CN106442665A - Preparation method of ratio-dependent adapter sensor for detecting antibiotic residues based on screen-printed electrodes - Google Patents

Abstract

The invention discloses a preparation method of a ratio type aptamer sensor for detecting antibiotic residues based on screen printing electrodes, belonging to the technical field of agricultural product safety detection. The present invention is to respectively modify nano-gold-chitosan and nano-carbon fiber-nano-gold composites prepared on the surface of screen printing electrodes after cleaning and activation, and then drop ferrocene-aptamers on the modified electrodes respectively. , aptamer, using ferrocene and carbon nanofibers as probes to obtain a ratiometric aptasensor for detecting antibiotics. The sensor preparation method uses tetracycline as a template, and the concentration of tetracycline is quantified by calculating the current change before and after the sensor is exposed to tetracycline. Detection of antibiotic residues.

Description

A Ratiometric Aptamer Sensing for Antibiotic Residue Detection Based on Screen-Printed Electrodes The preparation method of the device

technical field

The invention relates to a preparation method of a ratio-type aptamer sensor for detecting antibiotic residues based on screen-printed electrodes, and belongs to the technical field of agricultural product safety detection.

Background technique

Antibiotics are physiologically active substances that can inhibit or kill other microbial cells, mainly produced by microorganisms. Since the discovery of penicillin in the 1930s, more than 2,000 antibiotics have been discovered today. Antibiotics commonly used in dairy cattle mainly include aminoglycosides, β-lactams, tetracyclines, and macrolides. If humans eat animal foods containing antibiotic residues for a long time, the drugs will continue to accumulate in the body, which will have toxic effects on the human body, increase bacterial resistance, cause allergies and allergies in the human body, and even cause carcinogenesis, teratogenicity, and pathogenicity. mutagenesis. Although the maximum amount of antibiotic residues has been strictly limited internationally in recent years, there are still many illegal uses due to their growth-stimulating effect on certain crops. Therefore, it is very important to realize the detection of antibiotics.

Ordinary three-electrode systems are not easy to carry. The impedance change caused by the biometric identification process on the traditional electrode is very small, and the semi-infinite linear diffusion layer on the electrode surface is easy to cause the loss of reactants; although the microarray electrode can absorb the impedance change that occurs during the reaction Magnified, but it is easy to be worn, causing deviation when reused. Compared with traditional electrodes, screen-printed electrodes are lightweight, easy to carry, and can be used at one time. They have good advantages for on-site detection. Therefore, the combination of screen-printed electrodes and traditional detection systems has become a potential choice. The traditional pesticide residue detection method has the advantages of good selectivity, high sensitivity and high accuracy, and simultaneously detects multiple elements or compounds, but it requires expensive instruments and equipment, and the sample pretreatment process is cumbersome and time-consuming, and it is difficult for analysts. The technical level is very high, and it is not suitable for on-site rapid detection. Therefore, this paper attempts to prepare a ratiometric aptasensor based on two probes for the detection of antibiotic residues.

Contents of the invention

The purpose of the present invention is to provide a ratio-type aptamer sensor detection method for the detection of antibiotic residues that can overcome the defects of the above-mentioned method, and has high sensitivity, high specificity, integration and portability. The technical solution adopted is: use the integration and portability of screen printing electrodes, use ferrocene, that is, Fc, and nano-carbon fibers, that is, NCFs, to construct corresponding aptamer sensors respectively, based on the two basic sensors A ratiometric sensor is built on top of it to achieve the detection purpose of high sensitivity, high precision and reducing the difference between batches. Through the specific reaction between the antigen and the aptamer, the change of the current value on the electrode surface is detected, and the electrochemical performance of the sensor is studied.

The steps of the preparation method of the ratiometric aptasensor for detecting antibiotic residues based on screen-printed electrodes are as follows:

1) Preparation of nano-gold/nano-gold-chitosan composite, nano-carbon fiber, ferrocene-aptamer;

2) Clean and activate the screen-printed electrode to obtain a pretreated screen-printed electrode;

3) The nano-gold-chitosan composite and nano-carbon fiber/nano-gold prepared in step 1) are respectively modified on the screen-printed electrode pretreated in step 2) to obtain a modified screen-printed electrode;

4) Add ferrocene-aptamer and aptamer dropwise to the modified screen-printed electrode obtained in step 3), and dry naturally to obtain the basic aptamer sensor based on the screen-printed electrode;

5) Three experimental conditions for the basic aptasensor obtained in step 4);

6) Under the optimal conditions obtained in step 5), antibiotics such as tetracycline are detected.

The preparation method of the ratiometric aptamer sensor based on screen-printed electrodes to detect antibiotic residues is characterized in that, in step 1), the nano-gold-chitosan composite, nano-carbon fiber, and ferrocene-aptamer are respectively Using chitosan as a dispersant to disperse nano gold, take a certain concentration of nano carbon fiber solution, mix ferrocene and aptamer to obtain a uniformly dispersed suspension.

The preparation method of the ratiometric aptasensor based on screen-printed electrodes for detecting antibiotic residues is characterized in that, in step 3) the modification of the screen-printed electrodes is to firstly mix 7 μL nano-gold-chitosan The composite, 30% nano-carbon fiber solution and nano-gold solution were added dropwise to the pretreated screen-printed electrode, and dried at room temperature to obtain nano-gold-chitosan and nano-carbon fiber/nano-gold modified screen-printed electrodes, respectively.

The preparation method of the ratiometric aptasensor based on screen-printed electrodes to detect antibiotic residues is characterized in that, in step 4), 7 μL of ferrocene-aptamer complexes are respectively added dropwise on the modified electrodes , Aptamer solution, 7 μL of ferrocene-aptamer complex was added dropwise to the screen-printed electrode modified by nano-gold-chitosan, and 7 μL of aptamer solution was added dropwise to nano-carbon fiber/ On the screen-printed electrodes modified with gold nanoparticles, dry at 4°C to obtain two kinds of aptamer biosensors.

The preparation method of the ratiometric aptasensor based on screen-printed electrodes to detect antibiotic residues is characterized in that step 5) the three test conditions of the two aptasensors test the pH value of the bottom solution, the adaptation The aptamer concentration and incubation time were optimized respectively: the pH value was 7.0, the aptamer concentration was 6 μM, and the incubation time was 60 min.

The method for preparing a ratiometric aptasensor based on screen-printed electrodes to detect antibiotic residues is characterized in that, in step 6), tetracycline standard solutions of different concentrations are added dropwise, incubated for 60 min, and cyclic voltaic is carried out in the bottom solution. Security testing.

The method for preparing a ratiometric aptasensor based on screen-printed electrodes for detecting antibiotic residues is characterized in that the specific steps are as follows:

1) Preparation of nano-gold/nano-gold-chitosan composite, nano-carbon fiber, and ferrocene-aptamer: 100 mL of chloroauric acid with a mass-volume ratio of 0.01% was added dropwise to a beaker, and heated on an electric furnace , stirring while heating until boiling, then quickly added 2.5 mL of 1% sodium citrate solution, which quickly turned ruby color as the reaction progressed, indicating the formation of the indicated gold nanoparticles; vigorously stirred the solution for 1 Hours later, the prepared nano-gold solution was obtained; 0.5 g chitosan was weighed and placed in a beaker, and 1.0% acetic acid solution was added to stir and dissolve, and the dissolved solution was placed in a 250 mL volumetric flask and constant volume. The final solution was poured into a beaker, and magnetically stirred for 10 h under a magnetic stirrer to obtain a 0.2% chitosan solution; 20 mL of 1% chitosan acetic acid solution was stirred and added to the above nano-gold solution to obtain nano-gold- Chitosan complex; 1 g ferrocene was added to 100 mL ethanol solution and sonicated for 30 min to obtain a 1% ferrocene solution, then the aptamer solution was added to the ferrocene solution and stirred at 4 °C Uniform for 12 hours to obtain a ferrocene-aptamer complex;

2) Cleaning and activation of the screen-printed electrode: first, put the screen-printed carbon electrode into a small beaker filled with 1mM sodium hydroxide solution and ultrasonically clean it for 5 minutes, clean it with ultrapure water, and dry it with nitrogen gas. Put it into a small beaker containing 1mM hydrochloric acid solution for ultrasonic cleaning for 5 minutes, wash with ultrapure water, blow dry with nitrogen, then clean the electrode with absolute ethanol, blow dry with nitrogen, and finally conduct current in phosphate buffer solution with pH 5.0 - Time curve scanning for 300s, after that, cyclic voltammetry curve scanning until the performance is stable;

3) Modification of the screen-printed electrode: Add 7 μL of nano-gold-chitosan composite, 30% nano-carbon fiber solution and nano-gold solution to the pretreated screen-printed electrode, respectively. Dry at room temperature to obtain nano-gold-chitosan, nano-carbon fiber/nano-gold modified screen-printed electrodes respectively;

4) Immobilization of the aptamer: Add 7 μL of ferrocene-aptamer complex dropwise on the above-mentioned electrode to the screen-printed electrode modified by nano-gold-chitosan, and 7 μL of aptamer The solution was added dropwise to the screen-printed electrode modified by nano-carbon fiber/nano-gold, and dried at room temperature to obtain two kinds of aptamer biosensors, and the prepared electrodes were stored in a dry environment at 4 °C for later use;

5) Optimization of test conditions: Prepare a series of phosphate buffer solutions with pH values of 6.0, 6.5, 7.0, 7.5, and 8.0, and prepare a series of detection bottom solutions respectively. The current value of the sensor is detected, and the optimal pH value of 7.0 is screened out; the aptamers of 2 μM, 4 μM, 5 μM, 6 μM, and 8 μM are respectively loaded on the electrode, and the current value is detected to screen out the best fit The body concentration was 6 μM; the incubation time with the same concentration of tetracycline was controlled as 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, and 90 min, and the current value was detected to screen out the optimal incubation time 60min;

6) Tetracycline detection: Under optimal conditions: pH 7.0, aptamer concentration 6 μM, incubation time 60 min, current detection was performed on different concentrations of tetracycline on the two aptamer sensors, and established different The relationship curve between the tetracycline concentration and the current change of the screen printing electrode, and then the linear regression equation between the logarithmic value of different concentrations of tetracycline and the current peak ratio is obtained, and the concentration range is 10 ^-11 ~ 10 ^-9 g/mL y=-0.02854x - 0.02655, the correlation coefficient is 0.994; in the concentration range of 10 ^-9 ～10 ^-3 g/mL, y = -0.00225x + 0.20538, the correlation coefficient is 0.997.

The preparation method of the ratiometric aptasensor based on screen-printed electrodes to detect antibiotic residues is characterized in that the specific detection steps of antibiotic residues such as tetracycline in milk are as follows:

1) Pretreatment of milk samples: buy milk in a local supermarket, dilute the milk at a ratio of 1:10, then distribute it into centrifuge tubes, and centrifuge at 20,000 rpm for 90 minutes; after centrifugation, the milk is divided into Obviously three layers, the upper and lower layers are macromolecular substances such as fat and casein. In order to avoid the wrapping of antibiotics by macromolecular substances, we go to the middle layer of whey, collect the whey, and add tetracycline to the collected whey and other antibiotics, the concentrations are 5×10 ^-10 g/mL, 5×10 ^-9 g/mL, 5×10 ^-8 g/mL, 5×10 ^-7 g/mL;

2) Sample detection: Under optimal conditions: pH 7.0, aptamer concentration 6 μM, incubation time 60 min, milk samples were detected;

3) According to the established corresponding linear relationship, calculate the residual amount of antibiotics such as tetracycline in the corresponding sample.

The preparation method of the ratiometric aptasensor for detecting antibiotic residues based on screen-printed electrodes is characterized in that it is used for detecting antibiotic residues such as tetracycline.

The preparation method of the ratiometric aptasensor based on screen-printed electrodes to detect antibiotic residues is characterized in that the electrodes used are screen-printed electrodes, including a printed electrode substrate, and an external insulation printed on the substrate. Layer and three electrode lead wires, it is characterized in that three electrodes are also printed on the substrate, a working electrode: carbon electrode; a counter electrode: carbon electrode, diameter 3mm; and a reference electrode: Ag/AgCl electrode, each The electrodes are correspondingly connected with an electrode lead. The electrode has stable electrochemical performance and good uniformity. In the following description, the screen printing electrode is abbreviated as SPCE.

The method is characterized in that the sensor for detecting tetracycline using the ratiometric aptamer based on the screen-printed electrode prepared by the present invention has the advantages of simple operation, low cost, high detection sensitivity, high accuracy, and reduced batch-to-batch differences, etc. It has the advantages of short reaction time, less consumption of samples and reagents, and high stability. It can be used for on-site detection of actual samples, and meets the development and international requirements of rapid detection technology for tetracycline residues in my country.

Description of drawings

Figure 1 SEM images of ferrocene-aptamer, carbon nanofibers (NCFs), carbon nanofibers-nanogold (NCFs-AuNPs composites): A. Scanning electron micrographs of ferrocene-aptamer complexes at low magnification; B. SEM image of ferrocene-aptamer complex under high magnification; C. SEM image of carbon nanofiber; D. SEM image of carbon nanofiber-nanogold;

Fig.2 Nanogold-chitosan/ferrocene-aptamer/bovine serum albumin/tetracycline sensor assembly process in phosphate buffer containing 0.1mol/L KCl and 5 mmol/L potassium ferricyanide Cyclic voltammetry characterization diagram: a) Empty screen-printed electrode is SPCE; b) nano-gold-chitosan/SPCE; c) ferrocene-aptamer/nano-gold-chitosan/SPCE; d) bovine serum albumin/ferrocene-aptamer/nano-gold-chitosan/SPCE; e) tetracycline/bovine serum albumin/ferrocene-aptamer/nano-gold-chitosan/SPCE;

Fig.3 Cyclic voltammetry in phosphate buffer containing 0.1mol/L KCl and 5 mmol/L potassium ferricyanide during assembly of carbon nanofibers/gold nanoparticles/aptamer/bovine serum albumin/tetracycline sensor : a) empty screen-printed electrode; b) carbon nanofiber/SPCE; c) gold nanofiber/carbon nanofiber/SPCE; d) aptamer/gold nanofiber/carbon nanofiber/SPCE; e) serum albumin/aptamer/ Nanogold/nanocarbon fiber/SPCE; f) tetracycline/bovine serum albumin/nanogold/nanocarbon fiber/SPCE;

Fig. 4 The effect of the pH value of the bottom liquid on the current response of the sensor;

Fig. 5 Effect of aptamer concentration on sensor current response;

Fig. 6 Effect of incubation time on sensor current response;

Fig. 7 Differential pulse voltammetry curves of nano-gold-chitosan/ferrocene-aptamer/bovine serum albumin/tetracycline sensor after being incubated with different concentrations of tetracycline, tetracycline concentration: a-k: a. 0g/mL; b. 10-3g/mL; c.10-4g/mL; d.10-5g/mL; e.10-6g/mL; f.10-7g/mL; g.10-8g/mL; h.10- 9g/mL; i.10-10g/mL; j. 10-11g/mL; k. 10-12g/mL;

Figure 8. Differential pulse voltammetry curves of carbon nanofiber/gold nanoparticles/aptamer/bovine serum albumin/tetracycline sensor after being incubated with different concentrations of tetracycline, tetracycline concentration: a-j: a.0g/mL; b.10-3g/mL ; c.10-4g/mL; d.10-5g/mL; e.10-6g/mL; f.10-7g/mL; g.10-8g/mL; .10-10g/mL; j. 10-11g/mL;

Figure 9 The linear relationship between the current change and the logarithm of the tetracycline concentration after the two sensors were incubated with tetracycline;

Figure 10 The linear relationship between the current change ratio and the logarithm of the tetracycline concentration after the two sensors were respectively incubated with tetracycline;

Figure 11 Detection of the ratiometric aptasensor to the recovery rate of antibiotic spiked in actual samples.

detailed description

The present invention will be further described below in conjunction with embodiment, but the present invention is not limited by embodiment.

Example 1 Preparation steps of a ratiometric aptasensor based on screen-printed electrodes:

1) Preparation of nano-gold/nano-gold-chitosan composite, nano-carbon fiber, and ferrocene-aptamer: 100 mL of 0.01% chloroauric acid was added dropwise into a beaker, and placed on an electric furnace to heat, while heating Stir until boiling, then quickly add 2.5 mL of 1% sodium citrate solution, which quickly turns a ruby color as the reaction progresses, indicating the formation of the indicated gold nanoparticles; after vigorously stirring the solution for 1 hour, the obtained Prepared nano-gold solution; weigh 0.5 g of chitosan, i.e. CS, and place it in a beaker, add 1.0% acetic acid solution and stir to dissolve, place the dissolved solution in a 250 mL volumetric flask and constant volume, the solution after constant volume Pour it into a beaker and stir it magnetically for 10 h under a magnetic stirrer to obtain a 0.2% chitosan solution; stir and add 20 mL of 1% chitosan acetic acid solution into the above nano-gold solution to obtain a nano-gold-chitosan composite 1 g of ferrocene was added to 100 mL of ethanol solution and sonicated for 30 min to obtain a 1% ferrocene solution, then the aptamer solution was added to the ferrocene solution, stirred and mixed at 4 °C for 12 hours, Obtain a ferrocene-aptamer complex;

2) Cleaning and activation of the screen-printed electrode: First, put the screen-printed carbon electrode into a small beaker filled with 1mM NaOH solution and ultrasonically clean it for 5 minutes, clean it with ultrapure water, and dry it with nitrogen. Then, put the electrode into Ultrasonic cleaning in a small beaker containing 1mM HCl solution for 5 minutes, cleaning with ultrapure water, drying with nitrogen gas, after that, cleaning the electrode with absolute ethanol, drying with nitrogen gas, and finally, performing current- Scan the time curve for 300s, and then scan the cyclic voltammetry curve until the performance is stable;

3) Modification of the screen-printed electrode: Add 7 μL of nano-gold-chitosan composite, 30% nano-carbon fiber solution and nano-gold solution to the pretreated screen-printed electrode, respectively. Dry at room temperature to obtain nano-gold-chitosan, nano-carbon fiber/nano-gold modified screen-printed electrodes respectively;

4) Immobilization of aptamers: Add 7 μL of ferrocene-aptamer complex dropwise on the above-mentioned electrodes to the screen-printed electrodes modified by nano-gold-chitosan, and 7 μL of aptamers The solution was added dropwise to the carbon nanofiber/gold nanometer modified screen-printed electrode, and dried at room temperature to obtain two aptamer biosensors, and the prepared electrode was stored in a dry environment at 4 °C for future use.

Example 2 Electrochemical Characterization During Assembly of Aptamer Biosensor

1) Using a scanning electron microscope to characterize the microstructure of the screen-printed electrode modified with ferrocene-aptamer, carbon nanofibers, carbon nanofibers-nanogold, as shown in Figure 1, it can be seen that the nanomaterials have been successfully modified to the electrode surface;

2) Different electrodes in the phosphate buffer containing 0.1 mol/L KCl and 5 mmol/L potassium ferricyanide during the assembly process of nano-gold-chitosan/ferrocene-aptamer/bovine serum albumin/tetracycline The cyclic voltammetry curve in Figure 2 is shown in Figure 2. Curve a) in the figure is a characterization diagram of an empty screen-printed electrode, and we can see obvious redox peaks; as shown in curve b) in the figure, when the screen After the printed electrode is decorated with nano-gold-chitosan nanomaterials, the current is increased compared to the empty screen-printed electrode because the nano-gold has good conductivity; as shown in curve c), on this basis, the modified After adding ferrocene-aptamer, because ferrocene is also conductive, the current increases again; when 7 μL of bovine serum albumin is immobilized, because it is a macromolecular protein, it not only does not conduct electricity, but also It hinders the electron transfer at the interface, so the peak value of the current becomes smaller, as shown in curve d);

3) Cyclic voltammetry of different electrodes in phosphate buffer containing 0.1mol/L KCl and 5 mmol/L potassium ferricyanide during the assembly process of carbon nanofibers/gold nanoparticles/aptamer/bovine serum albumin/tetracycline The curve, as shown in Figure 2, the curve a) in the figure is the characterization diagram of the empty screen-printed electrode, we can see the obvious redox peak; as shown in the curve b) in the figure, when the screen-printed electrode is modified After nano-carbon fiber nanomaterials, due to the good conductivity of nano-carbon fibers, the current is increased compared to the empty screen-printed electrode; as shown in curve c), the current increases again after the nano-gold is modified on this basis; When 7 μL of the aptamer is immobilized, since the aptamer is a protein molecule, it not only does not conduct electricity, but also hinders the electron transfer at the interface, so the peak value of the current becomes smaller, as shown in curve d), which also proves that the aptamer body has been successfully immobilized on the electrode surface.

Example 3 Optimization of test conditions

1) Optimization of pH value

The different pH values of the test bottom solution have different effects on the activity of the aptamer, which in turn will affect the sensitivity of the sensor. Therefore, a series of phosphate buffer solutions with pH values were prepared in this experiment, and the pH values were 6.0, 6.5, 7.0, 7.5, and 8.0, and prepared a series of detection bottom solutions respectively; Figure 4 shows the current difference measured by cyclic voltammetry before and after the two sensors were incubated with aptamers and tetracycline in different pH value bottom solutions The size of the value. It can be seen from the figure that when the pH value is 7.0, the difference is the largest, which indicates that pH 7.0 is the optimal pH value of the sensor, and at this time, the aptamer can exert its activity better;

2) Optimization of aptamer concentration

In order to reduce the waste of aptamers in the experiment and make the performance of aptasensors more superior, some experimental conditions of aptasensors were optimized, and the modified electrodes were immersed in aptamer solutions of different concentrations, so that The aptamer was immobilized on the electrode, and then the prepared sensor was used to detect the same concentration of tetracycline, and the change of peak current was measured by differential pulse voltammetry. It can be clearly seen from Figure 5 that the value of ΔI increases continuously with the increase of aptamer concentration. When the concentration is greater than 6 μM, the value of ΔI basically remains in a stable state. This phenomenon shows that the aptamer concentration of 6 μM in this experiment is enough to cover the surface of the electrode, and the specific binding between the aptamer and tetracycline reaches saturation. Therefore, 6 μM aptamer is the best concentration;

3) Optimization of incubation time

Incubation time is an important criterion to measure the performance of the sensor. In order to determine the optimal incubation time, the prepared aptamer sensors were added dropwise with the same concentration of tetracycline, let it react for different times, and measured the ΔI at different times. The change of the value, as shown in Figure 6, the value of ΔI increases continuously with the increase of time, however, the value of ΔI no longer changes with time after the time is greater than 60 min, but remains at a stable level, This is mainly because the tetracycline captured by the aptamer immobilized on the electrode has reached saturation, so the optimal incubation time is 60 min.

Example 4 Application of the prepared ratiometric aptasensor

1) The linear relationship between the ratio of current change after incubation with tetracycline and the logarithm of tetracycline concentration

Configure a series of tetracycline standard solutions, and perform differential pulse voltammetry scans on the two aptamer sensors for different concentrations of tetracycline, as shown in Figure 7 and Figure 8, and establish different tetracycline concentrations and screen printing electrode current changes respectively. The relationship curve between them is shown in Figure 9, and then the linear regression equation between the logarithmic value and the current change ratio of tetracycline at different concentrations, that is, △ I _CNFs /△ I _Fc : in the concentration range of 10 ^-11 ~ 10 ^-9 g/mL y = -0.02854x - 0.02655, and the correlation coefficient is 0.994; within the concentration range of 10 ^-9 ~ 10 ^-3 g/mL, y = -0.00225x + 0.20538, and the correlation coefficient is 0.997, as shown in Figure 10;

2) Detection of antibiotic residues in real sample milk

Purchase milk at a local supermarket, dilute the milk at a ratio of 1:10, then dispense it into centrifuge tubes, and centrifuge at 20,000 rpm for 90 minutes. After the centrifugation, the milk is divided into three obvious layers. The upper and lower layers are macromolecular substances such as fat and casein. In order to avoid the macromolecular substances from wrapping tetracycline, we go to the middle layer of whey, collect the whey, and collect the whey. Add tetracycline to good whey, the concentrations are 5×10 ^-10 g/mL, 5×10 ^-9 g/mL, 5×10 ^-8 g/mL, 5×10 ^-7 g/mL; The milk samples were detected under the same conditions, and the concentration of tetracycline in the spiked samples was calculated according to the calibration curve, and the recovery rate could reach 95.98%-104.28%, as shown in Figure 11.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.

Claims (9)

1. a kind of preparation method and application of the Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, It is characterized in that, it is that the screen printing electrode surface after cleaning activation modifies the nanometer gold-shitosan of preparation, nanometer respectively Carbon fiber-nano-Au composite, then difference Deca ferrocene-aptamers, aptamers on the electrode modified, with ferrocene Obtain the Ratio-type aptamer sensor of detection antibiotic with carbon nano-fiber respectively as probe.

2. the system of the Ratio-type aptamer sensor of antibiotic remainss is detected according to claim 1 based on screen printing electrode Preparation Method it is characterised in that electrode used therein is screen printing electrode, include the substrate of a printed electrode, base printed on chip outward Portion's insulating barrier and each of three electrodes lead are it is characterised in that be also printed with three electrodes, a working electrode on described substrate：Carbon Electrode；One to electrode：Carbon electrode, diameter 3mm；With a reference electrode：Ag/AgCl electrode, each electrode pair should be connected with one Contact conductor, this electrode electro Chemical stable performance, homogeneity is good.

3. according to claim 1, the Ratio-type aptamer sensor of antibiotic remainss is detected based on screen printing electrode Preparation method is it is characterised in that step is as follows：

1）Nanometer gold/nanometer gold-chitosan complexes, carbon nano-fiber, the preparation of ferrocene-aptamers；

2）Cleaning activation screen printing electrode, obtains the screen printing electrode of pretreatment；

3）By step 1）Nanometer gold-the chitosan complexes preparing and carbon nano-fiber/nanometer gold modify step respectively 2）On the screen printing electrode of pretreatment, obtain the screen printing electrode modified；

4) ferrocene-aptamers, aptamers are added drop-wise to step 3 respectively）On the screen printing electrode modified of gained, from The basic aptamer sensor based on screen printing electrode is obtained after so drying；

5）Optimization step 4）Three kinds of experimental conditions of the basic aptamer sensor of gained；

6）In step 5）Under the optimal conditionss of gained, the antibiotic such as tetracycline are detected.

4. according to claim 3, the Ratio-type aptamer sensor of antibiotic remainss is detected based on screen printing electrode Preparation method is it is characterised in that step 1）Described nanometer gold-chitosan complexes, carbon nano-fiber, ferrocene-aptamers are divided It is not with shitosan for dispersant nanometer gold, take certain density carbon nano-fiber solution, ferrocene is mixed with aptamers Conjunction obtains finely dispersed suspension.

5. according to claim 3, the Ratio-type aptamer sensor of antibiotic remainss is detected based on screen printing electrode Preparation method is it is characterised in that step 3）The modification of described screen printing electrode electrode, is respectively first by 7 μ L nanometer gold-shell Polysaccharide complex, 30% carbon nano-fiber solution and nano-Au solution are added drop-wise on the screen printing electrode of pretreatment, under room temperature Dry, respectively obtain nanometer gold-shitosan, the screen printing electrode of carbon nano-fiber/decorated by nano-gold.

6. according to claim 3, the Ratio-type aptamer sensor of antibiotic remainss is detected based on screen printing electrode Preparation method is it is characterised in that step 4）Described Deca 7 μ L ferrocene-adaptation bluk recombination respectively on the electrode modified Thing, adaptation liquid solution, are that 7 μ L ferrocene-adaptor complex is added drop-wise to nanometer gold-chitosan-modified good silk screen printing On electrode, 7 μ L are adapted to liquid solution and are added drop-wise on the screen printing electrode of carbon nano-fiber/decorated by nano-gold, in 4 DEG C of conditions Lower drying, obtains two kinds of aptamers biosensors.

7. according to claim 3, the Ratio-type aptamer sensor of antibiotic remainss is detected based on screen printing electrode Preparation method is it is characterised in that step 5）Three kinds of experimental condition test bottom liquid pH value of described two aptamers biosensors, Adaptation bulk concentration, incubation time are optimized respectively：PH value is 7.0, and adaptation bulk concentration is 6 μM, and incubation time is 60 min.

8. according to claim 3, the Ratio-type aptamer sensor of antibiotic remainss is detected based on screen printing electrode Preparation method is it is characterised in that comprise the following steps that：

1）Nanometer gold/nanometer gold-chitosan complexes, carbon nano-fiber, the preparation of ferrocene-aptamers：100 mL mass Volume ratio is that 0.01% gold chloride is added drop-wise in beaker, is placed in heating on electric furnace, heats while stirring until boiling, Ran Houxun Speed adds 2.5 mL 1% sodium citrate solution, and this solution of carrying out with reaction has quickly become ruby color, and explanation refers to The formation of the golden nanometer particle showing；It is stirred vigorously after this solution continues 1 hour, obtain prepared nano-Au solution；Weigh 0.5 G shitosan is placed in beaker, adds the acetum stirring and dissolving of 1.0 %, the solution having dissolved is placed in 250 mL volumetric flasks In and constant volume, the solution after constant volume pours in beaker, magnetic agitation 10 h under magnetic stirring apparatuss, obtain 0.2% shitosan molten Liquid；The stirring of the shitosan acetic acid solution of 20 mL 1% is added in above-mentioned nano-Au solution and obtains nanometer gold-shitosan and be combined Thing；1 g ferrocene is added to ultrasonic 30 min in 100 mL ethanol solution, obtains 1% solution of ferrocene, then by aptamers Solution is added in solution of ferrocene, stirring and evenly mixing 12 hours at 4 DEG C, obtains ferrocene-adaptor complex；

2）The cleaning of screen printing electrode, activation：First, screen printing carbon electrode is put into and fill 1mM sodium hydroxide solution It is cleaned by ultrasonic 5 minutes in small beaker, ultra-pure water cleans, and nitrogen dries up, and then, puts the electrodes into and fills the little of 1mM hydrochloric acid solution It is cleaned by ultrasonic 5 minutes in beaker, ultra-pure water cleans, and nitrogen dries up, and afterwards, uses washes of absolute alcohol electrode, nitrogen dries up, Afterwards, carry out current versus time curve scanning 300s in the phosphate buffer of pH 5.0, afterwards, be circulated volt-ampere curve and sweep Retouch, until stable performance；

3）The modification of screen printing electrode：On screen printing electrode respectively Deca 7 μ L nanometer gold-chitosan complexes, 30% Carbon nano-fiber solution and nano-Au solution are added drop-wise on the screen printing electrode of pretreatment, dry, respectively obtain and receive under room temperature Meter Jin-shitosan, the screen printing electrode of carbon nano-fiber/decorated by nano-gold；

4）The fixation of aptamers：On above-mentioned electrode, Deca 7 μ L ferrocene-adaptor complex is added drop-wise to nanometer gold-shell and gathers On sugar-modified good screen printing electrode, 7 μ L are adapted to the screen printing that liquid solution is added drop-wise to carbon nano-fiber/decorated by nano-gold On brush electrode, it is dried at room temperature for, obtain two kinds of aptamers biosensors, and the electrode preparing is put in 4 DEG C of dryings Save backup in environment；

5）The optimization of experimental condition：Prepare a series of phosphate buffer of pH value, pH value be respectively 6.0,6.5,7.0,7.5, 8.0, and it has been made into a series of detection bottom liquid respectively, in these bottom liquid, sensor current value is detected, filter out Good pH value 7.0；Respectively to 2 μM of electrode load, 4 μM, 5 μM, 6 μM, 8 μM of aptamers, its current value is detected, Filter out optimal adaptation bulk concentration and be 6 μM；With the tetracycline incubation time of same concentration be controlled as 30 min, 40 min, 50 min, 60 min, 70 min, 80 min, 90 min, detect to its current value, and filtering out optimal incubation time is 60 min；

6）The detection of tetracycline：In optimal conditions：PH 7.0, is adapted to 6 μM of bulk concentration, incubation time 60 min, at two kinds In aptamer sensor, current detecting is carried out to the tetracycline of variable concentrations, and establish respectively the different tetracycline concentration of foundation with Relation curve between screen printing electrode curent change, and then obtain logarithm value and the current peak of the tetracycline of variable concentrations Equation of linear regression between ratio, in concentration range 10^-11～10^-9Y=-0.02854x -0.02655, phase is obtained in g/mL Closing coefficient is 0.994；In concentration range 10^-9～10^-3Y=- 0.00225x+0.20538, correlation coefficient is obtained in g/mL 0.997.

9. according to claim 1, the Ratio-type aptamer sensor of antibiotic remainss is detected based on screen printing electrode Preparation method it is characterised in that in milk the concrete detecting step of the antibiotic remainss such as tetracycline as follows：

1）The pre-treatment of milk sample：Buy milk in local supermarket, milk according to 1:10 ratio is diluted, Ran Houfen It is attached in centrifuge tube, with 20000 revolutions per seconds of centrifugation 90min；After centrifugation terminates, milk is divided into obvious three layers, upper and lower Layer is the macromolecular substances such as fat and casein, and in order to avoid the parcel to antibiotic for the macromolecular substances, we go middle one layer Milk surum, collect milk surum, the interpolation antibiotic such as tetracycline in the milk surum collected, concentration is 5 × 10 respectively^-10G/mL, 5 × 10^-9G/mL, 5 × 10^-8G/mL, 5 × 10^-7g/mL；

2）The detection of sample：In optimal conditions：PH 7.0, is adapted to 6 μM of bulk concentration, incubation time 60 min, to milk sample Product are detected；

3）Step 6 according to Claim 8）The corresponding linear relationship of middle foundation, calculates the antibiosis such as the tetracycline of respective sample Plain residual quantity.