CN106442665A - Preparation method of ratio-dependent adapter sensor for detecting antibiotic residues based on screen-printed electrodes - Google Patents
Preparation method of ratio-dependent adapter sensor for detecting antibiotic residues based on screen-printed electrodes Download PDFInfo
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Abstract
The invention discloses a preparation method of a ratio-dependent adapter sensor for detecting antibiotic residues based on screen-printed electrodes, and belongs to the technical field of safety detection of agricultural products. The preparation method includes the steps: respectively modifying prepared nano-gold-chitosan and carbon nano-fiber-nano-gold compounds on the surfaces of the cleaned and activated screen-printed electrodes; respectively dripping ferrocene-adapters and adapters on the decorated electrodes; respectively taking ferrocene and carbon nano-fibers as probes to obtain the ratio-dependent adapter sensor for detecting antibiotics. The preparation method of the sensor takes tetracycline as a template, the concentration of the tetracycline is quantitatively determined by calculating changes of current before and after the sensor contacts with the tetracycline, the sensor is high in sensitivity and accuracy and can be used for detecting residues of antibiotics such as tetracycline in practical samples, and difference between batches can be reduced.
Description
Technical field
The present invention relates to a kind of system of the Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode
Preparation Method, belongs to agricultural product security detection technique field.
Background technology
Antibiotic is a kind of biological active substanceies that can suppress or kill other microbial cells, is mainly produced by microorganism
Raw.Since the thirties in 20th century finds penicillin, nowadays it has been found that antibiosis have kind more than 2000.Often make in milch cow
Antibiotic mainly has aminoglycoside, beta-lactam, Tetracyclines and Macrolide etc..If the mankind eat for a long time
After the animal food containing antibiotic remainss, medicine is constantly accumulated in vivo, can produce toxic action to human body, increase antibacterial
Drug resistance, cause allergy and the allergy of human body, or even carcinogenic, teratogenesis, mutagenic action can be produced.Although state in recent years
The strict residual maximum limiting antibiotic on border, but because it has growth stimulation to some crops, so still having not
Few violating the regulations use phenomenon, therefore realize the detection to antibiotic it is critical that.
Common three-electrode system is not readily portable, and the impedance variation that in traditional electrode, biometric identification process causes is very
Little, the linear diffusion layer of semo-infinite of its electrode surface easily makes reactant be lost；Although tiny array electrode can be by course of reaction
The impedance variation occurring is amplified, but it is easily worn, and causes deviation when reusing.Compare traditional electrode, silk screen printing electricity
Extremely light and handy, easy to carry, can single use, for Site Detection, there is good advantage, therefore screen printing electrode and biography
The detecting system of system is combined into having potential selection.It is good, sensitive that traditional pesticide residue detection method has selectivity
Degree is high and accuracy is high, simultaneously the advantage of detection multiple element or compound, but it needs expensive instrument and equipment, before sample
Processing procedure is loaded down with trivial details, time-consuming, and the technical merit of analysis personnel is required very high, is unsuitable for field quick detection.Therefore originally
Literary composition is attempted preparing a kind of Ratio-type aptamer sensor of the detection antibiotic remainss based on two kinds of probes.
Content of the invention
It is an object of the invention to provide a kind of defect that can overcome said method, and sensitivity height, specificity are high, integrated
Change, the Ratio-type aptamer sensor detection method of the antibiotic element residue detection of portability.Using technical scheme be：Using
Integrated, the portability of screen printing electrode, using ferrocene, that is, Fc, carbon nano-fiber are that two kinds of probes of NCFs build respectively
Corresponding aptamer sensor, builds Ratio-type sensor on the basis of two kinds of pedestal sensors, high, smart to reach sensitivity
Exactness is high, testing goal that is reducing difference between batch.By the specific reaction between antigen and aptamers, detecting electrode surface
Electric current value changes, study the chemical property of this sensor.
The step of the preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode
Suddenly 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.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, step 1）Described nanometer gold-chitosan complexes, carbon nano-fiber, ferrocene-aptamers are to be respectively with shitosan
Dispersant nanometer gold, takes certain density carbon nano-fiber solution, ferrocene is mixed to get with aptamers and is uniformly dispersed
Suspension.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, step 3）The modification of described screen printing electrode electrode, be respectively first by 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.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, step 4）Described difference Deca 7 μ L ferrocene-adaptor complex, adaptation liquid solution on the electrode modified,
It is 7 μ L ferrocene-adaptor complex to be added drop-wise on nanometer gold-chitosan-modified good screen printing electrode, by 7 μ L
Adaptation liquid solution is added drop-wise on the screen printing electrode of carbon nano-fiber/decorated by nano-gold, is dried, obtains two under the conditions of 4 DEG C
Plant aptamers biosensor.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, step 5）Three kinds of experimental conditions of described two aptamers biosensors are tested bottom liquid pH value, adaptation bulk concentration, are incubated
Time of educating is optimized respectively：PH value is 7.0, and adaptation bulk concentration is 6 μM, and incubation time is 60 min.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, step 6）The tetracycline titer of described Deca variable concentrations, is incubated 60 min, is circulated volt-ampere in the liquid of bottom
Method detects.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, 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, illustrates to indicate
Golden nanometer particle formation；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
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 uses washes of absolute alcohol electrode afterwards, and nitrogen dries up, finally,
Carry out current versus time curve scanning 300s in the phosphate buffer of pH 5.0, afterwards, be circulated volt-ampere curve scanning,
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.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, in milk, the concrete detecting step of the antibiotic remainss such as tetracycline is 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）According to the corresponding linear relationship set up, calculate the antibiotic residual quantities such as the tetracycline of respective sample.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, for detecting the antibiotic remainss such as tetracycline.
The preparation method of the described Ratio-type aptamer sensor detecting antibiotic remainss based on screen printing electrode, it is special
Levy and be, electrode used therein is screen printing electrode, including the substrate of a printed electrode, base external insulation printed on chip and three
Root contact conductor is it is characterised in that be also printed with three electrodes, a working electrode on described substrate：Carbon electrode；One right
Electrode：Carbon electrode, diameter 3mm；With a reference electrode：Ag/AgCl electrode, each electrode pair should be connected with a contact conductor, should
Electrode electro Chemical stable performance, homogeneity is good, and in following description, this screen printing electrode is abbreviated as SPCE.
Methods described is it is characterised in that be adapted to health check-up using the Ratio-type based on screen printing electrode of present invention preparation
The sensor surveying tetracycline has that simple to operate, with low cost, detection sensitivity is high, degree of accuracy is high, reduces difference etc. between batch
The advantage and response time is short, sample and reagent consumption are few, and stability is high, can be used for the Site Detection of actual sample, meets me
State's tetracycline residue Fast Detection Technique development and internationalization require.
Brief description
Fig. 1 ferrocene-aptamers, carbon nano-fiber are NCFs, carbon nano-fiber-nanometer gold is that NCFs-AuNPs is combined
The scanning electron microscope (SEM) photograph of thing：A. under low range ferrocene-adaptor complex scanning electron microscope (SEM) photograph；B. ferrocene under high magnification-suitable
The scanning electron microscope (SEM) photograph of ligand complex；C. the scanning electron microscope (SEM) photograph of carbon nano-fiber；D. the scanning electricity of carbon nano-fiber-nanometer gold
Mirror figure；
0.1 is contained during Fig. 2 nanometer gold-shitosan/ferrocene-aptamers/bovine serum albumin/tetracycline sensor assembling
Cyclic voltammetric phenogram in the phosphate buffer of the potassium ferricyanide of mol/L KCl and 5 mmol/L：a）Empty silk screen printing electricity
Pole is SPCE；b）Nanometer gold-shitosan/SPCE；C) ferrocene-aptamers/nanometer gold-shitosan/SPCE；d）Sanguis Bovis seu Bubali is pure
Albumen/ferrocene-aptamers/nanometer gold-shitosan/SPCE；e）Tetracycline/bovine serum albumin/ferrocene-aptamers/receive
Meter Jin-shitosan/SPCE；
0.1 is contained during Fig. 3 carbon nano-fiber/nanometer gold/aptamers/bovine serum albumin/tetracycline sensor assembling
Cyclic voltammetric phenogram in the phosphate buffer of the potassium ferricyanide of mol/L KCl and 5 mmol/L：a）Empty silk screen printing electricity
Pole；b）Carbon nano-fiber/SPCE；C) nanometer gold/carbon nano-fiber/SPCE；d）Aptamers/nanometer gold/Nano carbon fibers/SPCE；
e）Serum albumin/aptamers/nanometer gold/carbon nano-fiber/SPCE；f）Tetracycline/bovine serum albumin/nanometer gold/nanometer
Carbon fiber/SPCE；
The impact that Fig. 4 bottom liquid pH value responds to sensor current；
Fig. 5 is adapted to the impact that bulk concentration responds to sensor current；
The impact that Fig. 6 brooding time responds to sensor current；
Fig. 7 nanometer gold-shitosan/ferrocene-aptamers/bovine serum albumin/tetracycline sensor is by variable concentrations tetracycline
Differential pulse voltammetry curve after incubation, 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；
Fig. 8 carbon nano-fiber/nanometer gold/aptamers/bovine serum albumin/tetracycline sensor is incubated by variable concentrations tetracycline
Differential pulse voltammetry curve after educating, 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；h.10-9g/mL；i.10-10g/mL；j. 10-11g/mL；
Two kinds of sensors of Fig. 9 be incubated tetracycline respectively after the logarithm of curent change and tetracycline concentration linear relationship；
Two kinds of sensors of Figure 10 be incubated tetracycline respectively after the logarithm of curent change ratio and tetracycline concentration linear pass
System；
This Ratio-type aptamer sensor detection to antibiotic recovery of standard addition in actual sample of Figure 11.
Specific embodiment
With reference to embodiment, the present invention will be further described, but the present invention should not be limited by the examples.
A kind of preparation process based on screen printing electrode Ratio-type aptamer sensor of embodiment 1：
1）Nanometer gold/nanometer gold-chitosan complexes, carbon nano-fiber, the preparation of ferrocene-aptamers：100 mL 0.01%
Gold chloride be added drop-wise in beaker, be placed on electric furnace heating, heat while stirring until boiling, be then rapidly added 2.5 mL
1% sodium citrate solution, this solution of carrying out with reaction has quickly become ruby color, and the golden nanometer particle indicating is described
Formation；It is stirred vigorously after this solution continues 1 hour, obtain prepared nano-Au solution；Weighing 0.5 g shitosan is that CS puts
In beaker, add the acetum stirring and dissolving of 1.0 %, the solution having dissolved be placed in 250 mL volumetric flasks and constant volume,
Solution after constant volume is poured in beaker, magnetic agitation 10 h under magnetic stirring apparatuss, obtains 0.2% chitosan solution；By 20
The shitosan acetic acid solution stirring of mL 1% is added in above-mentioned nano-Au solution and obtains nanometer gold-chitosan complexes；1 g bis-
Luxuriant ferrum is added to ultrasonic 30 min in 100 mL ethanol solution, obtains 1% solution of ferrocene, is then added to adaptation liquid solution
In solution of ferrocene, stirring and evenly mixing 12 hours at 4 DEG C, obtain ferrocene-adaptor complex；
2）The cleaning of screen printing electrode, activation：First, screen printing carbon electrode is put into the little burning filling 1mM NaOH solution
It is cleaned by ultrasonic 5 minutes in cup, ultra-pure water cleans, and nitrogen dries up, and then, puts the electrodes into the small beaker filling 1mM HCl solution
Middle ultrasonic cleaning 5 minutes, ultra-pure water cleans, and nitrogen dries up, and afterwards, uses washes of absolute alcohol electrode, nitrogen dries up, finally,
Carry out current versus time curve scanning 300s in the phosphate buffer of pH 5.0, be circulated volt-ampere curve scanning afterwards, 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
On the screen printing electrode that polysaccharide is modified, 7 μ L are adapted to the silk screen that liquid solution is added drop-wise to carbon nano-fiber/decorated by nano-gold
On printing electrode, it is dried at room temperature for, obtains two kinds of aptamers biosensors, and the electrode preparing is put in 4 DEG C of dryings
Environment in save backup.
Electrochemical Characterization in embodiment 2 aptamers biosensor assembling process
1）With scanning electron microscope to being modified with ferrocene-aptamers, carbon nano-fiber, carbon nano-fiber-nanometer gold
The micro-structure diagram of screen printing electrode is characterized, as shown in Figure 1 it can be seen that nano material successfully modifies electrode table
Face；
2）In nanometer gold-shitosan/ferrocene-aptamers/bovine serum albumin/tetracycline assembling process, Different electrodes are containing
Cyclic voltammetry curve in the phosphate buffer of the potassium ferricyanide of 0.1 mol/L KCl and 5 mmol/L, as shown in Fig. 2 figure
Middle curve a）It is the phenogram of sky screen printing electrode, we can see that obvious redox peaks；As in figure curve b）Institute
Show, after upper nanometer gold-chitosan nano-material is modified on screen printing electrode, because nanometer gold has good electric conductivity,
Therefore electric current has increased than empty screen printing electrode；As curve c）Shown, modified ferrocene-aptamers on this basis again
Afterwards, because ferrocene also has electric conductivity, electric current increases again；After fixing bovine serum albumin 7 μ L, because it is
Macro-molecular protein, it is not only non-conductive, but also can hinder the electron transmission at interface, so current peak diminishes, such as curve
d）Shown；
3）In carbon nano-fiber/nanometer gold/aptamers/bovine serum albumin/tetracycline assembling process, Different electrodes are containing 0.1
Cyclic voltammetry curve in the phosphate buffer of the potassium ferricyanide of mol/L KCl and 5 mmol/L, as shown in Fig. 2 in figure is bent
Line a）It is the phenogram of sky screen printing electrode, we can see that obvious redox peaks；As in figure curve b）Shown, when
After Nano carbon fibers dimension nano material is modified on screen printing electrode, because carbon nano-fiber has good electric conductivity, therefore
Electric current has increased than empty screen printing electrode；As curve c）Shown, after again having modified nanometer gold on this basis, electric current is again
Increase；After fixed adaptation body 7 μ L, because aptamers are protein molecules, it is not only non-conductive, but also can hinder interface
Electron transmission, so current peak diminishes, such as curve d）Shown, this also demonstrates aptamers and has successfully been fixed to electrode
Surface.
The optimization of embodiment 3 experimental condition
1）The optimization of pH value
The difference of test bottom liquid pH value, has different impacts to the activity of aptamers, and then can affect the sensitivity of sensor, institute
So that this experiment is prepared for a series of phosphate buffer of pH value, and pH value is respectively 6.0,6.5,7.0,7.5,8.0, and difference
It has been made into a series of detection bottom liquid；Fig. 4 is shown that two kinds of sensors aptamers in the liquid of different pH value bottoms are incubated with tetracycline
Educate the size of the current differential of the cyclic voltammetry carrying out in front and back.It can be seen that when pH value is 7.0, difference
Maximum, this shows, pH 7.0 is the optimal ph of this sensor, and now, aptamers can preferably play activity；
2）The optimization of adaptation bulk concentration
In order to reduce the waste of aptamers in experiment, make the performance of aptamer sensor more superior, to aptamer sensor
Some experiment conditions are optimized, and the electrode modified is immersed in the adaptation liquid solution of variable concentrations, make aptamers solid
Determine on electrode, then with preparing sensor, the tetracycline of same concentrations is detected, using the side of differential pulse voltammetry
Method measures the change of peak current.Can significantly find out from Fig. 5, the value of Δ I constantly increases with the increase of adaptation bulk concentration
Greatly.After concentration is more than 6 μM, the value of Δ I substantially remains in stable state.This phenomenon shows, aptamers in this experiment
Concentration takes 6 μM of surfaces having covered electrode enough, and aptamers reach saturation with the specific binding of tetracycline.Therefore, 6 μM
Aptamers be optimal concentration；
3）The optimization of brooding time
Incubation time is an important criteria weighing sensor performance, in order to determine optimal incubation time, will prepare
Aptamer sensor all Deca same concentrations tetracycline, allow its reaction different time, and measure the Δ I under different time
The change of value, as shown in fig. 6, the value of Δ I increases over time and constantly increases, however, the time is more than Δ I after 60 min
Value no longer changes over and changes, and is held in a stable level, and this is mainly secured to the aptamers on electrode
The tetracycline catching has had arrived at saturation, so, optimal incubation time selects 60 min.
The application of the Ratio-type aptamer sensor prepared by embodiment 4
1）The linear relationship of the logarithm of the curent change ratio after incubation tetracycline and tetracycline concentration
Configure a series of tetracycline standard solution of concentration, two kinds of aptamer sensor are carried out to the tetracycline of variable concentrations
Differential pulse voltammetry scanning is Fig. 7, Fig. 8, and establishes the different tetracycline concentration of foundation and the change of screen printing electrode electric current respectively
Relation curve between change is Fig. 9, and then to obtain the logarithm value of tetracycline of variable concentrations and curent change ratio be △I CNFs/
△I FcBetween equation of linear regression：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, i.e. Figure 10；
2）Antibiotic remainss in detection actual sample milk
Buy milk in local supermarket, milk according to 1:10 ratio is diluted, and is then dispensed in centrifuge tube, with
20000 revolutions per seconds of centrifugation 90min.After centrifugation terminates, milk is divided into obvious three layers, and upper and lower layer is fat and casein
Deng macromolecular substances, in order to avoid the parcel of macromolecular complex confrontation tetracycline, we go middle one layer of milk surum, collect milk surum,
Add 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；In optimal conditions milk sample is detected, in mark-on sample, the concentration of tetracycline is calculated according to calibration trace
Go out, its response rate can reach 95.98%-104.28%, as shown in figure 11.
Although the present invention is open as above with preferred embodiment, it is not limited to the present invention, any is familiar with this
The people of technology, without departing from the spirit and scope of the present invention, can do various changes and modify, the therefore protection of the present invention
Scope should be by being defined that claims are defined.
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.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727714A (en) * | 2017-09-10 | 2018-02-23 | 福建师范大学 | One kind is based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material |
| CN110146699A (en) * | 2019-05-31 | 2019-08-20 | 西北大学 | A kind of bi-component Ratio-type electrochemical immunosensor and preparation method thereof |
| CN110208343A (en) * | 2019-05-10 | 2019-09-06 | 江苏大学 | A kind of preparation method for the ratio electrochemica biological sensor detecting aflatoxin B1 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965766A (en) * | 2012-11-14 | 2013-03-13 | 同济大学 | New method for synthesizing nanometal particle-loaded carbon nanofiber |
| CN103792372A (en) * | 2014-02-20 | 2014-05-14 | 南京大学 | Dinucleotide-labelled ratio electrochemical immunosensor |
| KR20140098436A (en) * | 2013-01-31 | 2014-08-08 | 인하대학교 산학협력단 | Ratiometric fluorescent chemosensor including boronic acid binding to mercury ion selectively, preparation method thereof and detection method of mercury ion using the same |
| CN104502585A (en) * | 2014-12-30 | 2015-04-08 | 中山大学 | Nano-sensor for detecting antibiotics and preparation method and application of nano-sensor |
| CN104865216A (en) * | 2015-06-10 | 2015-08-26 | 常熟理工学院 | Method for colorimetrically detecting chloramphenicol based on nano-gold nucleic acid aptamer |
-
2016
- 2016-12-22 CN CN201611199136.5A patent/CN106442665B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102965766A (en) * | 2012-11-14 | 2013-03-13 | 同济大学 | New method for synthesizing nanometal particle-loaded carbon nanofiber |
| KR20140098436A (en) * | 2013-01-31 | 2014-08-08 | 인하대학교 산학협력단 | Ratiometric fluorescent chemosensor including boronic acid binding to mercury ion selectively, preparation method thereof and detection method of mercury ion using the same |
| CN103792372A (en) * | 2014-02-20 | 2014-05-14 | 南京大学 | Dinucleotide-labelled ratio electrochemical immunosensor |
| CN104502585A (en) * | 2014-12-30 | 2015-04-08 | 中山大学 | Nano-sensor for detecting antibiotics and preparation method and application of nano-sensor |
| CN104865216A (en) * | 2015-06-10 | 2015-08-26 | 常熟理工学院 | Method for colorimetrically detecting chloramphenicol based on nano-gold nucleic acid aptamer |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727714A (en) * | 2017-09-10 | 2018-02-23 | 福建师范大学 | One kind is based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material |
| CN107727714B (en) * | 2017-09-10 | 2019-06-28 | 福建师范大学 | One kind being based on carbon nanohorn and TiO2The preparation method of the Ratio-type electrochemical luminescence immunosensor of mesomorphic nano material |
| CN110208343A (en) * | 2019-05-10 | 2019-09-06 | 江苏大学 | A kind of preparation method for the ratio electrochemica biological sensor detecting aflatoxin B1 |
| CN110208343B (en) * | 2019-05-10 | 2021-04-20 | 江苏大学 | Preparation method of ratiometric electrochemical biosensor for detecting aflatoxin B1 |
| CN110146699A (en) * | 2019-05-31 | 2019-08-20 | 西北大学 | A kind of bi-component Ratio-type electrochemical immunosensor and preparation method thereof |
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