CN104034761B - A kind of device and method detecting OBP and pheromone cohesive process - Google Patents

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

A device and method for detecting the binding process of odorant binding protein and pheromone

technical field

The invention relates to an analysis method based on electrochemical impedance spectroscopy, in particular to a device and method for detecting the binding process of odorant binding protein and pheromone.

Background technique

In the field of biosensing, electrochemical impedance detection technology is widely used because of its low cost, easy signal processing and analysis, and real-time detection. Odor-binding protein is an extracellular low-molecular-weight hydrophobic protein that is easy to purify and can specifically bind to target molecules. In previous studies, the research on odorant-binding proteins mainly used fluorescent labeling and mass spectrometry. Fluorescent labeling requires labeling of the target, while mass spectrometry requires very expensive equipment. Utilizing the advantages of odorant-binding protein and electrochemical impedance detection technology, a method based on electrochemical impedance spectroscopy was developed to detect the binding process of Chinese bee odorant-binding protein (Acer-ASP2) with different concentrations of honeybee pheromone isoamyl acetate. The sensing device and method have important practical value and research significance.

Contents of the invention

The object of the present invention is to provide a device and method for detecting the binding process of odorant-binding protein and pheromone against the deficiencies of the prior art.

The purpose of the present invention is achieved through the following technical solutions: a device for detecting the binding process of odorant binding protein and pheromone in the present invention, the device includes the following parts: sample introduction system, measurement conversion system, counter electrode, reference electrode, Working electrode, measuring tank, oscillator, liquid outlet peristaltic pump, waste liquid tank, computer; sampling system consists of the first peristaltic pump, the second peristaltic pump, the third peristaltic pump, the first syringe, the second syringe, and the third syringe , controller, and the measurement conversion system is composed of a measurement circuit, a high-resistance electrometer, and a measurement circuit. The output end of the sampling system is connected to the input end of the measuring tank; the measuring ends of the counter electrode, reference electrode, and working electrode are respectively immersed in two-thirds of the test solution in the measuring tank; the output end of the measuring tank is connected to the liquid peristaltic pump and connected to the In the waste liquid tank; the measurement conversion system is composed of a measurement circuit, a high-resistance electrometer, and a measurement circuit; the measurement circuit is connected to the counter electrode through a wire; one end of the high-resistance electrometer is connected to the reference electrode through a wire; the other end of the high-resistance electrometer is connected to the reference electrode. After the measurement circuit is connected, it is connected to the working electrode through wires; the measured signal is converted by the measurement conversion system and then connected to the computer through the USB serial port.

A method for detecting the binding process of odorant binding protein and pheromone, characterized in that it comprises the following steps:

(1) Prepare different concentrations of honeybee pheromone isoamyl acetate standard solution, Chinese honeybee odorant binding protein (Acer-ASP2) standard solution, Chinese honeybee odorant binding protein (Acer-ASP2) and honeybee pheromone isoamyl acetate of different concentrations Ester mixed solution;

(2) Electrochemical impedance spectra of the three solutions in the detection step (1) at different times;

(3) The electrochemical impedance value-concentration relationship of the three solutions in the analysis step (1);

(4) Analyze the electrochemical impedance value-time relationship of the three solutions in step (1), and obtain the binding process of odorant binding protein and pheromone.

Further, step (4) is specifically: use the Randles impedance equivalent circuit to fit the electrochemical impedance spectra obtained in step (2) at different times to obtain the electron transfer resistance of each solution at different concentrations at different times, and use the respective The electron transfer resistance measured for the first time is the base, and the electron transfer resistance measured for the first time is subtracted from the electron transfer resistance measured for the first time, that is, R ₃ , R ₆ , R ₉ , R ₁₂ , R ₁₅ , R ₁₈ , R ₂₁ , R ₂₄ , R ₂₇ , R ₃₀ ; where R _i represents the electron transfer resistance measured at the i-th minute, i=3, 6, 9, 12, 15, 18, 21, 24, 27, 30; The electron transfer resistance measured for the first time is = R ₃ -R ₃ ; the electron transfer resistance measured for the second time is R ₆ -R ₃ ; and so on, the electron transfer resistance of the solution to be tested changes with time. Obtain the combination process of odorant binding protein and pheromone.

The beneficial effects of the present invention are: the present invention provides a method for detecting the binding process of the odorant binding protein and the pheromone without labeling, without immobilizing the protein, simple and easy, and with low cost. The method has high detection sensitivity and low detection limit.

Description of drawings

Fig. 1 is the schematic diagram of detection experiment device of the present invention;

Fig. 2 is the electrochemical impedance spectrum curve diagram of the honeybee pheromone isoamyl acetate standard solution whose concentration of the substance of the present invention is 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M;

Figure 3 is a mixed solution of honeybee pheromone isoamyl acetate with the concentration of ^10-9 M, ^10-8 M, ^10-7 M and ^10-6 M of the present invention The electrochemical impedance spectroscopy curve;

Figure 4 is the standard solution of isoamyl acetate of honeybee pheromone and Chinese bee odorant binding protein (Acer-ASP2) with the concentration of the substance of the present invention being 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M The relationship diagram of the electrochemical impedance spectrum curve of the honeybee pheromone isoamyl acetate mixed solution with the substance concentration of 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M;

Fig. 5 is the electrochemical impedance spectrum curve electron transfer resistance of the honeybee pheromone isoamyl acetate standard solution with the concentration of the substance of the present invention being 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M over time change the graph;

Figure 6 shows the standard solution of Apis mellifera odorant-binding protein (Acer-ASP2) of the present invention, the concentration of Apis cerana odorifera-binding protein (Acer-ASP2) and substances at 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 Electrochemical impedance spectroscopy curve of ^-6 M honey bee pheromone isoamyl acetate mixed solution, electron transfer resistance changing curve with time.

detailed description

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments, but the present invention is not limited.

The invention is a device for detecting the combination process of odorant-binding protein and pheromone, which is used to detect the combination process of Chinese bee odor-binding protein (Acer-ASP2) and honeybee pheromone isoamyl acetate. The whole system is composed of the following parts: sample injection System 1, measurement conversion system 2, counter electrode 3 (platinum wire as counter electrode 3), reference electrode 4 (Ag/AgCl electrode as reference electrode 4), working electrode 5 (gold electrode as working electrode 5) , a measuring tank 6, an oscillator 7, a liquid outlet peristaltic pump 8, a waste liquid tank 9, and a computer 10; the sampling system 1 is composed of a first peristaltic pump 11, a second peristaltic pump 12, a third peristaltic pump 13, and a first syringe 14. The second injector 15, the third injector 16, and the controller 17 are composed. The measurement conversion system 2 is composed of a measurement circuit 21, a high-resistance electrometer 22, and a measurement circuit 23. The output end of the sampling system 1 is connected to the input end of the measurement tank 6; the measurement ends of the counter electrode 3, reference electrode 4, and working electrode 5 are respectively immersed in two-thirds of the test solution in the measurement tank 6; the output end of the measurement tank 6 is connected to The liquid peristaltic pump 8 is connected to the waste liquid tank 9; the measurement conversion system 2 is composed of a measurement circuit 21, a high-resistance electrometer 22, and a measurement circuit 23; the measurement circuit 21 is connected to the counter electrode 3 through wires; the high-resistance electrometer 22 One end is connected to the reference electrode 4 through a wire; the other end of the high-resistance electrometer 22 is connected to the measuring circuit 23 and then connected to the working electrode 5 through a wire; the measured signal is converted by the measurement conversion system 2 and connected to the computer 10 through the USB serial port.

The sequence of the Apis cerana odoriferous binding protein (Acer-ASP2) is shown as SEQ ID NO.1 is shown.

The present invention is a method for detecting the binding process of odorant-binding protein and pheromone, based on electrochemical impedance spectroscopy analysis, detecting the binding process of Chinese honeybee odorant-binding protein (Acer-ASP2) with different concentrations of honeybee pheromone isoamyl acetate, including the following step:

(1) Solution preparation

In order to ensure the accuracy of the experimental results, all solutions were prepared and used immediately.

(1.1) Prepare the standard solution of bee pheromone isoamyl acetate: Dilute the standard stock solution of honeybee pheromone isoamyl acetate with a substance concentration of 0.01M to prepare the substance concentration of 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M standard solutions with four corresponding concentration gradients; the solvent is 0.1M phosphate buffer, pH=7.2, (0.1M refers to the molar concentration of phosphate in the phosphate buffer , the phosphate buffer used in this application refers to 0.1 M phosphate buffer with pH=7.2).

(1.2) Prepare standard solution of Apis mellifera odorant-binding protein (Acer-ASP2): Add 30 μl of Apis cerana odorifera-binding protein (Acer-ASP2) solution with a concentration of 500 μg/ml to every 540 μl of phosphate buffer.

(1.3) Prepare a mixed solution of Apis mellifera odorant binding protein (Acer-ASP2) and honeybee pheromone isoamyl acetate: the concentration of the substance is 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M In the standard solution of honey bee pheromone isoamyl acetate, add 30 μl of Chinese bee odorant binding protein (Acer-ASP2) solution with a concentration of 500 μg/ml for every 540 μl, Chinese bee odorant binding protein (Acer-ASP2) and honeybee pheromone acetate Isoamyl ester can specifically bind. In order to ensure that the combination process of the two can be detected, Chinese bee odorant binding protein (Acer-ASP2) was added to the corresponding concentration of honeybee pheromone isoamyl acetate standard solution before measurement.

(2) Electrochemical impedance spectra of the three solutions in the detection step (1).

(2.1) Detection of electrochemical impedance spectroscopy of different concentrations of bee pheromone isoamyl acetate standard solution:

Inject the redox couple solution (the redox couple solution contains 5mM potassium ferricyanide, 5mM potassium ferrocyanide and 0.1M KCl, and the solvent is deionized water) into the measuring tank 6 through the first injector 14 in Fig. 1, and turn on The first peristaltic pump 11, the perfusion rate is 10 μl/s, the first peristaltic pump 11 is turned off after the perfusion time is 40s, and then the honeybee pheromone isoamyl acetate standard solution with a substance concentration of 10 ⁻⁹ M is injected through the second syringe 15, The second peristaltic pump 12 is turned on, the perfusion rate is 10 μl/s, and the second peristaltic pump 12 is turned off after a perfusion time of 40 s. Then the counter electrode 3 , reference electrode 4 , and working electrode 5 are fixed in the measurement tank 6 , and connected to the computer 10 through the measurement conversion system composed of the measurement circuit 21 , the high-resistance electrometer 22 and the measurement circuit 23 . The specific test parameters are that the initial voltage is 0.23V, the AC voltage amplitude is 5mV, and the frequency sweep range is 1Hz~100KHz. The measurement was performed every 3 minutes for a total of 30 minutes, and the electrochemical impedance spectrum of the honeybee pheromone isoamyl acetate standard solution with a substance concentration of 10 ⁻⁹ M was obtained for 10 consecutive measurements.

After the measurement, the liquid outlet peristaltic pump 8 is turned on, the waste liquid is discharged to the waste liquid tank 9, and the liquid outlet peristaltic pump 8 is turned off after the waste liquid is exhausted. Then inject phosphate buffer solution through the third syringe 16, turn on the third peristaltic pump 13, the perfusion rate is 10 μl/s, and turn off the third peristaltic pump 13 after the perfusion time is 100s, for cleaning the residual redox pair and the amount of substances in the last measurement The effect of the concentration of 10 ^-9 M bee pheromone isoamyl acetate standard solution. Then repeat the measurement of the standard solution of isoamyl acetate of bee pheromone until the measurement of the standard solution of isoamyl acetate of bee pheromone with the concentration of 10 ⁻⁸ M, 10 ⁷ M and 10 ⁻⁶ M is completed. Finally, the electrochemical impedance spectra of the honeybee pheromone isoamyl acetate standard solution at different concentrations (10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M) were obtained, and the electrochemical impedance spectra were The curve composed of the real part of the impedance and the imaginary part of the impedance is taken from the electrochemical impedance spectrum measured last time at each concentration to make a curve, as shown in Figure 2.

(2.2) Detection of electrochemical impedance spectroscopy of standard solution of Apis mellifera odorant binding protein (Acer-ASP2):

After step (2.1), turn on the liquid outlet peristaltic pump 8, discharge the waste liquid to the waste liquid tank 9, and turn off the liquid outlet peristaltic pump 8 after the waste liquid is exhausted. Then inject phosphate buffer solution through the third syringe 16, turn on the third peristaltic pump 13, the perfusion rate is 10 μl/s, and turn off the third peristaltic pump 13 after the perfusion time is 100s, which is used to clean the residual redox pair and bee pheromone in the last measurement Effect of isoamyl acetate standard solution. Fix the counter electrode 3, reference electrode 4, and working electrode 5 in the measurement tank 6, inject the redox pair solution into the measurement tank 6 through the first syringe 14 in Figure 1, turn on the first peristaltic pump 11, and the perfusion rate is 10 μl/s , turn off the first peristaltic pump 11 after the perfusion time is 40s. Then the standard solution of Apis cerana odorifera binding protein (Acer-ASP2) was injected through the second syringe 15, the second peristaltic pump 12 was turned on, the perfusion rate was 10 μl/s, and the second peristaltic pump 12 was turned off after 40 s of perfusion time. Then fix the counter electrode 3 , reference electrode 4 , and working electrode 5 , and insert them into the measurement tank 6 , and connect them to the computer 10 through the measurement conversion system composed of the measurement circuit 21 , the high-resistance electrometer 22 and the measurement circuit 23 . The specific test parameters are that the initial voltage is 0.23V, the AC voltage amplitude is 5mV, and the frequency sweep range is 1Hz~100KHz. Measurements were made every 3 minutes for a total of 30 minutes, and the electrochemical impedance spectra of the standard solution of Apis mellifera odoriferous binding protein (Acer-ASP2) were finally obtained for 10 consecutive measurements.

(2.3) Detection of the electrochemical impedance spectroscopy of the mixed solution of Apis mellifera odorant-binding protein (Acer-ASP2) and different concentrations of honeybee pheromone isoamyl acetate:

After step (2.2), the liquid outlet peristaltic pump 8 is turned on, the waste liquid is discharged to the waste liquid tank 9, and the liquid outlet peristaltic pump 8 is turned off after the waste liquid is exhausted. Then inject phosphate buffer solution through the third syringe 16, turn on the third peristaltic pump 13, the perfusion rate is 10 μl/s, and turn off the third peristaltic pump 13 after the perfusion time is 100s, for cleaning the residual redox pair and Apis sinensis smell from the previous measurement Effect of standard solution of binding protein (Acer-ASP2).

Fix the counter electrode 3, reference electrode 4, and working electrode 5 in the measurement tank 6, inject the redox pair solution into the measurement tank 6 through the first syringe 14 in Figure 1, turn on the first peristaltic pump 11, and the perfusion rate is 10 μl/s , turn off the first peristaltic pump 11 after the perfusion time is 40s. Then inject the mixed solution of Apis mellifera odorant binding protein (Acer-ASP2) and honeybee pheromone isoamyl acetate with a substance concentration of 10 ^-9 M through the second syringe 15, turn on the second peristaltic pump 12, and the perfusion rate is 10 μl/s , turn off the second peristaltic pump 12 after the perfusion time is 40s. Then fix the counter electrode 3 , reference electrode 4 , and working electrode 5 , and insert them into the measurement tank 6 , and connect them to the computer 10 through the measurement conversion system composed of the measurement circuit 21 , the high-resistance electrometer 22 and the measurement circuit 23 . The specific test parameters are that the initial voltage is 0.23V, the AC voltage amplitude is 5mV, and the frequency sweep range is 1Hz~100KHz. Measured every 3 minutes for a total of 30 minutes, and finally obtained 10 consecutive measurements of the mixed solution of Chinese bee odorant binding protein (Acer-ASP2) and honeybee pheromone isoamyl acetate with a concentration of 10 ^-9 M Electrochemical impedance spectroscopy.

After the measurement, the liquid outlet peristaltic pump 8 is turned on, the waste liquid is discharged to the waste liquid tank 9, and the liquid outlet peristaltic pump 8 is turned off after the waste liquid is exhausted. Then inject PBS buffer solution through the third syringe 16, turn on the third peristaltic pump 13, the perfusion rate is 10 μ l/s, and close the third peristaltic pump 13 after the perfusion time is 100 s, for cleaning the residual redox pair and Apis sinensis smell from the last measurement Effects of the binding protein (Acer-ASP2) and the honeybee pheromone isoamyl acetate mixed solution with a concentration of 10 ^-9 M. Afterwards, repeat the measurement of the odorant mixed solution of Apis mellifera odorant-binding protein (Acer-ASP2) and different concentrations of isoamyl acetate until the concentration of the odorant-binding protein (Acer-ASP2) and substance in Apis mellifera is 10 ^-8 M, Measurement of 10 ⁷ M and 10 ^-6 M bee pheromone isoamyl acetate mixed solution. Finally, the mixed solution of honeybee pheromone isoamyl acetate under different concentrations (10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M) of odorant binding protein (Acer-ASP2) of Apis mellifera Electrochemical impedance spectrum, electrochemical impedance spectrum is a curve composed of impedance real part and impedance imaginary part, take the last measured electrochemical impedance spectrum at each concentration to make a curve, as shown in Figure 3.

(3) Electrochemical impedance spectroscopy analysis of bee pheromone isoamyl acetate standard solution, and electrochemical impedance spectroscopy analysis of mixed solutions of honeybee pheromone isoamyl acetate with different concentrations of odorant-binding protein (Acer-ASP2) and honeybee pheromone.

Using Randles impedance equivalent circuit to fit the electrochemical impedance spectra of the last measurement of the honeybee pheromone isoamyl acetate standard solution with different concentrations in step (2.1), and the odorant-binding protein (Acer-ASP2) of Apis mellifera Chinese bee (Acer-ASP2) in step (2.3) The electrochemical impedance spectrum of the last measurement of the mixed solution with different concentrations of bee pheromone isoamyl acetate, and the information of the bees with the concentration of 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M Amount of isoamyl acetate standard solution and Apis mellifera odorant binding protein (Acer-ASP2) with the concentration of 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M honeybee pheromone isoamyl acetate Electron transport resistance of ester mixed solution.

In order to visually show the linear trend of different concentrations of the two solutions in one graph, according to the principle that each point in the graph is divided by the same number without changing its slope, the concentration of the substance in the graph is 10 ^-9 M , 10 ^-8 M, 10 ^-7 M and 10 ^-6 M honey bee pheromone isoamyl acetate standard solution and Apis mellifera odorant binding protein (Acer-ASP2) with the concentration of 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M honey bee pheromone isoamyl acetate mixed solutions are divided by the electron transport resistance of the maximum substance concentration of 10 ^-6 M, respectively, to obtain the honeybee pheromone The relationship curve between the concentration of isoamyl acetate standard solution and the normalized impedance value and the amount of odorant binding protein (Acer-ASP2) of Apis mellifera and the concentration of substances are 10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 The relationship curve between the concentration of ^-6 M bee pheromone isoamyl acetate mixed solution and the normalized impedance value is shown in Figure 4.

(4) Electrochemical impedance spectroscopy of honeybee pheromone isoamyl acetate standard solution, electrochemical impedance spectroscopy of Chinese bee odorant-binding protein (Acer-ASP2) standard solution, Chinese bee odorant-binding protein (Acer-ASP2) with different concentrations of The electrochemical impedance spectroscopy analysis of the honeybee pheromone isoamyl acetate mixed solution over time, the specific analysis steps are as follows:

(4.1) Electrochemical impedance spectroscopy curve analysis of electron transfer resistance change with time of bee pheromone isoamyl acetate standard solution:

Honeybee pheromone isoamyl acetate standard solution with different substance concentrations (10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M) using Randles impedance equivalent circuit fitting step (2.1) 10 The electrochemical impedance spectrum measured for the first time was used to obtain the electron transfer resistance at different times at each solution concentration, and the electron transfer resistance measured for the first time was used as the base, and the electron transfer resistance measured later was subtracted from the first time. The obtained electron transfer resistance (for example, when the concentration of the substance is 10 ^-9 M is fitted to obtain R ₃ , R ₆ , R ₉ , R ₁₂ , R ₁₅ , R ₁₈ , R ₂₁ , R ₂₄ , R ₂₇ , R ₃₀ ; where R _i represents the electron transfer resistance measured at the i minute, i= 3, 6, 9, 12, 15, 18, 21, 24, 27, 30; the electron transfer resistance measured for the first time is =R ₃ -R ₃ ; the electron transfer resistance measured for the second time is R ₆ -R ₃ ; and so on), and finally the concentration of substances in the honeybee pheromone isoamyl acetate standard solution is 10 ^-9 M, 10 ^-8 M, 10 ^- The electron transfer resistance changes with time at ⁷ M and 10 ^-6 M. The electron transfer resistance changes with time in the honeybee pheromone isoamyl acetate standard solution at different concentrations as the ordinate, and the time point as the abscissa. Curve, as shown in Figure 5.

(4.2) Electrochemical impedance spectroscopy curve of the standard solution of Apis mellifera odorant binding protein (Acer-ASP2) electron transfer resistance change with time analysis:

Using Randles impedance equivalent circuit fitting step (2.2) to measure the electrochemical impedance spectrum of Chinese bee odorant-binding protein (Acer-ASP2) standard solution for 10 times, obtain the electron transfer resistance at different times, and measure it for the first time The electron transfer resistance is the base number, and the electron transfer resistance measured later is subtracted from the electron transfer resistance measured for the first time (for example, R ₃ , R ₆ , R ₉ , R ₁₂ , R ₁₅ , R ₁₈ , R ₂₁ , R ₂₄ , R ₂₇ , R ₃₀ ; where R _i represents the electron transfer resistance measured at the i-th minute, i=3, 6, 9, 12, 15, 18, 21, 24, 27, 30; the first time The measured electron transfer resistance is = R ₃ -R ₃ ; the second measured electron transfer resistance is R ₆ -R ₃ ; and so on), and finally the standard solution electron transfer Resistance changes value over time.

(4.3) Mixed solutions of Apis mellifera odorant binding protein (Acer-ASP2) and honeybee pheromone isoamyl acetate with different concentrations (10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M) Electrochemical impedance spectroscopy curve analysis of electron transfer resistance change with time:

Using the Randles impedance equivalent circuit fitting step (2.3) of the Chinese bee odorant-binding protein (Acer-ASP2) and the concentration of different substances (10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M) The electrochemical impedance spectrum of the honeybee pheromone isoamyl acetate mixed solution was used to obtain the electron transfer resistance of each mixed solution concentration at different times, and based on the electron transfer resistance measured for the first time, the electron transfer resistance measured later was The resistance is subtracted from the electron transfer resistance measured for the first time (for example, the concentration of the substance is 10 mixed ^-9 M to get R _{3 mixed solution} , R _{6 mixed solution} , R _{9 mixed solution} , R _{12 mixed solution} , R ₁₅ mixed solution _{Mixed solution} , R _{18 mixed solution} , R _{21 mixed solution} , R _{24 mixed solution} , R _{27 mixed solution} , R _{30 mixed solution} ; where R _{i mixed solution} represents the electron transfer resistance measured in the i minute under the mixed solution, i=3 , 6, 9, 12, 15, 18, 21, 24, 27, 30; the electron transfer resistance measured for the first time is R _{3 mixed solution} -R _{3 mixed solution} ; the electron transfer resistance measured for the second time is R _{6 mixed solution-} R _{3 mixed solution} ; and so on), to finally obtain the concentration of Chinese honeybee odorant binding protein (Acer-ASP2) and different substances (10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M) The electron transfer resistance of bee pheromone isoamyl acetate mixed solution changes with time. Electron transfer in honeybee pheromone isoamyl acetate mixed solution with different concentrations (10 ^-9 M, 10 ^-8 M, 10 ^-7 M and 10 ^-6 M) of odorant-binding protein (Acer-ASP2) of Apis mellifera The value of the resistance change with time is the ordinate, and the time point is the abscissa, and a dynamic time curve is drawn, where the blank is the electron transfer resistance change curve with time obtained in step (4.2), and 10 ^-9 M is the odorant binding protein of Apis chinensis ( Electrochemical impedance spectroscopy curve of the mixed solution of Acer-ASP2) and honeybee pheromone isoamyl acetate with a concentration of 10 ^-9 M, electron transfer resistance changing curves with time, 10 ^-8 M, 10 ^-7 M and 10 ^{- 6} M and so on, as shown in Figure 6.

SEQUENCE LISTING

<110> Zhejiang University

<120> A device and method for detecting the binding process of odorant binding protein and pheromone

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 142

<212> PRT

<213> Chinese bee

<400> 1

Met Asn Thr Leu Val Thr Val Thr Cys Leu Leu Ala Ala Leu Thr Val

1 5 10 15

Val Arg Gly Ile Asp Gln Asp Thr Val Val Ala Lys Tyr Met Glu Tyr

20 25 30

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 55 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

115 120 125

Gly Asn Lys Tyr Thr Asp Cys Tyr Ile Glu Lys Leu Phe Ser

130 135 140

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.