阅读说明：本技术 一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法 (Preparation method of aptamer sensor based on copper-gold bimetallic core-shell structure nanoparticles ) 是由 郭业民 黄靖程 孙霞 项耀东 李建森 孔倩倩 于 2021-08-20 设计创作，主要内容包括：一种基于铜金双金属核壳结构纳米粒子的适配体传感器的制备方法,涉及一种新的生物传感器制备方法。本发明利用铜金双金属核壳结构纳米粒子对电化学发光的增强作用,构建了一种检测有机磷农药的适配体传感器。具体制备过程如下：将发光试剂氯化三(2,2’-联吡啶)钌(II)六水合物滴加在多壁碳纳米管-壳聚糖改性的电极表面,然后加入水相中合成的铜金双金属核壳结构纳米粒子。铜金双金属核壳结构纳米粒子表面的纳米金可以催化三正丙胺的分解显著增加体系的电化学发光强度,同时,借助纳米铜大的比表面积特性有利于纳米金的固载。本发明适配体传感器制备方法简单、检测速度快、稳定性好、选择性高,可用于蔬菜中有机磷农药残留的高灵敏度检测。(A preparation method of an aptamer sensor based on copper-gold bimetallic core-shell structure nanoparticles relates to a new biosensor preparation method. The invention constructs an aptamer sensor for detecting organophosphorus pesticide by utilizing the enhancement effect of copper-gold bimetallic core-shell structure nanoparticles on electrochemiluminescence. The preparation process comprises the following steps: dripping a luminescent reagent, namely tris (2,2' -bipyridyl) ruthenium (II) chloride hexahydrate on the surface of the multiwalled carbon nanotube-chitosan modified electrode, and then adding the copper-gold bimetallic core-shell structure nano-particles synthesized in a water phase. The nanogold on the surface of the copper-gold bimetallic core-shell structure nanoparticle can catalyze the decomposition of tri-n-propylamine to obviously increase the electrochemical luminescence intensity of the system, and meanwhile, the immobilization of the nanogold is facilitated by virtue of the large specific surface area characteristic of the nanogold. The aptamer sensor disclosed by the invention is simple in preparation method, high in detection speed, good in stability and high in selectivity, and can be used for high-sensitivity detection of organophosphorus pesticide residues in vegetables.)

1. A preparation method of an aptamer sensor based on copper-gold bimetal core-shell structure nanoparticles is characterized in that the copper-gold bimetal core-shell structure nanoparticles are synthesized in a water phase, the characteristic of large specific surface area of nano-copper is favorable for immobilization of nano-gold, meanwhile, the electrochemical luminescence intensity of a system can be enhanced by catalyzing the oxidation process of tri-n-propylamine by the nano-gold, and the aptamer sensor is constructed by the copper-gold bimetal core-shell structure nanoparticles, and the specific preparation process is as follows:

(1) modifying the surface of the electrode by using a multi-walled carbon nanotube-chitosan composite material;

(2) dripping a luminescent reagent;

(3) dropwise adding copper-gold bimetallic core-shell structured nanoparticles;

(4) modification of the aptamer;

(5) coating a layer of bovine serum albumin solution to seal the non-specific binding sites, cleaning the surface with ultrapure water, drying with nitrogen, and completing the preparation of the aptamer sensor.

2. The preparation method of the aptamer sensor based on the copper-gold bimetallic core-shell structure nanoparticles, according to claim 1, is characterized in that the electrode is a platinum electrode, and the preparation method of the multiwalled carbon nanotube-chitosan composite material comprises the following steps: dissolving 0.05 g of chitosan powder in a mixed solution of 1 mL of glacial acetic acid and 99 mL of ultrapure water, stirring for more than 3 h to fully dissolve the chitosan powder, preparing 0.05% chitosan solution, weighing 25 mg of multi-walled carbon nanotubes, dispersing in 5 mL of the prepared chitosan solution, completing the preparation of the multi-walled carbon nanotube-chitosan composite material with the concentration of 5 mg/mL, and dripping on the surface of a bare electrode for modification.

3. The preparation method of the aptamer sensor based on the copper-gold bimetallic core-shell structure nanoparticles according to claim 1, is characterized in that the preparation method of the luminescent reagent comprises the following steps: weighing 7.5 mg of tris (2,2' -bipyridine) ruthenium (II) chloride hexahydrate powder, dissolving in 5 mL of prepared chitosan solution, fully dissolving, standing for 3 days, and dropwise coating the solution on the surface of the electrode modified by the multi-wall carbon nanotube-chitosan composite material.

4. The preparation method of the aptamer sensor based on the copper-gold bimetal core-shell structure nanoparticles according to claim 1, wherein the preparation method of the copper-gold bimetal core-shell structure nanoparticles comprises the following steps: under the ice bath condition, 0.6 mM sodium borohydride is added into 100 ml of mixed solution of 1 mM copper sulfate and 1 mM potassium iodide, vigorous stirring is carried out to obtain nano copper colloid, 25 ml of 1 mM trisodium citrate solution is added into the prepared nano copper colloid to be mixed evenly, 10 ml of 1 mM chloroauric acid is slowly added into the solution, vigorous stirring is needed in the adding process, the solution is centrifugally purified after the reaction is finished, and the prepared copper-gold bimetallic core-shell structure nano particles are dropwise added on the surface of an electrode coated with a luminescent reagent.

5. The preparation method of the aptamer sensor based on the copper-gold bimetallic core-shell structure nanoparticles, as claimed in claim 1, is characterized in that the aptamer is a broad-spectrum aptamer with a sulfydryl modified at one end, can specifically recognize four pesticides profenofos, isocarbophos, phorate and omethoate, is modified on the surface of an electrode dropwise added with the copper-gold bimetallic core-shell structure nanoparticles, and has an action time of 1.5 hours.

6. The preparation method of the aptamer sensor based on the copper-gold bimetallic core-shell structure nanoparticles, according to claim 1, is characterized in that the conditions of non-specific binding sites are sealed: coating a layer of bovine serum albumin solution on the surface of the electrode modified with the aptamer, reacting for 30 min, cleaning the surface with ultrapure water, drying with nitrogen, and completing the preparation of the aptamer sensor.

Technical Field

The invention relates to a preparation method of an aptamer sensor based on copper-gold bimetallic core-shell structure nanoparticles, belonging to the field of electrochemical luminescence biosensors.

Background

Organophosphorus pesticides are a class of compound pesticides containing phosphorus elements, and have the advantages of low cost and high efficiency in the aspects of crop protection and pest control, so the organophosphorus pesticides are widely applied to agricultural production, have good water solubility and easy absorbability, cannot completely remove pesticide components in the environment in a short time, finally enter human bodies through biological enrichment, and can also cause certain harm to respiratory systems and nervous systems of the human bodies even if the content of the pesticides is low, therefore, the detection of the content of the organophosphorus pesticides in the vegetables is very valuable, and the aptamer is a short oligonucleotide single chain screened in vitro, can be combined with a target object with high affinity, has strong specificity, and has the advantages of low cost, easy synthesis, good stability, easy modification and the like compared with enzymes and antibodies, is an ideal identification element for constructing a quick and stable biosensor.

In the last decade, many new methods for detecting pesticides have been developed, including electrochemical sensors, fluorescence sensors, colorimetric sensors, surface enhanced raman scattering sensors, etc., electrochemical luminescence is a novel technology combining electrochemistry and chemiluminescence, and due to the advantages of easy control, high detection sensitivity, fast response speed, etc., the novel technology rapidly becomes a research hotspot in the field of biological analysis, tris (2,2' -bipyridine) ruthenium (II) chloride hexahydrate is one of the commonly used luminescent reagents in electrochemical luminescence, and due to the characteristics of stable chemical properties, high luminescence efficiency and reversible electrochemical luminescence, the novel technology provides possibility for constructing sensors with high sensitivity and good stability.

With the continuous development of nanotechnology, various nanomaterials with different spatial structures and chemical properties are applied to the development of sensors, multi-walled carbon nanotubes are carbon materials with good electron transfer capacity, large specific surface area and extremely high chemical stability, and are widely applied to the construction of sensors, metal nanoparticles are widely concerned due to the characteristics of good catalytic performance, good conductive capacity, surface plasma resonance effect and the like, wherein the research on the synthesis and application of single-metal nanoparticles is more, and in recent years, the bimetallic nanoparticles have better catalytic, conductive and luminous effects than single metals due to the synergistic effect.

Disclosure of Invention

The copper-gold bimetallic core-shell structure nano particles are applied to an electrochemical luminescence system, so that the electrochemical luminescence intensity of the system is obviously improved, a novel electrochemical luminescence aptamer sensor is constructed, and the detection of organophosphorus pesticides in vegetables is successfully realized.

The technical scheme of the invention is as follows: the electrode surface is coated with a nano material, a luminescent reagent, an identification element, a sealing agent and a target object sequentially by a layer-by-layer self-assembly technology, and the preparation process comprises the following steps of (1) modifying the electrode surface by a multi-walled carbon nano tube-chitosan composite material, (2) dripping the luminescent reagent, (3) dripping copper-gold bimetal core-shell structure nano particles, (4) modifying an aptamer, (5) coating a layer of bovine serum albumin solution to seal a non-specific binding site, cleaning the surface by ultrapure water, drying by nitrogen, and completing the preparation of the aptamer sensor, and (6) detecting the organophosphorus pesticide.

Preferably, the electrode is a platinum electrode, and the preparation method of the multi-walled carbon nanotube-chitosan composite material comprises the following steps: dissolving 0.05 g of chitosan powder in a mixed solution of 1 mL of glacial acetic acid and 99 mL of ultrapure water, stirring for more than 3 h to fully dissolve the chitosan powder, preparing 0.05% chitosan solution, weighing 25 mg of multi-walled carbon nanotubes, dispersing in 5 mL of the prepared chitosan solution, completing the preparation of the multi-walled carbon nanotube-chitosan composite material with the concentration of 5 mg/mL, and dripping on the surface of a bare electrode for modification.

Preferably, the preparation method of the luminescent reagent comprises the following steps: weighing 7.5 mg of tris (2,2' -bipyridine) ruthenium (II) chloride hexahydrate powder, dissolving in 5 mL of prepared chitosan solution, fully dissolving, standing for 3 days, and dropwise coating the solution on the surface of the electrode modified by the multi-wall carbon nanotube-chitosan composite material.

Preferably, the preparation method of the copper-gold bimetallic core-shell structure nano particle comprises the following steps: under the ice bath condition, 0.6 mM sodium borohydride is added into 100 ml of mixed solution of 1 mM copper sulfate and 1 mM potassium iodide, vigorous stirring is carried out to obtain nano copper colloid, 25 ml of 1 mM trisodium citrate solution is added into the prepared nano copper colloid to be mixed evenly, 10 ml of 1 mM chloroauric acid is slowly added into the solution, vigorous stirring is needed in the adding process, the solution is centrifugally purified after the reaction is finished, and the prepared copper-gold bimetallic core-shell structure nano particles are dropwise added on the surface of an electrode coated with a luminescent reagent.

Preferably, the aptamer is a broad-spectrum aptamer with one end modified with a sulfydryl group, can specifically identify four pesticides including profenofos, isocarbophos, phorate and omethoate, is modified on the surface of an electrode dropwise added with the copper-gold bimetallic core-shell structure nanoparticles, and has the action time of 1.5 hours.

Preferably, the non-specific binding site blocking conditions: coating a layer of bovine serum albumin solution on the surface of the electrode modified with the aptamer, reacting for 30 min, cleaning the surface with ultrapure water, drying with nitrogen, and completing the preparation of the aptamer sensor.

Preferably, the detection process of the organophosphorus pesticide is as follows: and (3) dropwise adding an organophosphorus pesticide sample to be detected on the prepared sensor, testing the electrochemiluminescence intensity in a buffer solution containing tri-n-propylamine, and reflecting the concentration of the organophosphorus pesticide according to the intensity of the electrochemiluminescence.

Compared with the prior art, the invention has the beneficial effects that: the copper-gold bimetal core-shell structure nano particles are used for constructing the electrochemical luminescence aptamer sensor, the copper-gold bimetal core-shell structure nano particles fully utilize the space structure characteristics and the chemical properties of nano copper and nano gold, the high-efficiency catalytic action of the nano gold is ensured, meanwhile, the immobilization of more nano gold is realized by virtue of the large specific surface area of the nano copper, the electrochemical luminescence intensity is enhanced, and the detection sensitivity of the aptamer sensor to organophosphorus pesticides is improved.

Drawings

FIG. 1 Process for making aptamer sensors.

Figure 2 SEM characterization of nanomaterials.

FIG. 3 TEM characterization of nanomaterials.

Figure 4 AFM characterization of nanomaterials.

Figure 5 CV characterization of nanomaterials.

Fig. 6 ECL characterization of nanomaterials.

Figure 7 condition optimization of aptamer sensors.

FIG. 8 ECL strength for different concentrations of pesticide.

FIG. 9 is a standard graph of an aptamer sensor detecting organophosphorus pesticides.

Figure 10 the sensor detects the main parameters of different pesticides.

Figure 11 actual sample spiking recovery.

Figure 12 stability analysis of aptamer sensors.

Figure 13 reproducibility analysis of aptamer sensors.

FIG. 14 specificity analysis of aptamer sensors.

Detailed Description

The present invention will be described in further detail with reference to the following drawings and examples, which are not intended to limit the invention in any manner.

Example 1: preparation of aptamer sensor

FIG. 1 is a preparation process of an aptamer sensor, firstly, 3 uL of multi-walled carbon nanotube-chitosan solution is dripped on the surface of a bare electrode, the multi-walled carbon nanotube-chitosan solution is dried in the air, then 3 uL of a luminescent reagent is dripped on the electrode, the greenhouse is dried, thirdly, 3 uL of copper-gold bimetallic core-shell structure nano particles are dripped on the electrode, then, 3 uL of aptamer solution with a modified sulfydryl at one end is added on the electrode, the reaction is carried out for 1.5 h, finally, a layer of bovine serum albumin solution is coated on the surface of the electrode for reaction for 30 min, unbound materials are slightly washed away by ultrapure water, the performance of the aptamer sensor is verified, and 3 uL of organic phosphorus pesticide is dripped on the electrode to test the luminescent effect.

Example 2: characterization of nanomaterials

(1) SEM characterization

The surface appearance of the nano material is characterized by utilizing a scanning electron microscope (figure 2), the multi-wall carbon nano tube-chitosan is composed of a large number of thin tubular structures (figure 2 a), and the copper-gold bimetallic core-shell structure nano particles are uniformly distributed on the surface of the multi-wall carbon nano tube material (figure 2 b)

(2) TEM characterization

FIG. 3 is a transmission electron microscope image of Cu-Au bimetallic core-shell nanoparticles, from which it can be clearly seen that small-sized Au nanoparticles are grown on the surface of the Cu nanoparticles

(3) AFM characterization

An atomic force microscope represents surface morphology change in an electrode modification process, a multi-walled carbon nanotube-chitosan coated bare electrode has large fluctuation, the surface roughness is 0.745 nm (figure 4 a), figure 4b is an atomic force microscope image of copper-gold bimetallic core-shell structure nanoparticles dropping on the surface of the multi-walled carbon nanotube-chitosan, and compared with a multi-walled carbon nanotube-chitosan coating, the surface roughness is further increased to 6.528 nm

(4) CV characterization

As shown in fig. 5, the electrochemical properties of the aptamer sensor were analyzed using cyclic voltammetry, and the bare electrode had a pair of reversible redox peaks (curve a); dripping the multi-walled carbon nanotube-chitosan composite material on the surface of the electrode, and obviously increasing the current peak value of the electrode (curve b); continuing to drop the luminescent reagent solution, the current peak value of the electrode is reduced (curve c); after the copper-gold bimetallic core-shell structure nano particles are modified on the electrode, the current peak value of the electrode is improved (curve d); after the electrode surface is modified with the aptamer, the current response is remarkably reduced (curve e); after the bovine serum albumin is added, the current peak value of the electrode continues to decrease (curve f), and the bovine serum albumin successfully blocks the non-specific sites on the surface of the electrode; the peak current value of the electrode decreases again after the organophosphorus pesticide is added (curve g)

(5) ECL characterization

Fig. 6 shows the electrochemiluminescence behavior of electrodes modified by different nanomaterials, an electrochemiluminescence signal begins to appear in the system after a luminescent reagent is dripped on the surface of the electrode (curve a), and is greatly improved after a multiwalled carbon nanotube-chitosan nanomaterial is modified on the electrode (curve b), and the separate nanoparticles of nanogold and a bimetal core-shell structure of copper and gold are respectively modified on the surface of the electrode, and it is found that both materials have a certain promotion effect on the luminescent system, but the promotion effect of the nanoparticles of the bimetal core-shell structure of copper and gold (curve d) is higher than that of the separate nanoparticles of gold (curve c).

Example 3: condition optimization of aptamer sensors

The aptamer concentration, the incubation time, the base solution pH value and the tri-n-propylamine concentration are important factors influencing the electrochemiluminescence intensity of the aptamer sensor, as shown in figure 7a, the electrochemiluminescence intensity is reduced along with the increase of the aptamer concentration, when the aptamer concentration is 1 uM, the electrochemiluminescence intensity is reduced to the lowest value, then the luminescence intensity is increased along with the increase of the aptamer concentration, therefore, the aptamer concentration is selected from 1 uM to continue the subsequent experiment, the influence of the incubation time on the luminescence intensity of the aptamer sensor is shown in figure 7b, the electrochemiluminescence intensity is gradually reduced along with the increase of the incubation time, but the change becomes unobvious after 50 min, which shows that about 50 min is required for the complete combination of the aptamer and pesticide, the pH value of the base solution is also one of key factors influencing the electrochemiluminescence intensity, as shown in figure 7c, the optimal response is obtained when the pH value is 8.0, tri-n-propylamine was used as a co-reactant of the light-emitting system, the concentration of the tri-n-propylamine had a large influence on the electrochemiluminescence intensity, the electrochemiluminescence intensity was significantly increased with the increase of the concentration of the tri-n-propylamine in the solution (FIG. 7 d), and when the concentration of the tri-n-propylamine was increased to 8 mM, the electrochemiluminescence intensity was maximized and then remained unchanged, so that the concentration of the tri-n-propylamine was selected to be 8 mM.

Example 4: detection of organophosphorus pesticide by aptamer sensor

Under the best experimental conditions, the prepared sensor is used for detecting four organophosphorus pesticides of profenofos, isocarbophos, phorate and omethoate, the figure 8 is the relation between the concentration of the pesticide and the electrochemiluminescence intensity (figure 8a: profenofos; figure 8b: isocarbophos; figure 8c: phorate; figure 8d: omethoate), the electrochemiluminescence intensity is gradually reduced along with the increase of the concentration of the pesticide, figure 9 shows (figure 9e: profenofos; figure 9f: isocarbophos; figure 9g: phorate; figure 9h: omethoate), the electrochemiluminescence intensity and the logarithm of the concentration of the pesticide have good linear relation and have lower detection limit (S/N = 3) in a certain concentration range, the relevant detection parameters of the aptamer sensor are shown in figure 10, the detection platform is used for detecting isocarbophos in vegetables, the detection result is shown in figure 11, the recovery rate is 89.43-126.15%, and the relative standard deviation is less than 5.48%.

Example 5: performance analysis of aptamer sensors

(1) Stability test

FIG. 12 shows the results of stability tests of sensors, in which 12 electrodes were prepared under the same conditions and the concentration was 1X 10 immediately⁵ng/L isocarbophos is incubated on the surfaces of 4 electrodes, the electrochemical luminescence intensity of the electrodes is detected, the rest 8 electrodes are stored in a refrigerator with four degrees below zero, 4 electrodes are taken out every other week for detection, and the luminescence intensity of the electrodes after seven days is 97.95 percent of that of the electrodes before seven days (RSD =3.33 percent), and the luminescence intensity of 4 electrodes after fourteen days is only reduced by 7.32 percent (RSD =6.95 percent)

(2) Reproducibility test

The results of the sensor reproducibility test are shown in FIG. 13, and 4 electrodes were prepared under the same conditions for detecting a concentration of 1X 10³The relative standard deviation of 4 electrodes of the isocarbophos at the ng/L is 2.71 percent

(3) Specificity test

Four pesticides, namely acetamiprid, carbofuran, malathion and monocrotophos, are selected to analyze the specificity of the sensor, as shown in figure 14, under the same concentration, the four non-detection pesticides, whether a single system or a mixed system, can not effectively inhibit the intensity of electrochemiluminescence, and the luminous intensity is very high (FIG. 14 a: acetamiprid; FIG. 14 b: carbofuran; FIG. 14 c: malathion; FIG. 14 d: monocrotophos; FIG. 14 e: acetamiprid + carbofuran + malathion + monocrotophos), and the electrochemical luminescence intensity is obviously reduced once the components of the target object exist in the system (figure 14f: profenofos; figure 14g: isocarbophos; figure 14h: phorate; figure 14i: omethoate; figure 14j: profenofos + isocarbophos + phorate + omethoate; figure 14k: profenofos + isocarbophos + phorate + omethoate + acetamiprid + carbofuran + malathion + monocrotophos).