CN106442665B - A kind of preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue - Google Patents
A kind of preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue Download PDFInfo
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Abstract
The invention discloses a kind of preparation methods of Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, belong to agricultural product security detection technique field.The present invention is that nanogold-chitosan, the carbon nano-fiber-nano-Au composite prepared is modified on the screen printing electrode surface after cleaning activation respectively, then ferrocene-aptamers, aptamers are added dropwise respectively on the electrode modified, obtain the Ratio-type aptamer sensor of detection antibiotic using ferrocene and carbon nano-fiber as probe.The transducer production method is using tetracycline as template, the concentration for quantifying tetracycline by calculating the curent change before and after the sensor contacts tetracycline, the transducer sensitivity is high, accuracy is high, can reduce difference between batch, therefore can be used for the detection of the antibiotic residues such as tetracycline in actual sample.
Description
Technical field
The present invention relates to a kind of systems of Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue
Preparation Method belongs to agricultural product security detection technique field.
Background technique
Antibiotic is a kind of physiological activator that can inhibit or kill other microbial cells, is mainly produced by microorganism
It is raw.Since the 1930s finds penicillin, nowadays it has been found that antibiosis is known as more than 2000 kinds.Often make in milk cow
Antibiotic mainly has aminoglycoside, beta-lactam, Tetracyclines and macrolides etc..If the mankind eat for a long time
After the animal food containing antibiotic residue, drug is constantly accumulated in vivo, can be generated toxic effect to human body, be increased bacterium
Drug resistance, cause the allergy and allergy of human body, or even carcinogenic, teratogenesis, mutagenesis can be generated.Although state in recent years
The residual maximum of antibiotic is strictly limited on border, but since it has growth stimulation to certain crops, so still having not
It is few violating the regulations using phenomenon, therefore realizing is vital to the detection of antibiotic.
Common three-electrode system is not readily portable, and impedance variations caused by biometric identification process are very in traditional electrode
Small, reactant easily is lost in the linear diffusion layer of the semo-infinite of electrode surface；Although tiny array electrode can will be in reaction process
The impedance variations of generation are amplified, but it is easily worn, deviation when causing to utilize again.Compared to traditional electrode, silk-screen printing electricity
It is extremely light and handy, it is easy to carry, can be disposable, there is good advantage, therefore screen printing electrode and biography for on-site test
The detection system of system is combined into the potential selection of tool.Traditional pesticide residue detection method has selectivity good, sensitive
Degree is high and accuracy is high, while detecting the advantage of multiple element or compound, but the instrument and equipment that its needs is expensive, before sample
Treatment process is cumbersome, time-consuming, and very high to the technical level requirement of analysis personnel, is unsuitable for field quick detection.Therefore originally
Literary trial prepares a kind of Ratio-type aptamer sensor of detection antibiotic residue based on two kinds of probes.
Summary of the invention
The purpose of the present invention is to provide a kind of defects that can overcome the above method, and high sensitivity, specificity are high, integrated
Change, the Ratio-type aptamer sensor detection method of the antibiotic element residue detection of portability.The technical solution of use are as follows: utilize
Integrated, the portability of screen printing electrode, using ferrocene, i.e. two kinds of Fc, carbon nano-fiber, that is, NCFs probes construct respectively
Corresponding aptamer sensor constructs Ratio-type sensor, on the basis of two kinds of pedestal sensors to reach high sensitivity, essence
Exactness is high, reduces the testing goal of difference between batch.By the specific reaction between antigen and aptamers, detecting electrode surface
Current value variation, studies the chemical property of the sensor.
The step of the preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue
It is rapid as follows:
1) nanogold/nanogold-chitosan complexes, carbon nano-fiber, the preparation of ferrocene-aptamers；
2) cleaning activation screen printing electrode, obtains pretreated screen printing electrode；
3) nanogold-chitosan complexes and carbon nano-fiber/nanogold that step 1) is prepared are modified respectively and is arrived
On the pretreated screen printing electrode of step 2, the screen printing electrode modified is obtained；
4) ferrocene-aptamers, aptamers are added drop-wise to the resulting screen printing electrode modified of step 3) respectively
On, the basic aptamer sensor based on screen printing electrode is obtained after natural drying；
5) Optimization Steps 4) resulting basic aptamer sensor three kinds of experimental conditions；
6) under the resulting optimal conditions of step 5), the antibiotic such as tetracycline are detected.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is that nanogold-chitosan complexes, carbon nano-fiber described in step 1), ferrocene-aptamers are to be respectively with chitosan
Dispersing agent disperses nanogold, takes certain density carbon nano-fiber solution, ferrocene is mixed to get with aptamers and is uniformly dispersed
Suspension.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is that the modification of screen printing electrode electrode described in step 3) is respectively first by 7 μ L nanogold-chitosan complexes, 30%
Carbon nano-fiber solution and nano-Au solution are added drop-wise on pretreated screen printing electrode, are dried at room temperature, are respectively obtained and are received
Meter Jin-chitosan, carbon nano-fiber/decorated by nano-gold screen printing electrode.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is, 7 μ L ferrocene-adaptor complex, adaptation liquid solution is added dropwise described in step 4) respectively on the electrode modified,
It is that 7 μ L ferrocene-adaptor complex is added drop-wise on the chitosan-modified good screen printing electrode of nanogold-, by 7 μ L
Adaptation liquid solution is added drop-wise on carbon nano-fiber/decorated by nano-gold screen printing electrode, dry under the conditions of 4 DEG C, obtains two
Kind aptamers biosensor.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is that three kinds of experimental conditions test bottom liquid pH value of the described two aptamers biosensors of step 5), is incubated at adaptation bulk concentration
Educate the time is optimized respectively: pH value 7.0, and adaptation bulk concentration is 6 μM, and incubation time is 60 min.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is, the tetracycline titer of various concentration is added dropwise described in step 6), is incubated for 60 min, cyclic voltammetric is carried out in the liquid of bottom
Method detection.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is, the specific steps are as follows:
1) nanogold/nanogold-chitosan complexes, carbon nano-fiber, the preparation of ferrocene-aptamers: 100 mL
The gold chloride that mass volume ratio is 0.01% is added drop-wise in beaker, is placed on electric furnace and is heated, and is heated while stirring until boiling, so
After be rapidly added 2.5 mL, 1% sodium citrate solution, as the solution that carries out of reaction has quickly become ruby color, explanation
The formation of the gold nanoparticle of instruction；It is vigorously stirred after the solution continues 1 hour, obtains prepared nano-Au solution；It weighs
0.5 g chitosan is placed in a beaker, and the acetum stirring and dissolving of 1.0 % is added, and the solution dissolved is placed in 250 mL and is held
In measuring bottle and constant volume, the solution after constant volume is poured into beaker, 10 h of magnetic agitation under magnetic stirring apparatus, obtain 0.2% shell it is poly-
Sugar juice；The chitosan acetic acid solution stirring of 20 mL 1% is added in above-mentioned nano-Au solution and obtains nanogold-chitosan
Compound；1 g ferrocene is added to 30 min of ultrasound in 100 mL ethanol solutions, obtains 1% solution of ferrocene, then will fit
Ligand solution is added in solution of ferrocene, is stirred and evenly mixed 12 hours at 4 DEG C, is obtained ferrocene-adaptor complex；
2) cleaning, activation of screen printing electrode: firstly, screen printing carbon electrode is put into, to fill 1mM sodium hydroxide molten
It is cleaned by ultrasonic 5 minutes in the small beaker of liquid, ultrapure water cleaning is dried with nitrogen, then, puts the electrodes into and fill 1mM hydrochloric acid solution
Small beaker in be cleaned by ultrasonic 5 minutes, ultrapure water cleaning, be dried with nitrogen, later use washes of absolute alcohol electrode, be dried with nitrogen,
Finally, carrying out current versus time curve in the phosphate buffer of pH 5.0 scans 300s, later, cyclic voltammetry curve is carried out
Scanning, until performance is stablized；
3) it is compound that 7 μ L nanogold-chitosan the modification of screen printing electrode: is added dropwise respectively on screen printing electrode
Object, 30% carbon nano-fiber solution and nano-Au solution are added drop-wise on pretreated screen printing electrode, are dried at room temperature, respectively
Obtain nanogold-chitosan, carbon nano-fiber/decorated by nano-gold screen printing electrode；
4) fixation of aptamers: 7 μ L ferrocene-adaptor complex is added dropwise on above-mentioned electrode and is added drop-wise to nanogold-
On chitosan-modified good screen printing electrode, 7 μ L adaptation liquid solution is added drop-wise to carbon nano-fiber/decorated by nano-gold silk
It on wire mark brush electrode, is dried at room temperature for, obtains two kinds of aptamers biosensors, and the electrode prepared is put in 4 DEG C and is done
It is saved backup in dry environment；
5) optimization of experimental condition: preparing a series of phosphate buffer of pH value, pH value is respectively 6.0,6.5,7.0,
7.5,8.0, and it has been made into a series of detection bottom liquid respectively, sensor current value is detected in these bottom liquid, is screened
Optimal pH 7.0 out；Respectively to 2 μM of electrode load, 4 μM, 5 μM, 6 μM, 8 μM of aptamers, its current value is examined
It surveys, filtering out best adaptation bulk concentration is 6 μM；30 min, 40 are controlled as with the tetracycline incubation time of same concentration
Min, 50 min, 60 min, 70 min, 80 min, 90 min, detect its current value, filter out best incubation time
For 60 min；
6) detection of tetracycline: in optimal conditions: pH 7.0 is adapted to 6 μM of bulk concentration, 60 min of incubation time,
Current detecting is carried out to the tetracycline of various concentration in two kinds of aptamer sensors, and establishes that establish different tetracyclines dense respectively
Relation curve between degree and screen printing electrode curent change, and then obtain the logarithm and electric current of the tetracycline of various concentration
Equation of linear regression between peak ratio, in concentration range 10-11~10-9Y=- 0.02854x-is obtained in g/mL
0.02655, related coefficient 0.994；In concentration range 10-9~10-3Obtained in g/mL y=- 0.00225x+
0.20538, related coefficient 0.997.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is that the specific detecting step of the antibiotic residues such as tetracycline is as follows in milk:
1) pre-treatment of milk sample: milk is bought in local supermarket, milk is diluted according to the ratio of 1:10, so
After be dispensed into centrifuge tube, 90min is centrifuged with 20000 revolutions per seconds of speed；After centrifugation, milk is divided into apparent three layers,
Upper and lower layer is the macromolecular substances such as fat and casein, and in order to avoid macromolecular substances are to the package of antibiotic, we go to centre
One layer of whey collects whey, and it is 5 × 10 respectively that antibiotic, the concentration such as tetracycline are added into the whey collected-10 g/
ML, 5 × 10-9G/mL, 5 × 10-8G/mL, 5 × 10-7g/mL；
2) detection of sample: in optimal conditions: pH 7.0 is adapted to 6 μM of bulk concentration, 60 min of incubation time, to ox
Milk sample is detected；
3) according to the corresponding linear relationship of foundation, the antibiotic residual quantities such as the tetracycline of respective sample are calculated.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is, for detecting the antibiotic residues such as tetracycline.
The preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, it is special
Sign is that electrode used therein is screen printing electrode, substrate, base including a printed electrode external insulation and three printed on chip
Root contact conductor, it is characterised in that also there are three electrode, a working electrodes: carbon electrode for printing on the substrate；One right
Electrode: carbon electrode, diameter 3mm；With a reference electrode: Ag/AgCl electrode, each electrode have been correspondingly connected with a contact conductor, should
Electrode electro Chemical performance is stablized, and homogeneity is good, and in following description, which is abbreviated as SPCE.
The method, which is characterized in that physical examination is adapted to using the Ratio-type prepared by the present invention based on screen printing electrode
The sensor of tetracycline is surveyed with easy to operate, low in cost, detection sensitivity is high, accuracy is high, difference etc. between reduction batch
The advantage and reaction time is short, sample and reagent consumption are few, and stability is high, can be used for the on-site test of actual sample, meet me
The development of state's tetracycline residue Fast Detection Technique and internationalization require.
Detailed description of the invention
Fig. 1 ferrocene-aptamers, carbon nano-fiber, that is, NCFs, carbon nano-fiber-nanogold, that is, NCFs-AuNPs are compound
The scanning electron microscope (SEM) photograph of object: ferrocene-adaptor complex scanning electron microscope (SEM) photograph under A. low range；B. ferrocene-is suitable under high magnification
The scanning electron microscope (SEM) photograph of ligand complex；C. the scanning electron microscope (SEM) photograph of carbon nano-fiber；D. carbon nano-fiber-nanogold scanning electricity
Mirror figure；
Contain in Fig. 2 nanogold-chitosan/ferrocene-aptamers/bovine serum albumin(BSA)/tetracycline sensor assembling process
Cyclic voltammetric phenogram in the phosphate buffer of the potassium ferricyanide of 0.1 mol/L KCl and 5 mmol/L: a) empty screen printing
Brush electrode, that is, SPCE；B) nanogold-chitosan/SPCE；C) ferrocene-aptamers/nanogold-chitosan/SPCE；D) ox blood
Pure albumen/ferrocene-aptamers/nanogold-chitosan/SPCE；E) tetracycline/bovine serum albumin(BSA)/ferrocene-adaptation
Body/nanogold-chitosan/SPCE；
Contain 0.1 in Fig. 3 carbon nano-fiber/nanogold/aptamers/bovine serum albumin(BSA)/tetracycline sensor assembling process
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) nanogold/carbon nano-fiber/SPCE；D) aptamers/nanogold/Nano carbon fibers/SPCE；
E) seralbumin/aptamers/nanogold/carbon nano-fiber/SPCE；F) tetracycline/bovine serum albumin(BSA)/nanogold/nanometer
Carbon fiber/SPCE；
The influence that the bottom Fig. 4 liquid pH value responds sensor current；
The influence that Fig. 5 adaptation bulk concentration responds sensor current；
The influence that Fig. 6 brooding time responds sensor current；
Fig. 7 nanogold-chitosan/ferrocene-aptamers/bovine serum albumin(BSA)/tetracycline sensor is by various concentration four
Differential pulse voltammetry curve after the incubation of ring element, 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/nanogold/aptamers/bovine serum albumin(BSA)/tetracycline sensor is by various concentration Fourth Ring
Differential pulse voltammetry curve after element incubation, 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 are incubated for the linear relationship of the logarithm of the curent change after tetracycline and tetracycline concentration respectively；
Two kinds of sensors of Figure 10 are incubated for the linear of the logarithm of the curent change ratio after tetracycline and tetracycline concentration respectively
Relationship；
Figure 11 detection of the Ratio-type aptamer sensor to antibiotic recovery of standard addition in actual sample.
Specific embodiment
Below 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 step based on screen printing electrode Ratio-type aptamer sensor of embodiment 1:
1) nanogold/nanogold-chitosan complexes, carbon nano-fiber, the preparation of ferrocene-aptamers: 100 mL
0.01% gold chloride is added drop-wise in beaker, is placed on electric furnace and is heated, and is heated while stirring until boiling, is then rapidly added 2.5
1% sodium citrate solution of mL illustrates the gold nano of instruction as the solution that carries out of reaction has quickly become ruby color
The formation of particle；It is vigorously stirred after the solution continues 1 hour, obtains prepared nano-Au solution；Weigh 0.5 g chitosan i.e.
CS is placed in a beaker, and the acetum stirring and dissolving of 1.0 % is added, and the solution dissolved is placed in 250 mL volumetric flasks and is determined
Hold, the solution after constant volume pours into beaker, and 10 h of magnetic agitation under magnetic stirring apparatus obtains 0.2% chitosan solution；It will
The chitosan acetic acid solution stirring of 20 mL 1%, which is added in above-mentioned nano-Au solution, obtains nanogold-chitosan complexes；1
G ferrocene is added to 30 min of ultrasound in 100 mL ethanol solutions, obtains 1% solution of ferrocene, then adds adaptation liquid solution
Enter into solution of ferrocene, is stirred and evenly mixed at 4 DEG C 12 hours, obtain ferrocene-adaptor complex；
2) cleaning, activation of screen printing electrode: 1mM NaOH solution is filled firstly, screen printing carbon electrode is put into
It is cleaned by ultrasonic 5 minutes in small beaker, ultrapure water cleaning is dried with nitrogen, then, puts the electrodes into and fill the small of 1mM HCl solution
It is cleaned by ultrasonic 5 minutes in beaker, ultrapure water cleaning is dried with nitrogen, later, with washes of absolute alcohol electrode, is dried with nitrogen, most
Afterwards, current versus time curve is carried out in the phosphate buffer of pH 5.0 and scan 300s, carry out cyclic voltammetry curve later and sweep
It retouches, until performance is stablized；
3) it is compound that 7 μ L nanogold-chitosan the modification of screen printing electrode: is added dropwise respectively on screen printing electrode
Object, 30% carbon nano-fiber solution and nano-Au solution are added drop-wise on pretreated screen printing electrode, are dried at room temperature, respectively
Obtain nanogold-chitosan, carbon nano-fiber/decorated by nano-gold screen printing electrode；
4) fixation of aptamers: 7 μ L ferrocene-adaptor complex is added dropwise on above-mentioned electrode and is added drop-wise to nanometer
On gold-chitosan-modified good screen printing electrode, 7 μ L adaptation liquid solution is added drop-wise to carbon nano-fiber/decorated by nano-gold
Screen printing electrode on, be dried at room temperature for, obtain two kinds of aptamers biosensors, and the electrode prepared is put in 4
It is saved backup in DEG C dry environment.
Electrochemical Characterization in 2 aptamers biosensor assembling process of embodiment
1) with scanning electron microscope to being modified with ferrocene-aptamers, carbon nano-fiber, carbon nano-fiber-nanometer
The micro-structure diagram of the screen printing electrode of gold is characterized, as shown in Figure 1, it can be seen that nano material successfully modifies electrode
Surface；
2) Different electrodes exist in nanogold-chitosan/ferrocene-aptamers/bovine serum albumin(BSA)/tetracycline assembling process
Cyclic voltammetry curve in the phosphate buffer of the potassium ferricyanide containing 0.1 mol/L KCl and 5 mmol/L, as shown in Fig. 2,
Curve a) is the phenogram of sky screen printing electrode in figure, we can see that apparent redox peaks；Curve b) in such as figure
It is shown, after modifying upper nanogold-chitosan nano-material on screen printing electrode, since nanogold has good conduction
Property, therefore electric current is increased than empty screen printing electrode；As shown in curve c), it is suitable ferrocene-has been modified again on this basis
After ligand, because ferrocene is also conductive, electric current increases again；After fixed 7 μ L of bovine serum albumin(BSA), due to
It is macro-molecular protein, it is not only non-conductive, but also can hinder the electron transmission at interface, so current peak becomes smaller, such as
Shown in curve d)；
3) Different electrodes are containing in carbon nano-fiber/nanogold/aptamers/bovine serum albumin(BSA)/tetracycline assembling process
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) is the phenogram of sky screen printing electrode, we can see that apparent redox peaks；Such as curve b) institute in figure
Show, after modifying upper Nano carbon fibers dimension nano material on screen printing electrode, since carbon nano-fiber has good electric conductivity,
Therefore electric current is increased than empty screen printing electrode；As shown in curve c), electric current after nanogold has been modified again on this basis
Increase again；After 7 μ L of fixed adaptation body, since aptamers are protein molecules, it is not only non-conductive, but also can hinder
The electron transmission at interface, so current peak becomes smaller, as shown in curve d), this also demonstrates aptamers and is successfully fixed to
Electrode surface.
The optimization of 3 experimental condition of embodiment
1) optimization of pH value
The difference for testing bottom liquid pH value, has different influences to the activity of aptamers, and then will affect the sensitive of sensor
Degree, so, 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
It has been made into a series of detection bottom liquid respectively；Two kinds of sensors aptamers and Fourth Ring in the liquid of different pH value bottoms is shown in Fig. 4
Element is incubated for the size of the current differential for the cyclic voltammetry that front and back carries out.It can be seen from the figure that when pH value is 7.0,
Difference is maximum, this shows that pH 7.0 is the optimal ph of the sensor, at this point, aptamers can preferably play activity；
2) it is adapted to the optimization of bulk concentration
In order to reduce the waste of aptamers in experiment, makes the performance of aptamer sensor more superior, aptamers are sensed
Some experiment conditions of device are optimized, and the electrode modified is immersed in the adaptation liquid solution of various concentration, allow adaptation
Body is fixed on electrode, is then detected to the tetracycline of same concentrations with preparing sensor, is used differential pulse voltammetry
Method measurement peak current variation.Can significantly it find out from Fig. 5, the value of Δ I is continuous with the increase of adaptation bulk concentration
Ground increases.After concentration is greater than 6 μM, the value of Δ I substantially remains in stable state.This phenomenon shows to be adapted in this experiment
The concentration of body takes 6 μM of surfaces for having covered electrode enough, and the specific binding of aptamers and tetracycline reaches saturation.Therefore,
6 μM of aptamers are optimal concentration；
3) optimization of brooding time
Incubation time is that an important criteria of measurement sensor performance will be made to determine optimal incubation time
The tetracycline of same concentrations is all added dropwise in the aptamer sensor got ready, allows it to react the different time, and measure under different time
Δ I value variation, as shown in fig. 6, the value of Δ I constantly increases as time increases, however, after the time is greater than 60 min
The value of Δ I no longer changes over time and changes, and is held in a stable level, this is mainly fixed to suitable on electrode
The tetracycline that ligand captures has had arrived at saturation, so, optimal incubation time selects 60 min.
The application of Ratio-type aptamer sensor prepared by embodiment 4
1) linear relationship of the logarithm of curent change ratio and tetracycline concentration after being incubated for tetracycline
A series of tetracycline standard solution for configuring concentration, to the tetracycline of various concentration in two kinds of aptamer sensors
Carrying out differential pulse voltammetry scanning is Fig. 7, Fig. 8, and establishes establish different tetracycline concentrations and screen printing electrode electricity respectively
Relation curve, that is, Fig. 9 between rheology, and then obtain the logarithm and curent change ratio i.e. △ of the tetracycline of various concentrationI CNFs/△I FcBetween equation of linear regression: in concentration range 10-11~10-9Y=- 0.02854x-is obtained in g/mL
0.02655, related coefficient 0.994；In concentration range 10-9~10-3Obtained in g/mL y=- 0.00225x+
0.20538, related coefficient 0.997, i.e. Figure 10；
2) antibiotic residue in actual sample milk is detected
Milk is bought in local supermarket, milk is diluted according to the ratio of 1:10, is then dispensed into centrifuge tube, with
20000 revolutions per seconds of speed is centrifuged 90min.After centrifugation, milk is divided into apparent three layers, and upper and lower layer is fat and casein
Equal macromolecular substances, in order to avoid the package of macromolecular complex confrontation tetracycline, we go intermediate one layer of whey, collect whey,
Tetracycline is added into the whey collected, concentration is 5 × 10 respectively-10G/mL, 5 × 10-9G/mL, 5 × 10-8G/mL, 5 ×
10-7g/mL；Milk sample is detected in optimal conditions, the concentration of tetracycline is calculated according to calibration curve in mark-on sample
Out, the rate of recovery can achieve 95.98%-104.28%, as shown in figure 11.
Although the present invention has been disclosed in the preferred embodiment as above, it is not intended to limit the invention, any to be familiar with this
The people of technology can do various changes and modification, therefore protection of the invention without departing from the spirit and scope of the present invention
Range should subject to the definition of the claims.
Claims (9)
1. a kind of preparation method of the Ratio-type aptamer sensor based on screen printing electrode detection antibiotic residue, feature
It is, is that nanogold-chitosan, the Nano carbon fibers prepared are modified on the screen printing electrode surface after cleaning activation respectively
Then ferrocene-aptamers, aptamers are added dropwise, respectively on the electrode modified with ferrocene and nano-sized carbon in peacekeeping nanogold
Fiber obtains the Ratio-type aptamer sensor of detection antibiotic respectively as probe.
2. the system of the Ratio-type aptamer sensor according to claim 1 based on screen printing electrode detection antibiotic residue
Preparation Method, which is characterized in that electrode used therein is screen printing electrode, printed on chip outer of substrate, base including a printed electrode
Portion's insulating layer and each of three electrodes lead, it is characterised in that also there are three electrode, a working electrodes: carbon for printing on the substrate
Electrode, diameter 3mm；One to electrode；An electrode has been correspondingly connected with a reference electrode: Ag/AgCl electrode, each electrode
Lead, the electrode electro Chemical performance are stablized, and homogeneity is good.
3. the system of the Ratio-type aptamer sensor according to claim 1 based on screen printing electrode detection antibiotic residue
Preparation Method, which is characterized in that steps are as follows:
1) nanogold, nanogold-chitosan complexes, carbon nano-fiber, the preparation of ferrocene-aptamers；
2) cleaning activation screen printing electrode, obtains pretreated screen printing electrode；
3) nanogold-chitosan complexes and carbon nano-fiber/nanogold that step 1) is prepared are modified respectively to step
2) on pretreated screen printing electrode, the screen printing electrode modified is obtained；
4) ferrocene-aptamers, aptamers are added drop-wise to respectively on the resulting screen printing electrode modified of step 3), it is natural
The basic aptamer sensor based on screen printing electrode is obtained after drying；
5) Optimization Steps 4) resulting basic aptamer sensor three kinds of experimental conditions；
6) under the resulting optimal conditions of step 5), tetracycline is detected.
4. the system of the Ratio-type aptamer sensor according to claim 3 based on screen printing electrode detection antibiotic residue
Preparation Method, which is characterized in that nanogold-chitosan complexes, carbon nano-fiber described in step 1), ferrocene-aptamers system
Back-up be not using chitosan as dispersing agent disperse nanogold, take certain density carbon nano-fiber solution, by ferrocene be adapted to
Body is mixed to get finely dispersed suspension.
5. the system of the Ratio-type aptamer sensor according to claim 3 based on screen printing electrode detection antibiotic residue
Preparation Method, which is characterized in that the modification of screen printing electrode described in step 3) is first that 7 μ L nanogold-chitosan is compound respectively
Object, the carbon nano-fiber solution and nano-Au solution of 30 % are added drop-wise on pretreated screen printing electrode, dry at room temperature, point
Nanogold-chitosan, carbon nano-fiber/decorated by nano-gold screen printing electrode are not obtained.
6. the system of the Ratio-type aptamer sensor according to claim 3 based on screen printing electrode detection antibiotic residue
Preparation Method, which is characterized in that 7 μ L ferrocene-adaptation liquid solution is added dropwise on the electrode modified, is that 7 μ L ferrocene-are suitable
Ligand complex is added drop-wise on the chitosan-modified good screen printing electrode of nanogold-, and 7 μ L adaptation liquid solution is added drop-wise to and is received
It is dry under the conditions of 4 DEG C on rice carbon fiber/decorated by nano-gold screen printing electrode, obtain two kinds of aptamer sensors.
7. the system of the Ratio-type aptamer sensor according to claim 3 based on screen printing electrode detection antibiotic residue
Preparation Method, which is characterized in that the test bottom liquid pH value of aptamer sensor described in step 5), adaptation bulk concentration, incubation time point
Be not optimized: pH value 7.0, adaptation bulk concentration are 6 μM, and incubation time is 60 min.
8. the system of the Ratio-type aptamer sensor according to claim 3 based on screen printing electrode detection antibiotic residue
Preparation Method, which is characterized in that specific step is as follows:
1) nanogold, nanogold-chitosan complexes, carbon nano-fiber, the preparation of ferrocene-aptamers: 100mL mass volume
Than being added drop-wise in beaker for 0.01% gold chloride, it is placed on electric furnace and heats, heat while stirring until boiling, then adds rapidly
Enter 2.5 mL, 1% sodium citrate solution, as the solution that carries out of reaction has quickly become ruby color, illustrates the gold of instruction
The formation of nanoparticle is vigorously stirred after the solution continues 1 hour, obtains prepared nano-Au solution；Weigh 0.5 g shell
Glycan is placed in a beaker, and the acetum stirring and dissolving of 1.0 % is added, the solution dissolved is placed in 250 mL volumetric flasks
In and constant volume, the solution after constant volume pour into beaker, 10 h of magnetic agitation under magnetic stirring apparatus, the shell for obtaining 0.2 % is poly-
Sugar juice；The chitosan acetic acid solution stirring of 20ml 1% is added in above-mentioned nano-Au solution and obtains nanogold-chitosan
Compound；1 g ferrocene is added to 30 min of ultrasound in 100 mL ethanol solutions, obtains 1% solution of ferrocene, then will adaptation
Liquid solution is added in solution of ferrocene, is stirred and evenly mixed 12 hours at 4 DEG C, is obtained ferrocene-adaptor complex；
2) cleaning, activation of screen printing electrode: 1mM sodium hydroxide solution is filled firstly, screen printing carbon electrode is put into
It is cleaned by ultrasonic 5 minutes in small beaker, ultrapure water cleaning is dried with nitrogen, then, puts the electrodes into and fill the small of 1nM hydrochloric acid solution
It is cleaned by ultrasonic 5 minutes in beaker, ultrapure water cleaning is dried with nitrogen, later, with washes of absolute alcohol electrode, is dried with nitrogen, most
Afterwards, current versus time curve is carried out in the phosphate buffer of pH 5.0 and scan 300s, later, carry out cyclic voltammetry curve
Scanning, until performance is stablized；
3) modification of screen printing electrode, by 7 μ L nanogold-chitosan complexes, 30 % carbon nano-fiber solution and nanogold
Solution is added drop-wise to respectively on pretreated screen printing electrode, is dried at room temperature, and nanogold-chitosan, nano-sized carbon are respectively obtained
Fiber/decorated by nano-gold screen printing electrode；
4) 7 μ L ferrocene-adaptor complex the fixation of aptamers: is added drop-wise to the chitosan-modified silk-screen printing of nanogold-
On electrode, 7 μ L adaptation liquid solution is added drop-wise on carbon nano-fiber/decorated by nano-gold screen printing electrode, is done at room temperature
It is dry, two kinds of aptamers biosensors are obtained, and the electrode prepared is put in 4 DEG C of dry environment and is saved backup；
5) optimization of experimental condition, prepares a series of phosphate buffer of pH values, pH value is respectively 6.0,6.5,7.0,
7.5,8.0, it has been made into a series of detection bottom liquid respectively, sensor current value is detected in these bottom liquid, is filtered out
Best 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 examined
It surveys, filtering out best adaptation bulk concentration is 6 μM, is controlled as 30 min, 40 with the tetracycline incubation time of same concentration
Min, 50 min, 60min, 70 min, 80 min, 90 min, detect its current value, filter out best incubation time and are
60min；
6) detection of tetracycline: 7.0 pH in optimal conditions are adapted to 6 μM of bulk concentration, incubation time 60min, suitable at two kinds
With carrying out current detecting to the tetracycline of various concentration on body sensor, and different tetracycline concentrations and screen printing are established respectively
Relation curve between brush electrode curent change, so obtain the tetracycline of various concentration logarithm and current peak ratio it
Between equation of linear regression, in concentration range 10-11 ~10-9Y=- 0.02854x -0.02655, phase relation are obtained in g/ml
Number is 0.994；In concentration range 10-9~10-3Y=- 0.00225x+0.20538, related coefficient are obtained in g/ml
0.997。
9. the system of the Ratio-type aptamer sensor according to claim 1 based on screen printing electrode detection antibiotic residue
Preparation Method, which is characterized in that the specific detecting step of tetracycline residue is as follows in milk:
1) pre-treatment of milk sample is diluted in local supermarket purchase milk according to the ratio of 1:10, be then dispensed into from
In heart pipe, 90min is centrifuged with 20000 revolutions per seconds of speed, after centrifugation, milk is divided into apparent three layers, and upper and lower layer is big
Molecular substance fat and casein, in order to avoid macromolecular substances are to the package of antibiotic, we take intermediate one layer of whey, search
Collect whey, tetracycline is added into the whey collected, concentration is 5 × 10 respectively-10 G/mL, 5 × 10-9G/mL, 5
× 10-8G/mL, 5 x 10-7g/mL；
2) detection of sample is in optimal conditions, and: pH7.0 is adapted to 6 μM of bulk concentration, 60 min of incubation time, to milk sample into
Row detection；
3) according to the corresponding linear relationship established in claim 8 step 6), the Tetracycline Residues of respective sample are calculated.
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| 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 |
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| 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 |
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