectroscopy of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of variable concentrations, and concrete analysis step is as follows:
(4.1) the electrochemical impedance spectroscopy Curve Electron transfer resistance of Studies of Honeybee Pheromones isoamyl acetate standard solution changes over analysis:
Utilize the amount concentration (10 of the different material of Randles equivalent impedance circuit fit procedure (2.1)-9M、10-8M、10-7M and 10-6M) the electrochemical impedance collection of illustrative plates that Studies of Honeybee Pheromones isoamyl acetate standard solution is measured for 10 times, obtain the electron transfer resistance of different time under each solution concentration, and with the electron transfer resistance that records for respective 1st time as radix, the electron transfer resistance recorded below all deducts the 1st the electron transfer resistance recorded, and (such as substance withdrawl syndrome is 10-9M matching obtains R3、R6、R9、R12、R15、R18、R21、R24、R27、R30；Wherein RiRepresent i-th minute and measure the electron transfer resistance obtained, i=3,6,9,12,15,18,21,24,27,30;The electron transfer resistance that 1st time records is=R3-R3；The electron transfer resistance that 2nd time records is R6-R3；The like), finally giving Studies of Honeybee Pheromones isoamyl acetate standard solution substance withdrawl syndrome is 10-9M、10-8M、10-7M and 10-6Under M, electron transfer resistance changes over value, and under variable concentrations, Studies of Honeybee Pheromones isoamyl acetate standard solution electron transfer resistance changes over value as vertical coordinate, and time point is abscissa, makes dynamic time curve, as shown in Figure 5.
(4.2) the electrochemical impedance spectroscopy Curve Electron transfer resistance of apis cerana OBP (Acer-ASP2) standard solution changes over analysis:
Utilize the electrochemical impedance collection of illustrative plates that apis cerana OBP (Acer-ASP2) standard solution of Randles equivalent impedance circuit fit procedure (2.2) is measured for 10 times, obtain the electron transfer resistance of different time, and with the electron transfer resistance that records for respective 1st time as radix, the electron transfer resistance recorded below all deducts the electron transfer resistance that records the 1st time, and (such as matching obtains R3、R6、R9、R12、R15、R18、R21、R24、R27、R30；Wherein RiRepresent i-th minute and measure the electron transfer resistance obtained, i=3,6,9,12,15,18,21,24,27,30;The electron transfer resistance that 1st time records is=R3-R3；The electron transfer resistance that 2nd time records is R6-R3；The like), finally give apis cerana OBP (Acer-ASP2) standard solution electron transfer resistance and change over value.
(4.3) apis cerana OBP (Acer-ASP2) and the amount concentration (10 of different material-9M、10-8M、10-7M and 10-6M) Studies of Honeybee Pheromones isoamyl acetate mixed solution electrochemical impedance spectroscopy Curve Electron transfer resistance changes over analysis:
Utilize the amount concentration (10 of Randles equivalent impedance circuit fit procedure (2.3) apis cerana OBP (Acer-ASP2) and different material-9M、10-8M、10-7M and 10-6The electrochemical impedance collection of illustrative plates of Studies of Honeybee Pheromones isoamyl acetate mixed solution M), obtain the electron transfer resistance of different time under each mixed solution concentration, and with the electron transfer resistance that records for respective 1st time as radix, the electron transfer resistance recorded below all deducts the 1st the electron transfer resistance recorded, and (such as substance withdrawl syndrome is 10 mixing-9M matching obtains R3 Mixed solution、R6 Mixed solution、R9 Mixed solution、R12 Mixed solution、R15 Mixed solution、R18 Mixed solution、R21 Mixed solution、R24 Mixed solution、R27 Mixed solution、R30 Mixed solution；Wherein Ri Mixed solutionRepresent that mixed solution measures the electron transfer resistance obtained, i=3,6,9,12,15,18,21,24,27,30 in lower i-th minute;The electron transfer resistance that 1st time records is R3 Mixed solution-R3 Mixed solution；The electron transfer resistance that 2nd time records is R6 Mixed solution-R3 Mixed solution；The like), finally give the amount concentration (10 of apis cerana OBP (Acer-ASP2) and different material-9M、10-8M、10-7M and 10-6M) Studies of Honeybee Pheromones isoamyl acetate mixed solution electron transfer resistance changes over value.Amount concentration (10 with apis cerana OBP (Acer-ASP2) Yu different material-9M、10-8M、10-7M and 10-6M) it is vertical coordinate that Studies of Honeybee Pheromones isoamyl acetate mixed solution electron transfer resistance changes over value, and time point is abscissa, makes dynamic time curve, and its empty is that the electron transfer resistance obtained in step (4.2) changes over curve, 10-9M is apis cerana OBP (Acer-ASP2) and substance withdrawl syndrome is 10-9The electrochemical impedance spectroscopy Curve Electron transfer resistance of the Studies of Honeybee Pheromones isoamyl acetate mixed solution of M changes over curve, and 10-8M、10-7M and 10-6M the like, as shown in Figure 6.
SEQUENCE LISTING
<110>Zhejiang University
<120>a kind of device and method detecting OBP and pheromone cohesive process
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 142
<212> PRT
<213>apis cerana
<400> 1
Met Asn Thr Leu Val Thr Val Thr Cys Leu Leu Ala Ala Leu Thr Val
1
5
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Val Arg Gly Ile Asp Gln Asp Thr Val Val Ala Lys Tyr Met Glu Tyr
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Leu Met Pro Asp Ile Met Pro Cys Ala Asp Glu Leu His Ile Ser Glu
35
40
45
Asp Ile Ala Thr Asn Ile Gln Ala Ala Lys Asn Gly Ala Asp Met Lys
50
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60
Gln Leu Gly Cys Leu Lys Ala Cys Val Met Lys Arg Ile Asp Met Leu
65
70
75
80
Lys Gly Thr Glu Leu Asn Ile Glu Pro Val Tyr Lys Met Ile Glu Val
85
90
95
Val His Ala Gly Asn Ala Asp Asp Ile Gln Leu Val Arg Gly Ile Ala
100
105
110
Asn Glu Cys Ile Glu Asn Ala Lys Gly Glu Ala Asp Glu Cys Ser Ile
115
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Gly Asn Lys Tyr Thr Asp Cys Tyr Ile Glu Lys Leu Phe Ser
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Claims (3)
1. the device detecting OBP and pheromone cohesive process, it is characterised in that this device includes
Sampling system (1), measure converting system (2), to electrode (3), reference electrode (4), working electrode (5),
Measuring flume (6), agitator (7), go out liquid peristaltic pump (8), waste liquid tank (9), computer (10)；Sample introduction system
System (1) is by the first peristaltic pump (11), the second peristaltic pump (12), the 3rd peristaltic pump (13), the first injection
Device (14), the second syringe (15), the 3rd syringe (16), controller (17) form；Sampling system
(1) outfan accesses the input of measuring flume (6)；To electrode (3), reference electrode (4), work
Electrode (5) is measured end and is immersed respectively at measuring flume (6) build-in test liquid 2/3rds；Measuring flume (6) exports
End connects liquid peristaltic pump (8) and accesses in waste liquid tank (9)；Measure converting system (2) to be measured by first
Circuit (21), high resistant electrometer (22), the second measuring circuit (23) form；First measuring circuit (21)
It is connected by wire with to electrode (3)；High resistant electrometer (22) one end and reference electrode (4) are by leading
Line is connected；High resistant electrometer (22) other end and the second measuring circuit (23) are connected afterwards and working electrode (5)
It is connected by wire；Record signal by passing through USB serial ports and computer after measuring converting system (2) conversion
(10) it is connected.
2. device detection OBP and a method for pheromone cohesive process described in claim 1, it is special
Levy and be, comprise the following steps:
(1) the preparation Studies of Honeybee Pheromones isoamyl acetate standard solution of variable concentrations, apis cerana OBP
Standard solution, apis cerana OBP mix molten with the Studies of Honeybee Pheromones isoamyl acetate of variable concentrations
Liquid；
(2) in detecting step (1) 3 kinds of solution at the electrochemical impedance collection of illustrative plates of different time；
(3) relation of 3 kinds of solution electrochemistry resistance value concentration in analytical procedure (1)；
(4) relation of 3 kinds of solution electrochemistry resistance value times in analytical procedure (1), obtains abnormal smells from the patient and combines egg
White and pheromone cohesive process.
Detection OBP the most according to claim 2 and the method for pheromone cohesive process, its feature
Be, step (4) particularly as follows:
Utilize the electrochemical impedance figure of the different time that Randles equivalent impedance circuit fit procedure (2) obtains
Spectrum, obtains each solution electron transfer resistance of different time under variable concentrations；And survey with respective 1st time
The electron transfer resistance obtained is radix, and the electron transfer resistance recorded below all deducts the 1st electronics recorded
Transfer resistance, it is thus achieved that electron transfer resistance changes over value, makes solution electron transfer resistance to be measured at any time
Between change value change over relation curve, obtain OBP and pheromone cohesive process.
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