CN104237344A - Electrochemical aptamer electrode for kanamycin detection and preparation method of electrochemical aptamer electrode - Google Patents

Electrochemical aptamer electrode for kanamycin detection and preparation method of electrochemical aptamer electrode Download PDF

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CN104237344A
CN104237344A CN201410442491.5A CN201410442491A CN104237344A CN 104237344 A CN104237344 A CN 104237344A CN 201410442491 A CN201410442491 A CN 201410442491A CN 104237344 A CN104237344 A CN 104237344A
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electrode
graphene
compound
preparation
complexes
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黄加栋
徐伟
王玉
刘素
崔敏
李�杰
王虹智
郭玉娜
许颖
崔杰
邱婷婷
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University of Jinan
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Abstract

The invention relates to the technical field of detection sensors and particularly relates to an electrochemical aptamer electrode for kanamycin detection. Graphene-polyaniline compound, polyamide-amine dendritic high molecule-gold nano compound, kanamycin anti-body are modified on a glass carbon electrode in sequence from bottom to up; the surface of the electrode is sealed with BSA (Bovine Serum Albumin) and single chain DNA (Deoxyribose Nucleic Acid) containing 10 adenine nucleotide. The preparation method is simple, stable in performance and good in electrode repeating performance and is suitable for practical applications of kanamycin detection in food safety and bio-sensor industrialization; the prepared electrode is low in technical cost and is suitable for the requirement of low cost in industrialization; according to a sandwich type electrochemical sensing system fixed by using the glass carbon electrode as a fixing carrier and based on a DNA aptamer, rapid and online kanamycin detection in food is realized and the detection limit is 4.6x10<-6>mu gmL<-1>.

Description

For fit electrode of galvanochemistry that kanamycins detects and preparation method thereof
technical field
The present invention relates to detecting sensor technical field, particularly a kind of fit electrode of galvanochemistry detected for kanamycins, also relates to the preparation method of the fit electrode of described galvanochemistry.
background technology
Kanamycins (Ka) is a kind of aminoglycoside antibiotics, is to be produced by the fermentation of streptomycete, is widely used in infection and the transposition between Profilin matter synthesis phase indirectly for the treatment of mistranslation induction.Consistent with other aminoglycosides, Ka can accumulate and transfer in food chain in animal body, and this may cause potential danger to human health, such as occurs losing hearing, Toxicity of Kidney and drug anaphylaxis.European Union's tissue has determined the maximum residue limit of Ka in edible tissues and milk.In this respect, the method clinical diagnosis of a sensitive high selectivity is established and in Food Safety Analysis, detect Ka be vital.The method of the detection Ka of current report comprises high performance liquid chromatography, capillary electrophoresis, surface plasma body resonant vibration method and fluorescence resonance method.These methods have testing cost high, and instrumentation is complicated, needs the shortcomings such as professional operator.
summary of the invention
High for testing cost in existing detection method in order to solve in above prior art, instrumentation is complicated, needs the shortcoming of professional operator, devises the fit electrode of galvanochemistry detected for kanamycins of the Novel sandwich configuration based on nucleic acid aptamer.
Present invention also offers the preparation method of the fit electrode of described galvanochemistry.
The present invention is obtained by following steps:
A kind of fit electrode of galvanochemistry detected for kanamycins, glass-carbon electrode is modified with graphene-polyanilinecompound compound, polyamidoamine dendrimer-golden nano-complexes, kanamycins antibody from bottom to top successively, last enclosed-electrode surface be the single stranded DNA of BSA and 10 adenine base.
Described galvanochemistry is fit electrode, the thickness of preferred graphene-polyanilinecompound compound is at 100 ± 10nm, the thickness of polyamidoamine dendrimer-golden nano-complexes is 100-200 nm, the thickness of kanamycins antibody is 1 ± 0.1 μm, the thickness of the single stranded DNA of BSA and 10 adenine base and be 500 ± 20nm.
Described galvanochemistry is fit electrode, in preferred graphene-polyanilinecompound compound, the mass ratio of Graphene and aniline is 3.3:100.
Described galvanochemistry is fit electrode, in preferred polyamide-amine type dendrimer-golden nano-complexes, polyamidoamine dendrimer is 1:3 with the mol ratio of gold.
The preparation method of described galvanochemistry is fit electrode, preferably includes following steps:
(1) process washing is carried out to glass-carbon electrode, Graphene-polyaniline nano compound is added drop-wise to electrode surface, dry under room temperature, rinse with intermediate water;
(2) to immerse in polyamidoamine dendrimer-golden nano-complexes 3 hours, rinse with intermediate water, dry;
(3) the PBS damping fluid of kanamycins antibody is dripped on surface, overnight at room temperature;
(4) with the bovine serum albumin(BSA) of 0.1% and single stranded DNA enclosed-electrode 2 h of 10 adenine bases, to obtain final product.
Described preparation method, preferred Graphene-polyaniline nano compound is obtained by following steps:
(1) dag obtains graphene oxide through peroxidating,
(2) graphene oxide prepares Graphene-polyaniline nano compound with aniline according to mass ratio 3.3:100.
Described preparation method, in preferred polyamide-amine type dendrimer-golden nano-complexes, polyamidoamine dendrimer is 1:3 with the mol ratio of gold.
Described preparation method, drips 10 μ L and is dissolved with 5 μm of ol L on surface in preferred steps (3) -1the PBS damping fluid of kanamycins antibody (KAb), overnight at room temperature.
Described preparation method, preferred Graphene-polyaniline nano compound is obtained by following steps: 2.75 mL aniline are dissolved in 100 mL 1 mol L -1hCl in obtain aniline concentration be 0.3 mol L -1, add 100 mg graphene oxides, after ultrasonic 1 h, 0.17g (NH 4) 2s 4o 8with 10 mL 1 mol L -1hCl adds mixed liquor and vigorous agitation fast, and when color becomes bottle green, polymerization starts, and after using redistilled water filtration washing several times, obtains graphene-polyanilinecompound compound; Weighing 0.1 g graphene-polyanilinecompound compound is dissolved in the ultrapure water of 50 mL, reduces 1 h with the hydrazine hydrate of 0.1 mL80% at 95 DEG C, the hydrazine hydrate that washing removing is excessive; Compound is dispersed in 10 mL and contains 1 mol L -1hCl and 0.06 g (NH 4) 2s 4o 8, stir under room temperature and spend the night.With intermediate water, ethanol, methyl alcohol is washing copolymer progressively, collecting by filtration, and dry in the drying box of 60 DEG C, is dissolved in dimethyl formamide, obtains 1 mg mL -1homogeneous suspension.
Described preparation method, preferred polyamide-amine type dendrimer-golden nano-complexes is obtained by following steps:
Be 0.3 mmol L by 2.5 mL concentration -1hAuCl 4solution adds 2.5 mL 0.1 mmol L to -1polyamide-amide dendrimer aqueous solution in, stir 20 min and obtain mixed solution; By the 0.1 mmol L of 2.5 mL -1sodium citrate dropwise add in above-mentioned mixed solution, react 4 h, solution colour becomes peony, to obtain final product.
Principle of work of the present invention:
First glass-carbon electrode modifies synergistic matter GR-PANI and PAMAM-Au, not only can promote electrode surface electro transfer, and the connection of specific groups between synergistic matter can ensure their layer assembly.The amino of golden nanometer particle and kanamycins antibody (KAb), passes through Au-NH 2effect, antibody modification on electrode.Then, antibody (KAb) and kanamycins have single-minded recognition capability, and kanamycins just can successfully be modified on electrode.At the other end of kanamycins, biotin labeled aptamer (Bio-Apt), by the specific recognition capability of aptamer and object, is also successfully modified.Finally, the horseradish peroxidase (SA-HRP) being marked with Streptavidin is connected on electrode by the high forces between Bio-SA.In testing process, by the p-dihydroxy-benzene (HQ) in the HRP catalysis detection end liquid on electrode and hydrogen peroxide (H 2o 2) redox produce electric signal, connect electrochemical workstation, take Ag/AgCl as contrast electrode, be to electrode with Pt electrode, current potential is set to-0.2 to 0.6 V, pulse width 0.05V, and pulse width scanning is 0.06 S, adopt differential pulse voltammetry technology to read the change of electric signal, the size according to the electric current of electrode surface generation plays the effect detected object.The amount of HRP fixing on electrode and the amount of the detected material kanamycins of modification have direct relation, and detected material is more, and the amount of fixing HRP is also more, and the electric signal that catalysis produces is also stronger.
GR-PANI and the PAMAM-Au composite conductivity that the present invention adopts is strong, becomes the excellent material building sensor; Use horseradish peroxidase (HRP), by with H 2o 2with the reaction of HQ, amplifying signal; Have employed the detection model of sandwich type, introduce antibody and aptamers at the two ends detecting thing Ka respectively, detect more sensitive; The transducer sensitivity of preparation is high, and detection speed is fast; Detect the method for Ka, simple to operate, quick, sensitive, be convenient to Site Detection.
Beneficial effect of the present invention:
1, due to use glass-carbon electrode, its electrode is easy, miniaturization, portable, can repeatedly use.
2, responding layer uses surface modification technology fixing on the working electrode (s, and optimize the consumption and concentration that use material, the requirement of obtained sandwich type electrode pair environment temperature is not obvious, uses under room temperature.
3, preparation method is simple, stable performance, electrode reproducible, is applicable to the detection of kanamycins in food security and the practical application of biology sensor industrialization.
4, the process costs making electrode is low, is applicable to requirement inexpensive in industrialization.
5, take glass-carbon electrode as the sandwich type electrochemical sensing system that immobilization carrier fixes based on aptamer, can realize, to the quick online detection of kanamycins in food, detecting and being limited to 4.6 × 10 -6μ g mL -1.
accompanying drawing explanation
Fig. 1 is preparation technology's process flow diagram of the fit electrode of galvanochemistry.
Embodiment
Be further described the present invention below in conjunction with specific embodiment, following explanation is only exemplary, does not limit protection scope of the present invention.
First Graphene-polyaniline nano compound and polyamidoamine dendrimer-golden nano-complexes is prepared
1, the preparation of Graphene-polyaniline (GR-PANI) nano-complex:
(1) preparation of graphene oxide (GO):
The preparation of graphene oxide (GO) is the method according to classics.In brief, 1.0 g dags join containing 0.5 g sodium nitrate (NaNO 3) and 3.0 g potassium permanganate (KMnO 4) the 50 mL concentrated sulphuric acid (H of massfraction 98% 2sO 4) in.Temperature, lower than at 20 DEG C, reacts 1 h, is warmed up to 35 DEG C, continues stirring 30 min.Then slowly add the ionized water of about 100 mL, after Keep agitation 20 min, add 50 mL H 2o 2(30%), reduce residual oxygenant, solution becomes glassy yellow gradually; Filter while hot, wash until sulfate radical-free is detected in filtrate with HCl solution and deionized water.Sediment is dry in the constant temperature oven of 60 DEG C, finally obtains GO.
(2) synthesizing graphite alkene-polyaniline (GR-PANI) compound
2.75 mL aniline (99%) are dissolved in 100 mL 1 mol L -1hCl in obtain aniline concentration be 0.3 mol L -1, add the GO that 100 mg have been prepared.After ultrasonic 1 h, 0.17g (NH 4) 2s 4o 8with 10 mL 1 mol L -1hCl adds mixed liquor and vigorous agitation fast.When color becomes bottle green, polymerization starts.After using redistilled water filtration washing repeatedly, obtain Graphene-polyaniline (GO-PANI) compound.Weighing 0.1 g GO-PANI is dissolved in the ultrapure water of 50 mL, reduces 1 h with 0.1 mL hydrazine hydrate (80%) at 95 DEG C.Passed through to filter excessive hydrazine hydrate with intermediate water washing compound.
Finally, the compound of above-mentioned synthesis is dispersed in 10 mL and contains 1 mol L -1hCl and 0.06 g (NH 4) 2s 4o 8, stir under room temperature and spend the night.With intermediate water, ethanol, methyl alcohol is washing copolymer progressively, collecting by filtration, and dry in the drying box of 60 DEG C.
In order to prepare further experiment, the GR-PANI of preparation is dissolved in dimethyl formamide (DMF), and ultrasonic 1 h, obtain 1 mg mL -1homogeneous suspension.
2, the preparation of polyamidoamine dendrimer-golden nano-complexes (PAMAM-Au):
By 2.5 mL HAuCl 4solution (0.3 mmol L -1) add 2.5 mL 0.1 mmol L to -1in polyamide-amide dendrimer (PAMAM) (get 0.15 μ L dendrimer and add 2.5 mL water) solution, stir 20 min; By 2.5 mL sodium citrates (0.1 mmol L -1) dropwise add in above-mentioned solution, the reaction time is that after 4 h, solution colour becomes peony, forms zeroth order gold complex, to obtain final product.
embodiment 1
A preparation method for sandwich type electrochemical sensor of the present invention, comprises the following steps:
First a, glass-carbon electrode carry out polishing in the oxidation aluminium paste of 0.3 and 0.05 m, until in minute surface, rinse with intermediate water;
The GR-PANI that b, 10 μ L have been prepared drips at electrode surface, at room temperature dried overnight; The electrode modified is immersed in the PAMAM-Au solution of preparation, keeps 3 h;
After c, stand-by intermediate water rinse several times, 10 μ L dissolve 5 μm of ol L -1the PBS solution of kanamycins antibody (KAb) drips at electrode surface, keeps 90 min under room temperature, rinses electrode remove unconjugated KAb with intermediate water and PBS;
D, 10 μ L BSA (0.1%) and poly-(A) 10chain (single stranded DNA of 10 adenine bases, 5 μm of ol L -1) be used to enclosed-electrode surface there is no combined site, obtain sandwich type electrochemical sensor.
Detection method is as follows:
E, the electrode obtained are hatched, and are used PBS wash buffer fully stir cleaning in PBS damping fluid after in variable concentrations kanamycins to be detected (Ka) solution;
F, 10 μ L 5.5 × 10 -10mol L -1biotin labeled aptamer (Bio-KBA), by the strong recognition capability between object, is modified at electrode surface, hatches 4 h;
After g, PBS rinse, 10 μ L 0.96 μm ol L -1the horseradish peroxidase (SA-HRP) of marked by streptavidin is modified on electrode by acting force strong between SA and Bio;
H, be contrast electrode with Ag/AgCl, be to electrode with Pt electrode, current potential is set to-0.2 to 0.6 V, pulse width 0.05V, and pulse width scanning is 0.06 S, adopts differential pulse voltammetry technology to read the change of electric signal, detects object.
PBS damping fluid used in said method is prepared by method: take Na 2hPO 47.1 g, KCl 0.2 g and KH 2pO 46.8 g, KCl 0.2 g be dissolved in respectively in 500 mL intermediate waters, obtain two kinds of solution pH meter Mixed adjustments, obtaining pH value is 7.4, and concentration is 0.01 mol L -1pBS damping fluid.
embodiment 2
10 μ L dissolve 5 μm of ol L -1the PBS solution of kanamycins antibody (KAb) drips at electrode surface, room temperature changes incubation time (30 next time, 60,90,120,150,180 min), rinse electrode with intermediate water and PBS and remove unconjugated KAb, make Different electrodes, for selecting the incubation time of KAb the best, other are with embodiment 1.
embodiment 3
The biotin labeled aptamer (Bio-KBA) (1 × 10 of 10 μ L variable concentrations -12, 5.5 × 10 -12, 1 × 10 -11, 5.5 × 10 -11, 1 × 10 -10, 5.5 × 10 -10, 5.5 × 10 -9mol L -1) by the strong recognition capability between object, be modified at electrode surface, hatch 4 h, make Different electrodes, for selecting the optimum concentration of Bio-KBA, other are with embodiment 1.
embodiment 4
Taking Ag/AgCl as contrast electrode, is to electrode with Pt electrode, and current potential is set to-0.2 to 0.6 V, pulse width 0.05V, and pulse width scanning is 0.06 S, adopts differential pulse voltammetry technology to read the change of electric signal, detects object.To detect electric current for ordinate, with different kanamycins (Ka) concentration for horizontal ordinate, in order to drawing standard working curve.Other are with embodiment 1.
embodiment 5
Taking Ag/AgCl as contrast electrode, is to electrode with Pt electrode, and current potential is set to-0.2 to 0.6 V, pulse width 0.05V, and pulse width scanning is 0.06 S, adopts differential pulse voltammetry technology to read the change of electric signal, detects object.Adopt the method that mark-on reclaims, detect milk sample, replicate determination 3 times, in order to calculate the recovery, result is 97%-103%, illustrates within the scope of the milk medial error after process do not have kanamycins (Ka), belongs to qualified samples.Other are with embodiment 1.
embodiment 6
Taking Ag/AgCl as contrast electrode, is to electrode with Pt electrode, and current potential is set to-0.2 to 0.6 V, pulse width 0.05V, and pulse width scanning is 0.06 S, adopts differential pulse voltammetry technology to read the change of electric signal, detects object.Select different Ka concentration (5 × 10 -6, 4 × 10 -5, 4 × 10 -4, 4 × 10 -3, 4 × 10 -2μ g mL -1), other are with embodiment 1, draw different to detect electric current, and electrode prepared by the present invention is described, detection speed is fast, detection limit is low, can reach 4.6 × 10 -6μ g mL -1
。The signal detected is as shown in the table.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not by the restriction of embodiment; other is any do not deviate from Spirit Essence of the present invention and principle under make change, modification, combination, substitute, simplify and all should be equivalent substitute mode, be included within protection scope of the present invention.

Claims (10)

1. the fit electrode of galvanochemistry detected for kanamycins, it is characterized in that on glass-carbon electrode, be modified with graphene-polyanilinecompound compound, polyamidoamine dendrimer-golden nano-complexes, kanamycins antibody from bottom to top successively, last enclosed-electrode surface be the single stranded DNA of BSA and 10 adenine base.
2. the fit electrode of galvanochemistry according to claim 1, it is characterized in that the thickness of graphene-polyanilinecompound compound is at 100 ± 10nm, the thickness of polyamidoamine dendrimer-golden nano-complexes is 100-200 nm, the thickness of kanamycins antibody is 1 ± 0.1 μm, the thickness of the single stranded DNA of BSA and 10 adenine base and be 500 ± 20nm.
3. the fit electrode of galvanochemistry according to claim 1, is characterized in that the mass ratio of Graphene and aniline in graphene-polyanilinecompound compound is 3.3:100.
4. the fit electrode of galvanochemistry according to claim 1, is characterized in that in polyamidoamine dendrimer-golden nano-complexes, polyamidoamine dendrimer is 1:3 with the mol ratio of gold.
5. a preparation method for the galvanochemistry according to any one of claim 1-4 is fit electrode, is characterized in that comprising the following steps:
(1) process washing is carried out to glass-carbon electrode, Graphene-polyaniline nano compound is added drop-wise to electrode surface, dry under room temperature, rinse with intermediate water;
(2) to immerse in polyamidoamine dendrimer-golden nano-complexes 3 hours, rinse with intermediate water, dry;
(3) the PBS damping fluid of kanamycins antibody is dripped on surface, overnight at room temperature;
(4) with the bovine serum albumin(BSA) of 0.1% and single stranded DNA enclosed-electrode 2 h of 10 adenine bases, to obtain final product.
6. preparation method according to claim 5, is characterized in that Graphene-polyaniline nano compound is obtained by following steps:
(1) dag obtains graphene oxide through peroxidating,
(2) graphene oxide prepares Graphene-polyaniline nano compound with aniline according to mass ratio 3.3:100.
7. preparation method according to claim 5, is characterized in that in polyamidoamine dendrimer-golden nano-complexes, polyamidoamine dendrimer is 1:3 with the mol ratio of gold.
8. preparation method according to claim 5, is characterized in that dripping 10 μ L in step (3) on surface is dissolved with 5 μm of ol L -1the PBS damping fluid of kanamycins antibody (KAb), overnight at room temperature.
9. preparation method according to claim 5, is characterized in that Graphene-polyaniline nano compound is obtained by following steps: 2.75 mL aniline are dissolved in 100 mL 1 mol L -1hCl in obtain aniline concentration be 0.3 mol L -1, add 100 mg graphene oxides, after ultrasonic 1 h, 0.17g (NH 4) 2s 4o 8with 10 mL 1 mol L -1hCl adds mixed liquor and vigorous agitation fast, and when color becomes bottle green, polymerization starts, and after using redistilled water filtration washing several times, obtains graphene-polyanilinecompound compound; Weighing 0.1 g graphene-polyanilinecompound compound is dissolved in the ultrapure water of 50 mL, reduces 1 h with the hydrazine hydrate of 0.1 mL80% at 95 DEG C, the hydrazine hydrate that washing removing is excessive; Compound is dispersed in 10 mL and contains 1 mol L -1hCl and 0.06 g (NH 4) 2s 4o 8, stir under room temperature and spend the night,
With intermediate water, ethanol, methyl alcohol is washing copolymer progressively, collecting by filtration, and dry in the drying box of 60 DEG C, is dissolved in dimethyl formamide, obtains 1 mg mL -1homogeneous suspension.
10. preparation method according to claim 5, is characterized in that polyamidoamine dendrimer-golden nano-complexes is obtained by following steps:
Be 0.3 mmol L by 2.5 mL concentration -1hAuCl 4solution adds 2.5 mL 0.1 mmol L to -1polyamide-amide dendrimer aqueous solution in, stir 20min obtain mixed solution; By the 0.1 mmol L of 2.5 mL -1sodium citrate dropwise add in above-mentioned mixed solution, reaction 4h, solution colour becomes peony, to obtain final product.
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