阅读说明：本技术 一种基于双重信号放大的啶虫脒传感器及其检测方法 (A kind of Acetamiprid sensor and its detection method based on dual signal amplification ) 是由 郭业民 史孝杰 黄靖程 王豹 赵庆雪 孙霞 于 2019-08-28 设计创作，主要内容包括：一种基于双重信号放大的啶虫脒传感器及其检测方法,包括适配体修饰层、纳米粒子双重信号放大层和电极基体,其中所述的纳米粒子双重信号放大层为还原性氧化石墨烯纳米银粒子材料和电沉积普鲁士蓝-纳米金复合材料,其一方面能够增强传感器的导电性来加强信号输出,同时还增强传感器的氧化还原反应从而提高信号输出；所述的导电聚合物为复合材料；所述的电极基体为玻碳材质的电极基体；所述的适配体修饰层为啶虫脒适配体和牛血清白蛋白。本发明制备所得的传感器对现有机磷农药的检测线性范围为1pM–1μM,传感器对啶虫脒的检测限为0.136pM。(A kind of Acetamiprid sensor and its detection method based on dual signal amplification, including aptamers decorative layer, nanoparticle dual signal amplification layer and electrode matrix, wherein the nanoparticle dual signal amplification layer be reproducibility stannic oxide/graphene nano silver particles material and electro-deposition it is Prussian blue-nanogold composite material, its one side can enhance the electric conductivity of sensor to reinforce signal output, while enhance the redox reaction of sensor also to improve signal output；The conducting polymer is composite material；The electrode matrix is the electrode matrix of glass carbon materials matter；The aptamers decorative layer is Acetamiprid aptamers and bovine serum albumin(BSA).It is 1pM -1 μM to the detection range of linearity of existing machine phosphorus insecticide that the present invention, which prepares resulting sensor, and sensor is limited to 0.136pM to the detection of Acetamiprid.)

1. a kind of Acetamiprid sensor and its detection method based on dual signal amplification, it is characterised in that include the following steps:

(1) pretreatment of glass-carbon electrode；

(2) reproducibility graphene oxide-nano silver composite material (rGO-AgNPs) preparation；

(3) preparation of Prussian blue-nanogold (PB-AuNPs) laminated film deposition liquid and stabilizing solution；

(4) assembling of aptamer sensor；

(5) measurement of Acetamiprid pesticide.

2. a kind of Acetamiprid sensor and its detection method based on dual signal amplification according to claim 1, special Sign is, reproducibility graphene oxide described in step (2)-nano silver composite material (rGO-AgNPs) the preparation method comprises the following steps:

25mg graphene oxide is dissolved in 50ml deionized water, and ultrasound 90 minutes under conditions of 40KHz, 150W keep it completely molten Solution；Obtained graphene oxide dispersion is added in there-necked flask (250ml), the AgNO3 and 0.275g of 20mg are added when room temperature Sodium citrate react 30 minutes, 350 μ l ammonium hydroxide (35%) and 28mg hydrazine hydrate (80%) are added after being warming up to 98 degrees Celsius, stirring Reaction 4 hours, 3000rpm/min is centrifuged 15min while hot, collects sediment；Spend ethyl alcohol and deionized water be cleaned multiple times until PH is in neutrality.

3. a kind of Acetamiprid sensor and its detection method based on dual signal amplification according to claim 1, special Sign is that Prussian blue-nanogold (PB-AuNPs) laminated film described in step (3) deposits liquid and preparation method thereof are as follows:

0.05 g chitosan is dissolved in the acetum of 100 mL 1.0%, stirs 3 h at room temperature, is made into 0.05% shell Glycan solution, is completely dissolved chitosan；Weigh 0.0625 g FeCl3,0.0822 g K3 [Fe (CN) 6], 0.7455 g KCl and 1 mL concentrated hydrochloric acid are added in 100 mL chitosan solution obtained above, at room temperature ultrasonic disperse, until To stable dirty-green dispersion liquid；The nano Au colloid of 1 mL prepared is added again in the above-mentioned solution mixed, then 0.5 h of ultrasonic disperse is spare to being completely dissolved at room temperature.

4. a kind of Acetamiprid sensor and its detection method based on dual signal amplification according to claim 1, special Sign is, the assemble method of aptamer sensor described in step (4) are as follows:

Firstly, 6 μ L(1 are added dropwise in glassy carbon electrode surface) in the rGO-AgNPs for preparing be added drop-wise to electrode surface and at normal temperature It dries to obtain rGo-AgNPS/GCE；Glass-carbon electrode is immersed in the electro-deposition bottom liquid (deoxygenation) newly prepared, with cyclic voltammetry (CV) (scanning voltage is -0.6-0.2 V, and scanning speed is 0.05 V/s) is scanned to it, deposition circle number is 12；Later, electric Pole in stabilizing solution between -0.6 V -0.2 V scan cycle voltammogram until image stabilization obtain PB-AuNPs/rGo- AgNPS/GCE；Next, 0.5% bovine serum albumen solution (BSA) drop coating is incubated for 30 minutes in the electrode surface prepared with envelope Nonspecific binding site is closed, is gently rinsed with ultrapure water and removes extra BSA；Finally, by the BSA/Apt/rGO- of preparation CuNPs/SPCE biosensor is stored in 4 DEG C of temperature refrigerators, spare；The base sequence of the aptamer is 5'-SH-T GTAATTTGTCTGCAGCGGTTCTTGATCGCTGACACCATATTATGAAGA-3'。

5. a kind of Acetamiprid sensor and its detection method based on dual signal amplification according to claim 1, special Sign is, the continuous mode of Acetamiprid pesticide in step (5) are as follows: electro-chemical test condition be pH 7.4 the potassium ferricyanide it is molten It is scanned, is recorded using cyclic voltammetry in liquid (5 mmol/L of [Fe (CN) 6], 3/4,0.1 mol/L of KCl) The above-mentioned aptamer sensor prepared is incubated for the reduction peak value curent change of front and back by object solution, according to gained current difference The size of value reflects pesticide concentration.

Technical field

The present invention relates to a kind of Acetamiprid sensors and its detection method based on dual signal amplification, belong to electrochemical student Object sensor field.

Background technique

Pesticide Residue causes the extensive concern of people at present, because it has jeopardized human health and causes environment dirty Dye.Acetamiprid has become the substitute of organophosphorus pesticide or other common pesticides as a kind of anabasine insecticide, however its One of the potential hazard substance for still becoming human health is widely used.The method of detection Acetamiprid has at present: gas chromatography, Liquid chromatography, enzyme-linked immunization etc..However, every kind of method can all have following one or more disadvantages: equipment is expensive, square Method is complicated, stability is poor, time-consuming, sensitivity is not high, selectivity is general etc..Therefore, seek a kind of convenience, simplicity, quickly, oversoul Quick and high specific Acetamiprid.

Aptamer (Apt) with the diversity of its object, be easily manipulated and store, convenient for modification and signal amplification, The characteristics such as stability is good become system, simple, Sensitive Detection pesticide molecule powerful mean, can be for almost any pesticide Molecule carries out specific detection.Can use aptamer construct a kind of building it is easy, quickly, hypersensitive, high specific and Environmental-friendly electrochemical sensor.

Summary of the invention

The purpose of the present invention is to solve the above problem, provide it is a kind of prepare detection signal it is big, stability is high, selectivity is good Aptamer sensor, and be used for Acetamiprid detection.

The technical solution of the present invention is as follows: by the fixed reproducibility stannic oxide/graphene nano silver particles material of glass-carbon electrode and Electro-deposition is Prussian blue-nanogold composite material, then Acetamiprid aptamers are added dropwise, it realizes the building of sensor, has been subsequently used for The detection of machine phosphorus insecticide；Include the following steps: the pretreatment of (1) glass-carbon electrode；(2) reproducibility graphene oxide-nano silver is multiple The preparation of condensation material (rGO-AgNPs)；(3) Prussian blue-nanogold (PB-AuNPs) laminated film deposits liquid and stabilizing solution Preparation；(4) assembling of aptamer sensor；(5) measurement of Acetamiprid pesticide.

Preferably, electrode described in (1) is glass-carbon electrode, and needs grind 5min in aluminium oxide mud, use deionization The above-mentioned honed electrode of water repeated flushing is cleaned by ultrasonic 1-3 times, each 2-3 min, then uses 1:1 ethyl alcohol, 1 respectively again: 1 nitric acid and distilled water 5 min of ultrasound, are then placed in by 5 mmol/L of [Fe (CN) 6], 3/4 and 0.1 mol/L In the test bottom liquid of of KCl composition, scan that (scanning voltage is -0.6-+0.2 V, scanning with cyclic voltammetry (CV) to it Speed is 0.05 V/s), two measured spike potential differences obtain performance and stablize electrode less than 120 mV.

Preferably, the preparation process of described (2) rGO-AgNPs: 25mg graphene oxide is dissolved in 50ml deionized water, In Ultrasound 90 minutes, make it completely dissolved under conditions of 40KHz, 150W；There-necked flask is added in obtained graphene oxide dispersion In (250ml), the sodium citrate that the AgNO3 and 0.275g of 20mg is added when room temperature reacts 30 minutes, after being warming up to 98 degrees Celsius 350 μ l ammonium hydroxide (35%) and 28mg hydrazine hydrate (80%) is added, is stirred to react 4 hours, 3000rpm/min is centrifuged 15min while hot, receives Collect sediment；It spends ethyl alcohol and deionized water is cleaned multiple times until pH is in neutrality.

Preferably, 0.05 g chitosan the preparation process of deposition liquid in described (3): is dissolved in 100 mL's 1.0% In acetum, 3 h are stirred at room temperature, are made into 0.05% chitosan solution, are completely dissolved chitosan；Weigh 0.0625 g It is obtained above that 100 mL are added in FeCl3,0.0822 g K3 [Fe (CN) 6], 0.7455 g KCl and 1 mL concentrated hydrochloric acid In chitosan solution, ultrasonic disperse at room temperature, until obtaining stable dirty-green dispersion liquid；Again by 1 mL's prepared Nano Au colloid is added in the above-mentioned solution that has mixed, then 0.5 h of ultrasonic disperse is spare to being completely dissolved at room temperature.

Preferably, 200mL ultrapure water and 2mL nano Au colloid preparation method: are added in the vial Jing Guo silicidation Chlorauric acid solution (1%m/v), gently shakes up, then covers bottleneck, is careful not to tighten；Vial is placed in micro-wave oven, Boiling (about 4 min) is heated to high fire screen；Vial is taken out, is careful not to fall into dust and sundries, while rocking vial, And rapidly join 4.8 mL %1(m/v) citric acid three sodium solution, not allow solution to splash out when paying attention to rocking vial, be added Continue to rock mixing after trisodium citrate；Glass is covered into lid (being careful not to tighten) again, is placed in micro-wave oven, middle height fire 4min Continue to heat；Vial is taken out, product is claret nano-Au solution at this time, is cooled to room temperature and is arrived with ultrapure water constant volume 200ml。

Preferably, the assembling steps of the aptamer sensor in described (4): firstly, 6 μ L are added dropwise in glassy carbon electrode surface (1) rGO-AgNPs prepared in is added drop-wise to electrode surface and dries to obtain rGo-AgNPS/GCE at normal temperature；By glass carbon electricity Pole is immersed in the electro-deposition bottom liquid (deoxygenation) newly prepared, and scans that (scanning voltage is -0.6-+0.2 with cyclic voltammetry (CV) to it V, scanning speed are 0.05 V/s), deposition circle number is 12.Later, electrode in stabilizing solution between -0.6 V of V ~+0.2 Scan cycle voltammogram is until image stabilization obtains PB-AuNPs/rGo-AgNPS/GCE.Next, 0.5% bovine serum albumin is molten Liquid (BSA) drop coating is incubated for 30 minutes to close nonspecific binding site, is gently rushed with ultrapure water in the electrode surface prepared Extra BSA is removed in washout.Finally, the BSA/Apt/rGO-CuNPs/SPCE biosensor of preparation is stored in 4 DEG C of temperature ice Case, in case following experiment uses.

Preferably, the base sequence of described (5) the amplifying nucleic acid aptamers is 5'-SH-TGTAATTTGTCTGCAGCGGTTCTT GATCGCTGACACCAT ATTATGAAGA-3'。

Preferably, the nonspecific binding site sealing condition are as follows: drip 6 μ LBSA in electrode surface, be incubated for 30 at room temperature Min gently rinses electrode surface, is dried with nitrogen, and so far aptamer sensor preparation is completed.

Preferably, in (5) Acetamiprid pesticide continuous mode are as follows: electro-chemical test condition is the iron cyaniding in pH 7.4 It is scanned in potassium solution using cyclic voltammetry, records the above-mentioned aptamer sensor prepared by before the incubation of object solution Curent change afterwards reflects pesticide concentration according to the size of gained current differential.

Compared with prior art, the beneficial effects of the present invention are: the present invention provides one kind based on dual signal amplify Acetamiprid sensor and its detection method；It is compound using reproducibility stannic oxide/graphene nano silver particles/Prussian blue-nanogold The synergistic effect of material；One side reproducibility stannic oxide/graphene nano silver particles improve the electric conductivity of entire electrode and then improve defeated Current signal out；On the other hand enhance the redox reaction of entire reaction system using Prussian blue-nanogold to improve it Output signal.The sensor can be used for the detection of Acetamiprid pesticide in actual sample, mention for the fast slowdown monitoring in scene of supervision department For new method.

Detailed description of the invention

Fig. 1 is the preparation process with body sensor.

Fig. 2 is that the CV of aptamer sensor is characterized.

Fig. 3 is sensor electrodeposition time optimum results.

Fig. 4 is sensor pH optimum results.

Fig. 5 is sensor aptamers concentration optimization result.

Fig. 6 is sensor incubation time optimum results.

Fig. 7 is sensor specific detection result.

Fig. 8 is sensor actual sample testing result.

Invention is further described in detail with reference to the accompanying drawings and examples, but embodiment does not do any shape to the present invention The restriction of formula:

1: one step electrodeposition process of embodiment prepares aptamer sensor

(1) reproducibility graphene oxide-nano silver composite material (rGO-AgNPs) preparation

25mg graphene oxide is dissolved in 50ml deionized water, and ultrasound 90 minutes under conditions of 40KHz, 150W keep it completely molten Solution；Obtained graphene oxide dispersion is added in there-necked flask (250ml), the AgNO3 and 0.275g of 20mg are added when room temperature Sodium citrate react 30 minutes, 350 μ l ammonium hydroxide (35%) and 28mg hydrazine hydrate (80%) are added after being warming up to 98 degrees Celsius, stirring Reaction 4 hours, 3000rpm/min is centrifuged 15min while hot, collects sediment；Spend ethyl alcohol and deionized water be cleaned multiple times until PH is in neutrality.

(2) preparation of Prussian blue-nanogold (PB-AuNPs) electrodeposit liquid and Wen Ding liquid

0.05 g chitosan is dissolved in the acetum of 100 mL 1.0%, stirs 3 h at room temperature, is made into 0.05% shell Glycan solution, is completely dissolved chitosan；Weigh 0.0625 g FeCl3,0.0822 g K3 [Fe (CN) 6], 0.7455 g KCl and 1 mL concentrated hydrochloric acid are added in 100 mL chitosan solution obtained above, at room temperature ultrasonic disperse, until To stable dirty-green dispersion liquid；The nano Au colloid of 1 mL prepared is added again in the above-mentioned solution mixed, then 0.5 h of ultrasonic disperse is spare to being completely dissolved at room temperature;It is 0.1 mol/L KCl, 0.01 mol/L HCl that temperature, which determines liquid, Solution.

Nano Au colloid preparation method: 200mL ultrapure water is added in the vial Jing Guo silicidation and 2mL gold chloride is molten Liquid (1%m/v), gently shakes up, then covers bottleneck, is careful not to tighten；Vial is placed in micro-wave oven, with high fire screen It is heated to boiling (about 4 min)；Vial is taken out, is careful not to fall into dust and sundries, while rocking vial, and is quickly added Enter 4.8 mL %1(m/v) citric acid three sodium solution, not allow solution to splash out when paying attention to rocking vial, be added citric acid three Continue to rock mixing after sodium；Glass is covered into lid (being careful not to tighten) again, is placed in micro-wave oven, middle height fire 4min continues to add Heat；Vial is taken out, product is claret nano-Au solution at this time, is cooled to room temperature and with ultrapure water constant volume to 200ml.

(3) pretreatment of glass-carbon electrode electrode

Glass-carbon electrode (GCE) is ground into 5min in aluminium oxide mud, with the above-mentioned honed electrode of deionized water repeated flushing, is surpassed Sound cleans 1-3 times, each 2-3 min, then uses 1:1 ethyl alcohol, 1:1 nitric acid and distilled water 5 min of ultrasound respectively again, It is then placed in test bottom liquid, scans that (scanning voltage is -0.6-+0.2 V, scanning speed with cyclic voltammetry (CV) to it For 0.05 V/s), two measured spike potential differences obtain performance and stablize electrode less than 120 mV.

(4) preparation of aptamer sensor

Firstly, 6 μ L(1 are added dropwise in glassy carbon electrode surface) in the rGO-AgNPs for preparing be added drop-wise to electrode surface and at normal temperature It dries to obtain rGo-AgNPS/GCE；Glass-carbon electrode is immersed in the electro-deposition bottom liquid (deoxygenation) newly prepared, with cyclic voltammetry (CV) (scanning voltage is -0.6-+0.2 V, and scanning speed is 0.05 V/s) is scanned to it, deposition circle number is 12.Later, electric Pole in stabilizing solution between -0.6 V of V ~+0.2 scan cycle voltammogram until image stabilization obtain PB-AuNPs/rGo- AgNPS/GCE.Next, 0.5% bovine serum albumen solution (BSA) drop coating is incubated for 30 minutes in the electrode surface prepared with envelope Nonspecific binding site is closed, is gently rinsed with ultrapure water and removes extra BSA.Finally, by the BSA/Apt/rGO- of preparation CuNPs/SPCE biosensor is stored in 4 DEG C of temperature refrigerators, in case following experiment uses.Fig. 1 shows electrodeposition process With aptamer sensor packaging technology.

(5) electrodeposited material CV is characterized

The different composite material of electro-deposition on the electrode carries out electrochemical Characterization (Fig. 2) by CV technology.Relative to naked GCE(Fig. 2 Middle f curve), rGo-AgNPS is fixed on (e curve in Fig. 2) and shows higher peak current.As PB-AuNPs/rGo-AgNPS It is all fixed that obtained PB-AuNPs/rGo-AgNPS/GCE can produce more high current on the electrode (a curve in Fig. 2), this Show that conductive CuNPs is successfully modified on the surface rGO.(Fig. 2 after further adding aptamers in electric depositing solution Middle b curve), since negatively charged aptamers increase electronics transfer resistance, redox peak is decreased obviously.Ox blood is added Albumin, especially poorly conductive, therefore electric current further decreases (c curve in Fig. 2).Above-mentioned material Morphological Characterization is the result shows that a step It is feasible that electrodeposition process, which prepares aptamer sensor,.In addition to this, we in fixed PB-AuNPs also to individually being surveyed Amount, than two kinds composite material effects of discovery are far short of what is expected.Described in summary, the biggish current signal input of the sensor that we construct, Amplify strategy compared to only with substance signal.

(6) aptamer sensor condition optimizing

Electrodeposition time is an important factor for influencing sensor PB-AuNPs material property.We are by changing cyclic voltammetric Cycle-index optimizes electrodeposition time.Fig. 3 is the result shows that the best electrochemical deposition time is 12 CV circulations.

Since pesticide and the binding affinity of aptamers can reduce under relatively low or higher pH value, aptamers are passed Acidity and alkalinity environment locating for sensor is to influence the vital factor of its detection performance.In optimization in this section, use is had studied Influence of the potassium ferricyanide solution of phosphate buffer solution (PBS) configuration of different pH to Pesticides Testing result；As shown in Figure 4 The discovery of PH optimum results, when pH value is 7.5, CV peak point current difference is minimum, shows the adaptation to form stable structure Body-pesticide compound is most.

Fig. 5 depicts influence of the adaptation bulk concentration to sensor.The result shows that when suitable containing 4 μM in electric depositing solution When ligand, CV current differential reaches maximum, but with the increase of aptamers content, current differential sharply declines.Because excessive Aptamers may cause their intermolecular hybridization, and the fixed amount of aptamers on the electrode is caused to reduce.Therefore, in the aptamers It is adapted to bulk concentration in the preparation of sensor and is set as 4 μM.

Object and aptamers specific binding incubation time be influence aptamer sensor performance it is another it is important because Element, we carry out to it the study found that result is as shown in Figure 6.Current differential is basically unchanged after 40 min.When being optimal incubation Between.