Preparation of aptamer functionalized coral-like gold loaded carbon-magnesium composite and application thereof in chemiluminescence sensing

Abstract

The application discloses a kind of aptamer functionalized coral-like gold load carbon magnesium composite material and its application technology in chemiluminescence sensing, main technical features are: carbon magnesium oxide composite material is prepared, and gold nanoparticle is decorated on its surface, and after gold nanoparticle modification, the surface of composite material is decorated thrombin aptamer, thus obtain aptamer functionalized gold nanoparticle / carbon magnesium oxide composite material, and the composite material preparation process is simple, condition is easy to control, and production cost is low;The application simultaneously provides a kind of new method for detecting thrombin, and the aptamer functionalized coral-like gold load carbon magnesium composite material prepared is applied to chemiluminescence sensing detection thrombin, with low detection limit, good selectivity, instrument equipment operation simple and the like advantages, for application in human serum sample detection provides the possibility, has important biological significance in human health.

Description

TECHNICAL FIELD

[0001] The present application relates to the preparation of aptamer functionalized coral-like composite materials and its application in chemiluminescence sensing technology, belonging to the field of photochemical sensing technology, specifically relates to the preparation of coral-like gold nanoparticles / magnesium oxide carbide composite materials and its surface aptamer functionalization, and its application to the detection of thrombin in chemiluminescence sensor. BACKGROUND

[0002] Carbon materials have excellent chemical, mechanical, thermal stability, and because of high specific surface area and good adsorption capacity are widely used in many fields. Nanostructured metal oxides have a special morphology, and this composite material can still maintain the original morphology after carbonization, and its special morphology and pore structure can provide a large specific surface area, thereby improving the catalytic and adsorption properties of the material. Gold nanomaterials have good photoelectricity, electrical conductivity, and catalytic activity, and are widely concerned in the fields of environment, biology, food, and drug analysis. By utilizing the high stability and good catalytic activity of gold nanoparticles, they can be loaded onto the surface of other materials, thereby playing a synergistic catalytic role and improving the overall catalytic performance of the material.

[0003] Thrombin (THR) is a serine protease and is also the main effector protease in the blood coagulation cascade, showing procoagulant and anticoagulant properties. Thrombin can convert fibrinogen into fibrin. After local application, it acts on the blood on the surface of the lesion to form a stable blood clot quickly, which is used to control capillary and venous bleeding, or as an adhesive and fixing agent for skin and tissue grafts. Imbalance of thrombin in the body can lead to bleeding or thrombotic diseases. Thrombin can also be used as a biomarker for the diagnosis and treatment of cardiovascular diseases, tumor cell growth, inflammation, and coagulation. Therefore, sensitive detection of thrombin is particularly important for human health. Currently, common methods for detecting thrombin include fluorescence, colorimetry, electrochemistry, and electrochemiluminescence. However, these methods have their own problems, such as poor reproducibility, complex operation, and narrow linear range. Therefore, there is an urgent need to develop a method for detecting thrombin with high sensitivity and selectivity.

[0004] Compared with traditional chemical analysis method, flow injection-chemiluminescence method can obtain information that uniform equilibrium system cannot provide, and can be well applied to quantitative analysis of unstable reaction, which is a revolution of analysis technology. The technology greatly improves analysis rate, selectivity and sensitivity of analysis method, and has the advantages of small volume of instrument device, simple operation, rapid analysis, high sensitivity, good repeatability of results and low detection limit. However, chemiluminescence method has a great disadvantage of poor selectivity, because various coexisting substances can easily cause change of chemiluminescence intensity, and this disadvantage can be overcome by introducing some specific recognition materials, such as molecular imprinting material, antigen-antibody material and aptamer material.

[0005] In the patent, gold nanoparticle / magnesium oxide carbide composite material is prepared, and thrombin aptamer is modified on the surface of the material to obtain coral-shaped composite material with easy separation, excellent immobilization performance and high specific recognition ability. The functionalized coral-shaped composite material is combined with flow injection-chemiluminescence technology to construct a chemiluminescence sensor for high-sensitivity and high-selectivity detection of thrombin, and a new method for simple, sensitive and selective detection of thrombin is invented. SUMMARY

[0006] One of the purposes of the present application is to provide a preparation method of aptamer functionalized coral-shaped composite material, mainly to prepare gold nanoparticle / magnesium oxide carbide composite material, and modify thrombin aptamer on the surface of the material to obtain coral-shaped composite material with high specific recognition ability to thrombin molecules;

[0007] The purpose of the present application is realized by the following technical solutions.

[0008] (1) Preparation of magnesium oxide carbide composite material: 0.25~0.30 g polyvinylpyrrolidone is weighed and added to 40 mL ethylene glycol, then 0.46~0.50 g urea is added to the above solution, and ultrasonic is applied for 15~20 min until it is uniformly dispersed; 180~220 μL of 30% ammonia water and 0.40~0.44 g of magnesium acetate tetrahydrate are sequentially added to the above solution, and stirring is carried out at room temperature for 20~40 min until the solution becomes clear; the clarified solution is transferred to a reaction kettle, and reaction is carried out at 175~195℃ for 5 h; after reaction is completed and cooling, the product is transferred to a centrifuge tube, and then centrifugation is carried out at a speed of 8000 r / min for 6 min, and the supernatant is removed; finally, vacuum drying is carried out at 60°C to obtain glycolated magnesium precursor; under the protection of nitrogen, the obtained precursor is carbonized in a tube furnace at 800 °C for 2~4 h, and finally the magnesium oxide carbide composite material is obtained;

[0009] (2) Preparation of coral-like gold nanoparticles / magnesium carbide composite materials: Weigh 0.8-1.0 g of magnesium carbide composite materials, add them to 50 mL of deionized water, and sonicate for 10-15 min until they are evenly dispersed; pipette 150-160 μL of 0.117 mol / L chloroauric acid solution and add it to the above solution, stir magnetically for 10-15 min, and then continue to heat the solution until it boils slightly; add 2-4 mL of 1% sodium citrate solution, stop heating, continue stirring for 10-15 min until the solution cools to room temperature, and then centrifuge at 8000 r / min for 5 min to remove the supernatant; finally, the centrifuged product is vacuum-dried at 60°C to obtain a gold nanoparticles / magnesium carbide composite material;

[0010] (3) Preparation of aptamer-functionalized gold nanoparticles / magnesium oxide carbide composite materials: 0.1-0.2 g of gold nanoparticles / magnesium oxide carbide composite materials were weighed and added to 5 mL of 0.02 mol / L phosphate buffer (pH = 7.4), and then 10-30 nmol of thrombin aptamer was added to the centrifuge tube; the centrifuge tube was shaken for 15-25 min, and then incubated at 20˚C for 36 h; finally, the tube was centrifuged at 10,000 r / min for 6 min, and the supernatant was removed to obtain the aptamer-functionalized gold nanoparticles / magnesium oxide carbide composite materials.

[0011] Another object of the present invention is to use an aptamer-functionalized gold nanoparticle / magnesium oxide carbide composite material in a chemiluminescence sensor for detecting thrombin. The prepared aptamer-functionalized gold nanoparticle / magnesium oxide carbide composite material is fixed on aptamer complementary chain-functionalized magnetic graphene oxide. When thrombin is present, the thrombin molecules are specifically recognized and combined with their aptamers, causing the gold nanoparticles / magnesium oxide carbide to be released. The released gold nanoparticles / magnesium oxide carbide catalyze the luminol-hydrogen peroxide chemiluminescence system, causing a change in chemiluminescence intensity, thereby detecting thrombin. The chemiluminescence sensor for detecting thrombin has the advantages of high sensitivity, good selectivity, and simple and easy operation of the instrument. In the construction of the chemiluminescence sensor, the immobilization performance of the synthetic material is studied, the chemiluminescence conditions are optimized, and the analytical performance is tested. Finally, the sensor is used to detect thrombin in human serum samples.

[0012] The advantages and effects of the present invention are:

[0013] (1) The coral-shaped gold nanoparticle / magnesium oxide carbide composite material is prepared, and the aptamer is functionalized, then the aptamer is fixed on the aptamer complementary chain functionalized magnetic graphene oxide by using the principle of base complementary pairing, so as to construct a chemiluminescence sensor; the magnesium oxide carbide has the advantages of large specific surface area and strong adsorption, which provides the possibility for the loading of gold nanoparticles; the coral-shaped gold nanoparticle / magnesium oxide carbide composite material has rich functional groups and ionic bonds, and provides a large number of action sites for the aptamer, and the saturated fixed amount of the aptamer functionalized gold nanoparticle / magnesium oxide carbide on the aptamer complementary chain functionalized magnetic graphene oxide is 3 mg / mg;

[0014] (2) The aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material prepared by the method has the advantages of simple preparation process, easy to control conditions, strong catalytic performance and specific recognition ability of the material;

[0015] (3) The aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material prepared by the method can be applied to the chemiluminescence sensor for detecting thrombin, which provides the possibility for practical detection, and has important biological significance in human health. Embodiment Example 1

[0016] (1) Preparation of magnesium oxide carbide composite material: 0.25 g of polyvinylpyrrolidone is weighed and added to 40 mL of ethylene glycol, then 0.46 g of urea is added to the above solution, and ultrasonic is performed for 15 min until it is uniformly dispersed; 180 μL of 30% ammonia water and 0.40 g of magnesium acetate tetrahydrate are sequentially added to the above solution, and stirring is performed at room temperature for 20 min until the solution becomes clear; the clear solution is transferred to a reaction kettle, and reaction is performed at 175°C for 5 h; after the reaction is completed and cooled, the product is transferred to a centrifuge tube, and then centrifugation is performed at a speed of 8000 r / min for 6 min, and the supernatant is removed; finally, vacuum drying is performed at 60°C to obtain glycolated magnesium precursor; the obtained precursor is carbonized in a tube furnace at 800 °C for 2 h under the protection of nitrogen, and finally the magnesium oxide carbide composite material is obtained;

[0017] (2) Preparation of coral-like gold nanoparticle / magnesium oxide carbonate composite: 0.8 g of magnesium oxide carbonate composite was weighed and added to 50 mL of deionized water, and ultrasonicated for 10 min until uniformly dispersed; 150 mL of 0.117 mol / L chloroauric acid solution was removed and added to the above solution, and magnetically stirred for 10 min, then the solution was continuously heated to micro-boiling; 2 mL of 1% sodium citrate solution was added, the heating was stopped, and the stirring was continued for 10 min until the solution cooled to room temperature, then centrifuged at a speed of 8000 r / min for 5 min, and the supernatant was removed; finally, the centrifuged product was vacuum dried at 60°C to obtain a gold nanoparticle / magnesium oxide carbonate composite;

[0018] (3) Preparation of aptamer functionalized gold nanoparticle / magnesium oxide carbonate composite: 0.1 g of gold nanoparticle / magnesium oxide carbonate composite was weighed and added to 5 mL of 0.02 mol / L phosphate buffer solution (pH=7.4), then 10 nmol of thrombin aptamer was added to the centrifuge tube; the centrifuge tube was shaken for 15 min, then incubated at 20°C for 36 h; finally, centrifuged at a speed of 10000 r / min for 6 min, and the supernatant was removed to obtain an aptamer functionalized gold nanoparticle / magnesium oxide carbonate composite. Example 2

[0019] (1) Preparation of magnesium oxide carbonate composite: 0.27 g of polyvinylpyrrolidone was weighed and added to 40 mL of ethylene glycol, then 0.48 g of urea was added to the above solution and ultrasonicated for 17 min until uniformly dispersed; 200 μL of 30% ammonia water and 0.42 g of magnesium acetate tetrahydrate were sequentially added to the above solution, and stirred at room temperature for 30 min until the solution became clear; the clear solution was transferred to a reaction kettle and reacted at 185°C for 5 h, after the reaction was completed and cooled, the product was transferred to a centrifuge tube, then centrifuged at a speed of 8000 r / min for 6 min, and the supernatant was removed; finally, vacuum dried at 60°C to obtain glycolated magnesium precursor; under the protection of nitrogen, the obtained precursor was carbonized in a tube furnace at 800°C for 3 h, and finally the magnesium oxide carbonate composite was obtained;

[0020] (2) Preparation of coral-like gold nanoparticle / magnesium oxide carbonate composite: 0.9 g of magnesium oxide carbonate composite was weighed and added to 50 mL of deionized water, and ultrasonic dispersion was performed for 12 min until uniform dispersion was achieved; 155 mL of 0.117 mol / L chloroauric acid solution was removed and added to the above solution, and magnetic stirring was performed for 12 min, and then the solution was continuously heated to micro-boiling; 3 mL of 1% sodium citrate solution was added, heating was stopped, and continuous stirring was performed for 12 min until the solution cooled to room temperature, and then centrifugation was performed at a speed of 8000 r / min for 5 min, and the supernatant was removed; finally, the centrifuged product was vacuum dried at 60°C to obtain a gold nanoparticle / magnesium oxide carbonate composite;

[0021] (3) Preparation of aptamer functionalized gold nanoparticle / magnesium oxide carbonate composite: 0.15 g of gold nanoparticle / magnesium oxide carbonate composite was weighed and added to 5 mL of 0.02 mol / L phosphate buffer solution (pH = 7.4), and then 20 nmol of thrombin aptamer was added to the centrifuge tube; the centrifuge tube was shaken for 20 min, and then incubated at 20°C for 36 h; finally, centrifugation was performed at a speed of 10000 r / min for 6 min, and the supernatant was removed to obtain an aptamer functionalized gold nanoparticle / magnesium oxide carbonate composite. Example 3

[0022] (1) Preparation of magnesium oxide carbonate composite: 0.30 g of polyvinylpyrrolidone was weighed and added to 40 mL of ethylene glycol, and then 0.50 g of urea was added to the above solution, and ultrasonic dispersion was performed for 20 min until uniform dispersion was achieved; 220 μL of 30% ammonia water and 0.44 g of magnesium acetate tetrahydrate were sequentially added to the above solution, and stirring was performed at room temperature for 40 min until the solution became clear; the clear solution was transferred to a reaction kettle, and reaction was performed at 195°C for 5 h; after the reaction was completed and cooled, the product was transferred to a centrifuge tube, and then centrifugation was performed at a speed of 8000 r / min for 6 min, and the supernatant was removed; finally, vacuum drying was performed at 60°C to obtain glycolated magnesium precursor; under the protection of nitrogen, the obtained precursor was carbonized in a tube furnace at 800°C for 4 h, and finally a magnesium oxide carbonate composite was obtained;

[0023] (2) Preparation of coral-like gold nanoparticle / magnesium oxide carbide composite material: 1.0 g of magnesium oxide carbide composite material was weighed and added to 50 mL of deionized water, and ultrasonicated for 15 min until uniformly dispersed; 160 μL of 0.117 mol / L chloroauric acid solution was removed and added to the above solution, and magnetically stirred for 15 min, then the solution was continuously heated to micro-boiling; 4 mL of 1% sodium citrate solution was added, the heating was stopped, and the stirring was continued for 15 min until the solution cooled to room temperature, then centrifuged at a speed of 8000 r / min for 5 min, and the supernatant was removed; finally, the centrifuged product was vacuum dried at 60°C to obtain the gold nanoparticle / magnesium oxide carbide composite material;

[0024] (3) Preparation of aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material: 0.2 g of gold nanoparticle / magnesium oxide carbide composite material was weighed and added to 5 mL of 0.02 mol / L phosphate buffer solution (pH=7.4), then 30 nmol of thrombin aptamer was added to the centrifuge tube; the centrifuge tube was shaken for 25 min, then incubated at 20°C for 36 h; finally, it was centrifuged at a speed of 10000 r / min for 6 min, and the supernatant was removed to obtain the aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material. Example 4

[0025] Method for applying aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material to chemiluminescence sensor for detecting thrombin: the prepared aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material was fixed on the aptamer complementary chain functionalized magnetic graphene oxide, when thrombin was present, the thrombin molecules and their aptamers specifically recognized and combined together, so that the gold nanoparticle / magnesium oxide carbide was released, the released gold nanoparticle / magnesium oxide carbide catalyzed the luminol-hydrogen peroxide chemiluminescence system, causing a change in the chemiluminescence intensity, realizing the detection of thrombin, the specific operation process was as follows:

[0026] (1) Study on the fixing performance of aptamer complementary chain functionalized magnetic graphene oxide on aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material: an equal amount of aptamer complementary chain functionalized magnetic graphene oxide was accurately weighed and placed in a 50 mL cuvette, and an appropriate amount of known concentration of aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material solution was added, and the chemiluminescence intensity was measured by flow injection-chemiluminescence instrument, according to the concentration of gold nanoparticle / magnesium oxide carbide composite material solution when the chemiluminescence intensity suddenly changed, the fixed amount of aptamer complementary chain functionalized magnetic graphene oxide on aptamer functionalized gold nanoparticle / magnesium oxide carbide composite material could be calculated Q ;

[0027] (2) Chemiluminescence condition optimization: By controlling the single variable method, the pump speed of the main and auxiliary pumps, the concentration of luminol, hydrogen peroxide and sodium hydroxide were optimized respectively. According to the highest point of chemiluminescence intensity, the optimal chemiluminescence condition was obtained;

[0028] The aptamer functionalized gold nanoparticles / magnesium oxide carbon composite material was applied to the chemiluminescence sensor for the detection of thrombin. The saturation fixation amount of the aptamer complementary chain functionalized magnetic graphene oxide on the aptamer functionalized gold nanoparticles / magnesium oxide carbon composite material was 3 mg / mg. The optimal experimental conditions were as follows: the main pump speed was 35 r / min, the auxiliary pump speed was 40 r / min, the concentration of NaOH was 0.3 mol / L, the concentration of H2O2 was 0.04 mol / L, and the concentration of luminol was 5.0×10 -5 mol / L. The working curve could be drawn, which provided the possibility for the detection of thrombin in human serum.

Claims

1. A method for preparing an aptamer-functionalized coral-shaped gold-loaded carbon-magnesium composite material, characterized in that The method has the following process steps: (1) Preparation of carbonized magnesium oxide composite material: Weigh 0.25-0.30 g of polyvinyl pyrrolidone and add it to 40 mL of ethylene glycol solution, then add 0.46-0.50 g of urea to the ethylene glycol solution, and ultrasonicate for 15-20 min until it is evenly dispersed; add 180-220 μL of 30% ammonia water and 0.40-0.44 g of magnesium acetate tetrahydrate to the ethylene glycol mixed solution in sequence, and stir at room temperature for 20-40 min until the solution becomes clear; the clarified solution is transferred to a reactor and reacted at 175-195 ° C for 5 h. After the reaction is completed and cooled, the product is transferred to a centrifuge tube, and then centrifuged at a speed of 8000 r / min for 6 min, and the supernatant is removed; finally, vacuum drying is performed at 60 ° C to obtain an ethanolated magnesium precursor; Under nitrogen protection, the obtained precursor was carbonized in a tube furnace at 800°C for 2 to 4 hours to obtain a carbonized magnesium oxide composite material. (2) Preparation of coral-like gold nanoparticles / magnesium carbide composite materials: Weigh 0.8-1.0 g of magnesium carbide composite materials, add them to 50 mL of deionized water, and sonicate for 10-15 min until they are evenly dispersed; pipette 150-160 μL of 0.117 mol / L chloroauric acid solution, add it to the above solution, magnetically stir for 10-15 min, and then continue to heat the solution until it boils slightly; add 2-4 mL of 1% sodium citrate solution, stop heating, continue stirring for 10-15 min until the solution cools to room temperature, then centrifuge at 8000 r / min for 5 min, and remove the supernatant; finally, the centrifuged product is vacuum-dried at 60°C to obtain coral-like gold nanoparticles / magnesium carbide composite materials; (3) Preparation of aptamer-functionalized coral-like gold nanoparticles / magnesium carbide composite materials: 0.1-0.2 g of gold nanoparticles / magnesium carbide composite materials were weighed and added to 5 mL of 0.02 mol / L phosphate buffer (pH = 7.4), and then 10-30 nmol of thrombin aptamer was added to the centrifuge tube; the centrifuge tube was shaken for 15-25 min, and then incubated at 20°C for 36 h; finally, the tube was centrifuged at 10,000 r / min for 6 min, and the supernatant was removed to obtain the aptamer-functionalized coral-like gold nanoparticles / magnesium carbide composite materials.

2. The method for preparing an aptamer-functionalized coralline gold-supported carbon-magnesium composite material according to claim 1, characterized in that: In step (1), the powder dried in vacuum at 60° C. is placed in a tubular furnace and carbonized at a high temperature of 800° C. under nitrogen protection to obtain a carbonized magnesium oxide composite material, which makes the material structure more stable and improves its adsorption performance.

3. The method for preparing an aptamer-functionalized coral-shaped gold-loaded carbon-magnesium composite material according to claim 1, characterized in that: In step (3), the coral-like gold nanoparticle / magnesium oxide carbide composite material is modified with aptamer functionalization, so that the base complementary pairing effect between the complementary double strands of oligonucleotides can be used to combine it with the magnetic graphene oxide functionalized with the aptamer complementary chain, thereby being used to construct a chemiluminescent sensor.

4. A method for using the aptamer-functionalized coral-shaped gold-loaded carbon-magnesium composite material prepared by the method for preparing the aptamer-functionalized coral-shaped gold-loaded carbon-magnesium composite material according to claim 1 for chemiluminescent sensing detection of thrombin, characterized in that The chemiluminescence sensor was constructed using the flow injection-chemiluminescence method as follows: the prepared aptamer-functionalized coral-shaped gold nanoparticles / magnesium oxide carbide composite material was fixed on the aptamer complementary chain-functionalized magnetic graphene oxide. When thrombin was present, the thrombin molecules specifically recognized and bound to its aptamer, causing the gold nanoparticles / magnesium oxide carbide to be released. The released gold nanoparticles / magnesium oxide carbide catalyzed the luminol-hydrogen peroxide chemiluminescence system, causing a change in the chemiluminescence intensity, thereby realizing the detection of thrombin.