CN113219016A - Preparation method of electrochemical immunosensor based on uracil modified graphite-like phase carbon nitride - Google Patents
Preparation method of electrochemical immunosensor based on uracil modified graphite-like phase carbon nitride Download PDFInfo
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Abstract
The invention relates to a preparation method of an electrochemical immunosensor based on uracil modified graphite-like phase carbon nitride. According to the invention, in a manner of constructing the sandwich type electrochemical sensor, uracil-doped modified graphite-like carbon nitride is combined with Cyfra21-1 primary antibody to form a primary antibody marker, and the aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material is combined with Cyfra21-1 secondary antibody to form a secondary antibody marker, so that the construction process is greatly simplified. The uracil-doped modified graphite-like phase carbon nitride is used as a substrate luminescent material, and a very strong and stable electrochemical luminescence signal is provided. The aminated dendritic fiber nano-silica is used for providing high specific surface area and high-load copper sulfide quantum dots, high quenching efficiency is realized based on resonance energy transfer, ultra-sensitive detection of Cyfra21-1 is realized, and the detection limit is 20.3 fg mL‑1。
Description
Technical Field
The invention relates to a preparation method of an electrochemical immunosensor based on uracil-modified graphite-like carbon nitride, in particular to a sandwich quenching type electrochemical immunosensor for detecting Cyfra21-1, which is prepared by taking uracil-doped modified graphite-like carbon nitride as a substrate luminescent material and taking an aminated dendritic fiber nano silicon dioxide loaded copper sulfide quantum dot composite material as a marker for marking a secondary antibody of a Cyfra21-1 recognition antibody, and belongs to the technical field of novel functional materials and biosensing detection.
Background
Lung cancer is one of the most lethal cancers known, causing massive death worldwide, and is largely divided into Small Cell Lung Cancer (SCLC) and non-small cell lung cancer (NSCLC). Cyfra21-1 is currently considered to be a tumor marker mainly used for detecting lung cancer, and has important value particularly for diagnosing non-small cell lung cancer (NSCLC). The serum concentration level of Cyfra21-1 is positively correlated with the clinical stage of tumor, and can be used as effective index for tracking early recurrence after lung cancer operation and radiotherapy and chemotherapy. Electrochemiluminescence is a new product combining electrochemistry and luminescence, has the advantages of low background, wide dynamic range, simple and convenient instrument and equipment, sensitive detection and the like, and is widely concerned in the fields of biological analysis, food safety analysis, environmental pollution monitoring and the like.
Graphite-like phase carbon nitride is a planar two-dimensional sheet material with good biocompatibility, and is commonly used as a catalyst or a good carrier due to its simple preparation, good chemical stability and adjustable electronic structure. In recent years, many scholars focus on the electrochemical luminescence property of graphite-like carbon nitride and apply the electrochemical luminescence property to the field of electrochemical immunosensors, but the original graphite-like carbon nitride has weak chemiluminescence and unstable signals, so that the application of the original graphite-like carbon nitride to the immunosensors is greatly limited, and most common methods for improving the luminescence property of graphite-like carbon nitride are doped with noble metal or heteroatom and semiconductor, so that the preparation process is complex and the price is high. The invention improves the electrochemiluminescence property of the graphite-like carbon nitride by doping uracil, changes the electron distribution of the graphite-like carbon nitride network structure by copolymerizing uracil and melamine with different proportions, does not introduce heteroatoms, endows the modified graphite-like carbon nitride with excellent electrochemiluminescence behavior, and greatly expands the application prospect of the modified graphite-like carbon nitride.
An electrochemical immunosensor is a type of detection device that determines the concentration of an analyte based on the electrical-to-optical signal transduction characteristics of a substance. Common construction processes require layer-by-layer modification, are complex and affect the biological activity of immune molecules.
The invention constructs a novel electrochemical luminescence sensor based on uracil modified graphite-like carbon nitride, and the novel electrochemical luminescence sensor is used for detecting Cyfra 21-1. The uracil modified graphite-like carbon nitride is used as a substrate luminescent material, has good biocompatibility, stable signal and strong anti-interference capability, and is connected with the primary antibody of Cyfra21-1 to form a primary antibody marker. In addition, the aminated dendritic fiber nano silica-loaded copper sulfide quantum dots are used as quenching materials, so that a good luminescent signal quenching effect can be realized. The reason is that the ultraviolet absorption spectrum of the aminated dendritic fiber nano-silica-supported copper sulfide quantum dot is greatly overlapped with the electrochemical luminescence emission spectrum of the modified graphite-like phase carbon nitride, so that quenching can be realized based on the resonance energy transfer principle. The secondary antibody of the aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot for labeling Cyfra21-1 is used as a secondary antibody marker, so that the sandwich quenching type electrochemical immunosensor is constructed, the ultrasensitive detection of Cyfra21-1 is realized, and the detection limit is 20.3 fg mL-1. Compared with the conventional sensor, the electrochemical immunosensor constructed by the invention has the advantages that the number of modified layers is greatly reduced, and based on the excellent performance of materials and a simplified detection method, the electrochemical immunosensor has high sensitivity, low detection limit and good stability. Based on the above findings, the inventors have completed the present invention.
Disclosure of Invention
One of the purposes of the invention is to adopt uracil modified graphite-like phase carbon nitride as a substrate luminescent material, which has good biocompatibility, stable electrochemical signal and strong anti-interference capability, so that the uracil modified graphite-like phase carbon nitride is connected with a primary antibody of Cyfra21-1 to form a primary antibody marker, thereby simplifying the construction process of the sensor.
The other purpose of the invention is to use the aminated dendritic fiber nano-silica-loaded copper sulfide quantum dots as quenching materials, so that a good luminescent signal quenching effect can be realized, and because the ultraviolet absorption spectrum of the aminated dendritic fiber nano-silica-loaded copper sulfide quantum dots is greatly overlapped with the electrochemical luminescence emission spectrum of the modified graphite-like carbon nitride, the quenching can be realized based on the resonance energy transfer principle, and a new resonance energy transfer donor-acceptor pair is provided for constructing an electrochemical immunosensor.
The invention also aims to provide a preparation method of the novel electrochemical immunosensor based on resonance energy transfer, the method respectively marks primary antibody and secondary antibody of the substance to be detected with the substrate luminescent material and the quenching material, and the prepared sensor is more convenient and simpler, and has strong specificity, good stability, high sensitivity and good reproducibility.
The fourth purpose of the invention is to realize the construction of the electrochemical immunosensor and sensitively detect Cyfra21-1, and the detection limit is 20.3 fg mL-1The purpose of the electrochemiluminescence sensor in detecting Cyfra21-1 is achieved.
Technical scheme of the invention
1. A preparation method of an electrochemical immunosensor based on uracil modified graphite-like phase carbon nitride is characterized by comprising the following steps:
(1) preparation of uracil modified graphite-like phase carbon nitride
The preparation method comprises the steps of preparing a modified graphite-like phase carbon nitride sample by copolymerizing melamine and uracil, uniformly mixing 1 g of melamine and 10-100 mg of uracil in an agate mortar for full grinding, then putting the mixture into a covered ceramic crucible, and placing the mixture in a muffle furnace at 5 ℃ for min-1The temperature rising rate is increased to 550 ℃ and kept for 4 h to obtain uracil-modified graphite-like carbon nitride, and when uracil is not doped, other conditions are the same, so that the original graphite-like carbon nitride is prepared;
(2) preparation of modified graphite-like phase carbon nitride combined Cyfra21-1 recognition antibody primary anti-label
5-20 microgram mL-1The primary antibody of Cyfra21-1 is activated by 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with uracil modified graphite-like carbon nitride for 6 h at 4 ℃ to obtain a modified graphite-like carbon nitride-Cyfra 21-1 recognition antibody primary antibody marker;
(3) preparation of dendritic fiber nano-silica
Dissolving 2.5 g tetraethyl silicate in a mixed solution of 30 mL cyclohexane and 1.5 mL amyl alcohol, and fully stirring to obtain a solution A; dissolving 1 g of cetyl pyridine bromide and 0.6 g of urea in 30 mL of deionized water, and fully stirring to form a solution B; adding the solution B into the solution A, fully stirring to form a mixed reaction solution, placing the reaction solution into a polytetrafluoroethylene reaction kettle, reacting for 4 hours at 120 ℃ in the hydrothermal reaction kettle, centrifugally separating a product after the reaction is finished, washing the product for 3 times by using deionized water and acetone respectively, drying for 12 hours in a vacuum drying oven, calcining the obtained product in a muffle furnace at 550 ℃ for 6 hours, and removing a template agent to obtain the dendritic fiber nano-silica;
(4) preparation of aminated dendritic fiber nano-silica
Adding 50-100 mg of the prepared dendritic fiber nano-silica into a solution containing 10 mL of deionized water and 0.5 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, cooling to room temperature, centrifuging, and drying in vacuum to obtain aminated dendritic fiber nano-silica;
(5) preparation of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material
Dissolving 50-100 mg of aminated dendritic fiber nano silicon dioxide powder in 300 mL of deionized water for ultrasonic treatment for 1 h, then dissolving 50-100 mg of copper chloride and 60-120 mg of trisodium citrate in the suspension, magnetically stirring for 5 min, and then dissolving 3-10 mL and 8 mg mL of trisodium citrate-1Slowly dripping the sodium sulfide nonahydrate solution into the suspension, and heatingKeeping the temperature at 90 ℃ for 30 min to obtain the aminated dendritic fiber nano silicon dioxide loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody
20-100. mu.L of a secondary antibody (10. mu.g mL) of Cyfra21-1 was added-1) Adding the mixture into 2 mL of 1-3 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in the amination dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material solution, centrifuging, and dispersing the obtained product in 1 mL PBS to obtain a secondary antibody marker solution of the amination dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody;
(8) preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and then polishing 6 muL and 0.5-2.5 mg mL-1The primary-antibody marker solution of the uracil-modified graphite-phase carbon nitride combined Cyfra21-1 recognition antibody is dripped on the surface of the electrode and dried at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-50 ng mL-1A series of Cyfra21-1 antigens with different concentrations are applied to the surface of the electrode, incubated for 2 h, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding a second antibody marker solution of 6 mu L of aminated dendritic fiber nano-silica loaded copper sulfide quantum dot composite material combined with the Cyfra21-1 recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting Cyfra 21-1.
2. The detection method of the electrochemical immunosensor comprises the following steps:
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation is connected with the chemiluminescence detector, the high voltage of a photomultiplier is set to be 500-800V, and the electrochemical luminescence sensor contains 20-100 mmoL L-1The test was performed in a PBS buffer solution of potassium persulfate;
(2) detecting Cyfra21-1 standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.6-0V;
(3) observing the electrochemiluminescence intensity of the sensor before and after Cyfra21-1 is added, recording the linear relation between the electrochemiluminescence intensity value and the concentration of Cyfra21-1, and drawing a working curve;
(4) and replacing Cyfra21-1 antigen standard solution with the Cyfra21-1 antigen sample solution to be detected for detection.
The linear range of the sensor for detecting Cyfra21-1 antigen is 0.00005-50 ng mL-1The detection limit is 20.3 fg mL-1。
The chemicals required for the synthesis were all purchased at the local reagent store and were not reprocessed.
Advantageous results of the invention
(1) The invention adopts uracil modified graphite-like carbon nitride as a substrate luminescent material, the doping of uracil increases the structural defects of the modified graphite-like carbon nitride network, greatly improves the electronic transmission of the whole material, greatly overcomes the defects that the electrochemical luminescent signal of the original graphite-like carbon nitride is weak and unstable, greatly improves the electroluminescent performance, and has important significance for the expanded application of the graphite-like carbon nitride.
(2) The invention adopts a novel donor-acceptor pair for resonance energy transfer, takes uracil-modified graphite-like phase carbon nitride as a substrate luminescent material, and takes aminated dendritic fiber nano-silica-loaded copper sulfide quantum dots as a quenching material to construct a sandwich quenching type electrochemical immunosensor.
(3) The invention provides a novel method for constructing an electrochemical immunosensor, a primary antibody and a secondary antibody of an object to be detected are respectively marked by a substrate luminescent material and a quenching material, and the prepared sensor is more convenient and simpler, and has strong specificity, good stability, high sensitivity and good reproducibility.
(4) The electrochemical immunosensor prepared by the invention sensitively detects Cyfra21-1 within the detection range of 0.00005-50 ng mL-1The detection limit is 20.3 fg mL-1And the detection with simplicity, convenience, rapidness, high sensitivity, high specificity and high stability can be realized.
Detailed Description
EXAMPLE 1 preparation of electrochemical immunosensor
(1) Preparation of uracil modified graphite-like phase carbon nitride
A modified graphite-like phase carbon nitride sample is prepared by copolymerizing melamine and uracil, 1 g of melamine and 10 mg of uracil are uniformly mixed in an agate mortar and sufficiently ground, and then the mixture is placed in a covered ceramic crucible and heated in a muffle furnace at 5 ℃ for min-1The temperature rising rate is increased to 550 ℃ and kept for 4 h to obtain uracil-modified graphite-like carbon nitride, and when uracil is not doped, other conditions are the same, so that the original graphite-like carbon nitride is prepared;
(2) preparation of modified graphite-like phase carbon nitride combined Cyfra21-1 recognition antibody primary anti-label
5 microgram mL-1The primary antibody of Cyfra21-1 is activated by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with uracil modified graphite-like carbon nitride for 6 h at 4 ℃ to obtain a modified graphite-like carbon nitride-Cyfra 21-1 recognition antibody primary antibody marker;
(3) preparation of dendritic fiber nano-silica
Dissolving 2.5 g tetraethyl silicate in a mixed solution of 30 mL cyclohexane and 1.5 mL amyl alcohol, and fully stirring to obtain a solution A; dissolving 1 g of cetyl pyridine bromide and 0.6 g of urea in 30 mL of deionized water, and fully stirring to form a solution B; adding the solution B into the solution A, fully stirring to form a mixed reaction solution, placing the reaction solution into a polytetrafluoroethylene reaction kettle, reacting for 4 hours at 120 ℃ in the hydrothermal reaction kettle, centrifugally separating a product after the reaction is finished, washing the product for 3 times by using deionized water and acetone respectively, drying for 12 hours in a vacuum drying oven, calcining the obtained product in a muffle furnace at 550 ℃ for 6 hours, and removing a template agent to obtain the dendritic fiber nano-silica;
(4) preparation of aminated dendritic fiber nano-silica
Adding 50 mg of the prepared dendritic fiber nano-silica into a solution containing 10 mL of deionized water and 0.5 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, cooling to room temperature, centrifuging and drying in vacuum to obtain aminated dendritic fiber nano-silica;
(5) preparation of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material
Dissolving 50 mg of aminated dendritic fiber nano-silica powder in 300 mL of deionized water, performing ultrasonic treatment for 1 h, then dissolving 50 mg of copper chloride and 60 mg of trisodium citrate in the suspension, performing magnetic stirring for 5 min, and then dissolving 3 mL of 8 mL-1Slowly dripping the sodium sulfide nonahydrate solution into the suspension, heating to 90 ℃ and keeping for 30 min to obtain the aminated dendritic fiber nano silicon dioxide loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody
mu.L of a secondary antibody to Cyfra21-1 (10. mu.g mL)-1) Added to 2 mL of 1 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in the amination dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material solution, centrifuging, and dispersing the obtained product in 1 mL PBS to obtain a secondary antibody marker solution of the amination dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody;
(8) preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 muL and 0.5 mg mL-1The primary-antibody marker solution of the uracil-modified graphite-phase carbon nitride combined Cyfra21-1 recognition antibody is dripped on the surface of the electrode and dried at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-50 ng mL-1A series of Cyfra21-1 antigens with different concentrations are applied to the surface of the electrode, incubated for 2 h, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding a second antibody marker solution of 6 mu L of aminated dendritic fiber nano-silica loaded copper sulfide quantum dot composite material combined with the Cyfra21-1 recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting Cyfra 21-1.
EXAMPLE 2 preparation of electrochemical immunosensor
(1) Preparation of uracil modified graphite-like phase carbon nitride
A modified graphite-like phase carbon nitride sample is prepared by copolymerizing melamine and uracil, 1 g of melamine and 30 mg of uracil are uniformly mixed in an agate mortar and sufficiently ground, and then the mixture is placed in a covered ceramic crucible and heated in a muffle furnace at 5 ℃ for min-1The temperature rising rate of the reaction is increased to 550 ℃ and kept for 4 hours to obtain uracilThe modified graphite-like phase carbon nitride is not doped with uracil, and other conditions are the same, so that the original graphite-like phase carbon nitride is prepared;
(2) preparation of modified graphite-like phase carbon nitride combined Cyfra21-1 recognition antibody primary anti-label
10 microgram mL-1The primary antibody of Cyfra21-1 is activated by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with uracil modified graphite-like carbon nitride for 6 h at 4 ℃ to obtain a modified graphite-like carbon nitride-Cyfra 21-1 recognition antibody primary antibody marker;
(3) preparation of dendritic fiber nano-silica
Dissolving 2.5 g tetraethyl silicate in a mixed solution of 30 mL cyclohexane and 1.5 mL amyl alcohol, and fully stirring to obtain a solution A; dissolving 1 g of cetyl pyridine bromide and 0.6 g of urea in 30 mL of deionized water, and fully stirring to form a solution B; adding the solution B into the solution A, fully stirring to form a mixed reaction solution, placing the reaction solution into a polytetrafluoroethylene reaction kettle, reacting for 4 hours at 120 ℃ in the hydrothermal reaction kettle, centrifugally separating a product after the reaction is finished, washing the product for 3 times by using deionized water and acetone respectively, drying for 12 hours in a vacuum drying oven, calcining the obtained product in a muffle furnace at 550 ℃ for 6 hours, and removing a template agent to obtain the dendritic fiber nano-silica;
(4) preparation of aminated dendritic fiber nano-silica
Adding 80 mg of the prepared dendritic fiber nano-silica into a solution containing 10 mL of deionized water and 0.5 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, cooling to room temperature, centrifuging and drying in vacuum to obtain aminated dendritic fiber nano-silica;
(5) preparation of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material
80 mg of aminated dendritic fiber nano-silica powder was dissolved in 300 mL of deionized water for sonication for 1 h, and then 80 mg of cupric chloride and 90 mg of trisodium citrate were dissolved in the suspensionIn solution, magnetically stir for 5 min, then add 5 mL and 8 mg mL-1Slowly dripping the sodium sulfide nonahydrate solution into the suspension, heating to 90 ℃ and keeping for 30 min to obtain the aminated dendritic fiber nano silicon dioxide loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody
50 μ L of Cyfra21-1 secondary antibody (10 μ g mL)-1) Added to 2 mL of 2 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in the amination dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material solution, centrifuging, and dispersing the obtained product in 1 mL PBS to obtain a secondary antibody marker solution of the amination dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody;
(8) preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 mu L and 1.5 mg mL-1The primary-antibody marker solution of the uracil-modified graphite-phase carbon nitride combined Cyfra21-1 recognition antibody is dripped on the surface of the electrode and dried at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-50 ng mL-1A series of Cyfra21-1 antigens with different concentrations are applied to the surface of the electrode, incubated for 2 h, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding a second antibody marker solution of 6 mu L of aminated dendritic fiber nano-silica loaded copper sulfide quantum dot composite material combined with the Cyfra21-1 recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting Cyfra 21-1.
EXAMPLE 3 preparation of electrochemical immunosensor
(1) Preparation of uracil modified graphite-like phase carbon nitride
A modified graphite-like phase carbon nitride sample is prepared by copolymerizing melamine and uracil, 1 g of melamine and 100 mg of uracil are uniformly mixed in an agate mortar and sufficiently ground, and then the mixture is placed in a covered ceramic crucible and heated in a muffle furnace at 5 ℃ for min-1The temperature rising rate is increased to 550 ℃ and kept for 4 h to obtain uracil-modified graphite-like carbon nitride, and when uracil is not doped, other conditions are the same, so that the original graphite-like carbon nitride is prepared;
(2) preparation of modified graphite-like phase carbon nitride combined Cyfra21-1 recognition antibody primary anti-label
20 microgram mL-1The primary antibody of Cyfra21-1 is activated by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with uracil modified graphite-like carbon nitride for 6 h at 4 ℃ to obtain a modified graphite-like carbon nitride-Cyfra 21-1 recognition antibody primary antibody marker;
(3) preparation of dendritic fiber nano-silica
Dissolving 2.5 g tetraethyl silicate in a mixed solution of 30 mL cyclohexane and 1.5 mL amyl alcohol, and fully stirring to obtain a solution A; dissolving 1 g of cetyl pyridine bromide and 0.6 g of urea in 30 mL of deionized water, and fully stirring to form a solution B; adding the solution B into the solution A, fully stirring to form a mixed reaction solution, placing the reaction solution into a polytetrafluoroethylene reaction kettle, reacting for 4 hours at 120 ℃ in the hydrothermal reaction kettle, centrifugally separating a product after the reaction is finished, washing the product for 3 times by using deionized water and acetone respectively, drying for 12 hours in a vacuum drying oven, calcining the obtained product in a muffle furnace at 550 ℃ for 6 hours, and removing a template agent to obtain the dendritic fiber nano-silica;
(4) preparation of aminated dendritic fiber nano-silica
Adding 100 mg of the prepared dendritic fiber nano-silica into a solution containing 10 mL of deionized water and 0.5 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, cooling to room temperature, centrifuging and drying in vacuum to obtain aminated dendritic fiber nano-silica;
(5) preparation of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material
Dissolving 100 mg of aminated dendritic fiber nano-silica powder in 300 mL of deionized water, performing ultrasonic treatment for 1 h, then dissolving 100 mg of copper chloride and 120 mg of trisodium citrate in the suspension, magnetically stirring for 5 min, and then adding 10 mL of 8 mL-1Slowly dripping the sodium sulfide nonahydrate solution into the suspension, heating to 90 ℃ and keeping for 30 min to obtain the aminated dendritic fiber nano silicon dioxide loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody
mu.L of Cyfra21-1 secondary antibody (10. mu.g mL)-1) Added to 2 mL of 3 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in the amination dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material solution, centrifuging, and dispersing the obtained product in 1 mL PBS to obtain a secondary antibody marker solution of the amination dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody;
(8) preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and polishing by using 6 mu L and 2.5 mg mL-1The primary-antibody marker solution of the uracil-modified graphite-phase carbon nitride combined Cyfra21-1 recognition antibody is dripped on the surface of the electrode and dried at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-50 ng mL-1A series of Cyfra21-1 antigens with different concentrations are applied to the surface of the electrode, incubated for 2 h, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding a second antibody marker solution of 6 mu L of aminated dendritic fiber nano-silica loaded copper sulfide quantum dot composite material combined with the Cyfra21-1 recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting Cyfra 21-1.
Example 4 detection of Cyfra21-1
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 500V, and the electrochemical luminescence sensor contains 20 mmoL L-1The test was performed in potassium persulfate in PBS buffer;
(2) detecting Cyfra21-1 standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.6-0V;
(3) the electrochemiluminescence intensity of the sensor before and after Cyfra21-1 is added is observed, then the linear relation between the electrochemiluminescence intensity value and the concentration of Cyfra21-1 is recorded, and a working curve is drawn.
Example 5 detection of Cyfra21-1
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, and electrochemical engineering is carried outThe working station is connected with a chemiluminescence detector, the high voltage of the photomultiplier is set to 600V and the photomultiplier contains 50 mmoL L-1The test was performed in potassium persulfate in PBS buffer;
(2) detecting Cyfra21-1 standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.5-0V;
(3) the electrochemiluminescence intensity of the sensor before and after Cyfra21-1 is added is observed, then the linear relation between the electrochemiluminescence intensity value and the concentration of Cyfra21-1 is recorded, and a working curve is drawn.
Example 6 detection of Cyfra21-1
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 700V, and the electrochemical luminescence sensor contains 80 mmoL L-1The test was performed in potassium persulfate in PBS buffer;
(2) detecting Cyfra21-1 standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.4-0V;
(3) the electrochemiluminescence intensity of the sensor before and after Cyfra21-1 is added is observed, then the linear relation between the electrochemiluminescence intensity value and the concentration of Cyfra21-1 is recorded, and a working curve is drawn.
Example 7 detection of Cyfra21-1
(1) Ag/AgCl is used as a reference electrode, a platinum wire is used as a counter electrode, the prepared electrochemical luminescence sensor is used as a working electrode and is correctly connected in a cassette of a chemiluminescence detector, an electrochemical workstation and the chemiluminescence detector are connected together, the high voltage of a photomultiplier is set to be 800V, and the electrochemical luminescence sensor contains 100 mmoL L-1The test was performed in potassium persulfate in PBS buffer;
(2) detecting Cyfra21-1 standard solution by an electrochemical luminescence method, wherein the voltage test range is-1.3-0V;
(3) the electrochemiluminescence intensity of the sensor before and after Cyfra21-1 is added is observed, then the linear relation between the electrochemiluminescence intensity value and the concentration of Cyfra21-1 is recorded, and a working curve is drawn.
Claims (1)
1. A preparation method of an electrochemical immunosensor based on uracil modified graphite-like phase carbon nitride is characterized by comprising the following steps:
(1) preparation of uracil modified graphite-like phase carbon nitride
The method comprises the steps of preparing a modified graphite-like phase carbon nitride sample by copolymerizing melamine and uracil, uniformly mixing 1 g of melamine and 10-100 mg of uracil in an agate mortar for full grinding,
the mixture was then placed in a covered porcelain crucible in a muffle furnace at 5 ℃ for min-1The temperature rising rate is increased to 550 ℃ and kept for 4 hours to obtain uracil modified graphite-like carbon nitride,
when the uracil is not doped, the other conditions are the same, and the original graphite-like phase carbon nitride is prepared;
(2) preparation of modified graphite-like phase carbon nitride combined Cyfra21-1 recognition antibody primary anti-label
5-20 microgram mL-1The primary antibody of Cyfra21-1 is activated by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at 4 ℃, and then is incubated with uracil modified graphite-like carbon nitride for 6 h at 4 ℃ to obtain a modified graphite-like carbon nitride-Cyfra 21-1 recognition antibody primary antibody marker;
(3) preparation of dendritic fiber nano-silica
2.5 g of tetraethyl silicate is dissolved in a mixture of 30 mL of cyclohexane and 1.5 mL of amyl alcohol, the solution A is obtained after full stirring,
dissolving 1 g of cetyl pyridine bromide and 0.6 g of urea in 30 mL of deionized water, fully stirring to form a solution B,
adding the solution B into the solution A, fully stirring to form a mixed reaction solution, placing the reaction solution into a polytetrafluoroethylene reaction kettle, reacting for 4 hours at 120 ℃ in the hydrothermal reaction kettle, centrifugally separating a product after the reaction is finished, washing the product for 3 times by using deionized water and acetone respectively, then drying for 12 hours in a vacuum drying oven,
calcining the obtained product in a muffle furnace at 550 ℃ for 6 h to remove the template agent, and obtaining the dendritic fiber nano-silica;
(4) preparation of aminated dendritic fiber nano-silica
Adding 50-100 mg of the prepared dendritic fiber nano-silica into a solution containing 10 mL of deionized water and 0.5 mL of aminopropyltriethoxysilane, refluxing for 1.5 h at 70 ℃, cooling to room temperature, centrifuging, and drying in vacuum to obtain aminated dendritic fiber nano-silica;
(5) preparation of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material
Dissolving 50-100 mg of aminated dendritic fiber nano silicon dioxide powder in 300 mL of deionized water for ultrasonic treatment for 1 h, then dissolving 50-100 mg of copper chloride and 60-120 mg of trisodium citrate in the suspension, magnetically stirring for 5 min, and then dissolving 3-10 mL and 8 mg mL of trisodium citrate-1Slowly dripping the sodium sulfide nonahydrate solution into the suspension, heating to 90 ℃ and keeping for 30 min to obtain the aminated dendritic fiber nano silicon dioxide loaded copper sulfide quantum dot composite material;
(6) preparation of PBS buffer solution
11.94 g of disodium hydrogen phosphate dodecahydrate were taken out and dissolved in a 500 mL volumetric flask so as to be prepared into a solution having a concentration of 1/15 mol L-1As an aqueous solution of (a); 4.54 g of monopotassium phosphate is taken and fixed to a 500 mL volumetric flask, and the volume is configured to be 1/15 mol L-1As solution b; mixing the solution A and the solution B in proportion to prepare a series of PBS (phosphate buffer solution) with the pH value of 6.0-8.0;
(7) preparation of secondary antibody marker of aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody
20-100. mu.L of a secondary antibody (10. mu.g mL) of Cyfra21-1 was added-1) Adding the mixture into 2 mL of 1-3 mg mL-1Carrying out oscillation incubation for 12 h at 4 ℃ in an aminated dendritic fiber nano silicon dioxide loaded copper sulfide quantum dot composite material solution, and centrifuging to obtain the nano copper sulfide quantum dot composite materialDispersing the product in 1 mL of PBS to obtain a second antibody marker solution of the aminated dendritic fiber nano-silica-loaded copper sulfide quantum dot composite material combined with Cyfra21-1 recognition antibody;
(8) preparation of electrochemical immunosensor
1) Polishing a glassy carbon electrode with the diameter of 4 mm by using aluminum oxide polishing powder, cleaning by using ultrapure water, and then polishing 6 muL and 0.5-2.5 mg mL-1The primary-antibody marker solution of the uracil-modified graphite-phase carbon nitride combined Cyfra21-1 recognition antibody is dripped on the surface of the electrode and dried at room temperature;
2) continuously dropwise adding bovine serum albumin solution with the mass fraction of 0.1% to the surface of the electrode, cleaning with ultrapure water, and airing at room temperature;
3) continuously dropwise adding 6 muL and 0.00005-50 ng mL-1A series of Cyfra21-1 antigens with different concentrations are applied to the surface of the electrode, incubated for 2 h, rinsed with ultrapure water and dried at room temperature;
4) and finally, dropwise adding a second antibody marker solution of 6 mu L of aminated dendritic fiber nano-silica loaded copper sulfide quantum dot composite material combined with the Cyfra21-1 recognition antibody, washing with ultrapure water, and airing at room temperature to obtain the electrochemical immunosensor for detecting Cyfra 21-1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114100665A (en) * | 2021-12-06 | 2022-03-01 | 合肥工业大学 | Preparation method and application of carbonyl functionalized graphite phase carbon nitride |
| CN114177289A (en) * | 2021-12-15 | 2022-03-15 | 华南师范大学 | Composite nano material for photodynamic and photothermal combined treatment and preparation method and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114100665A (en) * | 2021-12-06 | 2022-03-01 | 合肥工业大学 | Preparation method and application of carbonyl functionalized graphite phase carbon nitride |
| CN114100665B (en) * | 2021-12-06 | 2024-03-29 | 合肥工业大学 | Preparation method and application of carbonyl functionalized graphite phase carbon nitride |
| CN114177289A (en) * | 2021-12-15 | 2022-03-15 | 华南师范大学 | Composite nano material for photodynamic and photothermal combined treatment and preparation method and application thereof |
| CN114177289B (en) * | 2021-12-15 | 2023-10-20 | 华南师范大学 | Composite nano material for photodynamic and photothermal combined treatment and preparation method and application thereof |
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