CN110133075B - A sialic acid immunosensor based on photothermal effect of silver iodide nanoparticles induced in situ signal amplification - Google Patents
A sialic acid immunosensor based on photothermal effect of silver iodide nanoparticles induced in situ signal amplification Download PDFInfo
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- SQVRNKJHWKZAKO-UHFFFAOYSA-N beta-N-Acetyl-D-neuraminic acid Natural products CC(=O)NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO SQVRNKJHWKZAKO-UHFFFAOYSA-N 0.000 title claims abstract description 68
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Abstract
本发明公开一种基于碘化银纳米粒子光热效应诱导原位信号放大的唾液酸免疫传感器的制备及应用。将聚苯胺与螺旋碳纳米管复合作为基底固定唾液酸抗体，壳聚糖修饰的碘化银纳米粒子为探针标记唾液酸二抗，在唾液酸存在的情况下，通过抗原与抗体之间特异性识别构建出一种夹心型免疫传感器。由于碘化银自身发生氧化还原反应能产生一定强度的电化学信号，在808nm红外激光器的照射下，基于碘化银纳米粒子的光热效应，传感界面迅速升温，电化学信号随温度升高显著增强，从而实现了传感界面的原位信号放大。在光热条件下，随着唾液酸浓度的不断升高，通过免疫反应结合的碘化银信号探针增多，电化学信号随之增强，从而实现对唾液酸的高灵敏检测。
The invention discloses the preparation and application of a sialic acid immunosensor based on the photothermal effect of silver iodide nanoparticles to induce in-situ signal amplification. The composite of polyaniline and helical carbon nanotubes is used as a substrate to immobilize sialic acid antibodies, and chitosan-modified silver iodide nanoparticles are used as probes to label sialic acid secondary antibodies. A sandwich-type immunosensor was constructed. Because the redox reaction of silver iodide itself can generate a certain intensity of electrochemical signal, under the irradiation of 808 nm infrared laser, based on the photothermal effect of silver iodide nanoparticles, the sensing interface heats up rapidly, and the electrochemical signal increases significantly with the increase of temperature. In situ signal amplification at the sensing interface is achieved. Under photothermal conditions, with the continuous increase of the sialic acid concentration, the number of silver iodide signal probes bound by the immune reaction increased, and the electrochemical signal was enhanced accordingly, thus realizing the highly sensitive detection of sialic acid.
Description
Technical Field
The invention belongs to the technical field of novel functional materials and biosensing detection, and particularly relates to preparation and application of a sialic acid immunosensor based on silver iodide nanoparticle photothermal effect induction in-situ signal amplification.
Background
An electrochemical detection method is a method for converting various physical and chemical signals generated before and after a target object is combined with a molecular recognition element into electrochemical signals, has the advantages of simple structure, low cost, high response speed, high stability and the like, and is widely applied to the field of analytical chemistry. The electrochemical analysis method is combined with the photothermal effect, so that the analysis performance of the electrochemical immunosensor can be obviously improved, and the signal amplification is realized. The photothermal effect is a phenomenon that light acts on a material and converts a part of energy into heat energy, compared with a traditional heating method, 808nm near-infrared laser irradiation can locally heat a sensing interface, a non-isothermal system with a temperature gradient is established around a sensor, the overall temperature of a solution is basically not influenced, and the method has the advantages of simple equipment, convenience in operation, accurate temperature control and the like. The material with the photothermal effect is used as the photothermal agent, 808nm near infrared light capable of generating strong penetrating power is used as a light source, the photothermal agent is introduced into the sensor interface, the near infrared light rapidly generates heat energy through the photothermal effect, the local temperature of the sensor interface is rapidly raised, the convective mass transfer of a body solution is accelerated, the electronic transfer of the sensor interface is accelerated, and the electrochemical signal amplification is realized.
Sialic Acid (SA) is a derivative of a 9-carbon monosaccharide, and elevated concentrations in humans are closely related to various diseases, such as diabetes, cardiovascular and neurological diseases. In addition, SA is over-expressed in serum of some malignant tumors, so that SA is considered as an index of high-sensitivity tumor marker auxiliary diagnosis, and the determination of the content of serum sialic acid has important clinical significance for early diagnosis, curative effect observation and prognosis judgment of malignant tumors. Immunoassay is a technical means for detection based on antigen and antibody specific reaction, and has high selectivity and low detection limit. The electrochemical immunoassay method is an immunoassay method combining immunoassay and an electrochemical analysis technology, and can remarkably improve analysis sensitivity, so that development of a new signal amplification strategy is important for construction of an electrochemical immunosensor. The core of the method is that the photo-thermal material silver iodide nanoparticle labeled antibody is used as a signal probe, which can be used as an electrochemical signal source and can realize signal amplification by using the self photo-thermal effect. By applying the irradiation of a near infrared light source with 808nm, the rapid and sensitive detection of the target object can be realized in situ at the interface of the sensor. Therefore, compared with the traditional electrochemical immunosensor, the sensor adopts a signal probe with dual functions of a signal source and a photothermal effect, the excellent affinity of the sensor avoids electrode pollution, the phenomenon of signal instability caused by signal source leakage does not exist in the detection process, and the analysis performance of the sensor is obviously improved.
The polyaniline-spiral carbon nanotube composite is used as a sensor substrate, the spiral carbon nanotube shows good conductivity which is beneficial to electron transmission, and in addition, the unique spiral three-dimensional structure has a larger specific surface area, so that the loading capacity of polyaniline on a sensor interface is improved, and more active sites can be provided for the fixation of an antibody. The chitosan-modified silver iodide nanoparticles have better film-forming property, are beneficial to immobilization of signal probes, and have the advantages of difficult leakage of signal sources, good biocompatibility and the like. A sandwich type immunosensor is constructed through specific recognition between an antigen and an antibody, and electrochemical signals are amplified in situ based on the photothermal effect of silver iodide nanoparticles under the irradiation of a near-infrared light source with the wavelength of 808 nm. Along with the increase of the sialic acid concentration, the electrochemical signal is continuously enhanced and has a linear relation in a certain range, so that the high-sensitivity detection of the sialic acid is realized.
Disclosure of Invention
The invention aims to provide a sialic acid immunosensor based on silver iodide nanoparticle photothermal effect induction in-situ signal amplification and a preparation method and application thereof.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
(1) pretreatment of GCE: firstly, mechanically grinding and polishing a chamois leather paved with alumina powder by GCE, washing residual powder on the surface by secondary water, then moving the chamois leather into an ultrasonic water bath for cleaning until the chamois leather is cleaned, and finally completely washing the chamois leather by ethanol, dilute acid and water in sequence;
（2） PANI-HCNT/Ab1/SA/Ab2preparation of @ SICNPs modified electrode: dissolving 5 mg of Polyaniline (PANI) powder in 1 mL of N, N-Dimethylformamide (DMF), dissolving 5 mg of Helical Carbon Nanotube (HCNT) powder in 1 mL of N, N-Dimethylformamide (DMF), mixing 5 mg/mL of PANI solution and 5 mg/mL of HCNT solution according to the volume ratio of 5:1, dripping 3 mu L of PANI-HCNT mixed solution on the surface of a clean glassy carbon electrode, drying in an oven, cooling to room temperature, dripping 3 mu L of 2.5 wt.% of glutaraldehyde aqueous solution on the electrode, activating for 40 min at 4 ℃, soaking the obtained modified electrode in 10 ng/mL of sialic acid primary antibody (Ab) for 40 min at 10 ng/mL1) (purchased from Wuhan purity Biotech Ltd.) solution, incubated at 4 ℃ for 50 min, followed by removal of excess Ab with phosphate buffered pH7.41Finally, the electrode was immersed in 1.0 wt.% BSA aqueous solution for 1 h to block Ab2Surface non-specific active sites are washed away to obtain PANI-HCNT/Ab1Modifying the electrode; the electrodes were immersed in 5 μ L of Sialic Acid (SA) standard solutions of different concentrations (purchased from wuhan purity biotechnology limited) and incubated in a refrigerator for 40 min; washing the surface of the electrode with a phosphoric acid buffer solution with pH7.4, and naturally drying at room temperature to obtain PANI-HCNT/Ab1a/SA modified electrode; finally, 3. mu.L Ab was taken2@ SICNPs solution was droplet coated on PANI-HCNT/Ab1Modification of electrode surface by SA, incubation for 50 min at 4 deg.C, washing with pH7.4 phosphoric acid buffer solution to remove residual liquid, and naturally drying at room temperature to obtain PANI-HCNT/Ab1/SA/Ab2@ SICNPs modified electrodes;
(3) detection of sialic acid: the measurement is carried out by adopting a three-electrode system and using PANI-HCNT/Ab1/SA/Ab2The @ SICNPs modified electrode is a working electrode, the Ag/AgCl is a reference electrode, and the platinum wire electrode is an auxiliary electrodeSialic acid was detected on an electrochemical workstation, setting the initial potential to-0.5V and the final potential to 0.6V, using Differential Pulse Voltammetry (DPV) on a 1X 10 solution in phosphate buffered saline at pH7.4-4Measuring the electrochemical behavior of a series of sialic acid standard solutions with different concentrations in ng/mL-1 ng/mL on a modified electrode, and drawing a working curve by recording an electrochemical signal generated after 808nm laser irradiation; and replacing the sialic acid standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
Preparation of the above spiral carbon nanotube (HCNT)
Quartz plate (50 mm × 30 mm × 2 mm) as substrate, ultrasonic cleaning in ethanol for 30 min, washing with secondary water, and passing through 0.1M ferric nitrate (Fe (NO)3)·9H2O) drying at room temperature after the aqueous solution treatment; then placing the quartz plate in a horizontal quartz tube furnace, heating to 800 ℃ and keeping the temperature at H2After reacting for 90 min in the atmosphere, the obtained sample is placed in N2Slowly cooling to room temperature in the atmosphere to obtain the helical carbon nanotube;
the preparation of the chitosan modified silver iodide nanoparticles (SICNPs) comprises the following steps:
under dark conditions, 10 mL of 0.1M AgNO was added to 10 mL of a 0.5 wt.% Chitosan (CS) solution3Stirring the solution for 30 min to obtain a mixed solution; slowly adding 10 mL of 0.15M KI solution into the mixed solution, stirring for 3h, centrifuging the obtained SICNPs compound at the rotating speed of 6000 rpm for 20min, washing with secondary water and ethanol, drying at 60 ℃ for 12h, and dispersing the obtained product with 50 mL of secondary water to obtain 2 mM SICNPs stock solution;
chitosan-modified silver iodide nanoparticle (SICNPs) labeled secondary antibody (Ab)2@ SICNPs) preparation of the solution:
50 μ L of 1 ng/mL sialic acid secondary antibody (Ab)2) (purchased from Wuhan Biotech Ltd.) solution was mixed with 50. mu.L of 2.5 wt.% glutaraldehyde aqueous solution for 40 min, 300. mu.L of 2 mM stock solution of SICNPs was added, incubation was performed at 4 ℃ for 2h with gentle stirring, 50. mu.L of 1.0 wt.% BSA aqueous solution was added and the reaction was allowed to proceed for 1 h, and blocking was performedAb2Surface non-specific active sites, centrifuging the mixed solution at 6000 rpm for 20min, washing with secondary water and ethanol, and removing supernatant to obtain Ab2@ SICNPs complex, dispersed in phosphate buffer pH7.4, and then 20. mu.L of BSA aqueous solution was added thereto, and the mixture was left at 4 ℃ for further use.
The sensor for detecting sialic acid based on silver iodide nanoparticle photothermal effect induced in-situ signal amplification comprises a working electrode, a platinum wire electrode as a counter electrode and Ag/AgCl as a reference electrode, and is characterized in that the working electrode adopts PANI-HCNT/Ab1/SA/Ab2@ SICNPs modified electrode, the PANI-HCNT/Ab1/SA/Ab2The @ SICNPs modified electrode is prepared by the following method: 1) polishing of glassy carbon electrode: firstly, mechanically grinding and polishing a glassy carbon electrode on chamois paved with alumina powder, washing residual powder on the surface by using secondary water, then moving the chamois into an ultrasonic water bath for cleaning until the chamois is cleaned, and finally, thoroughly washing the chamois by using ethanol, dilute acid and water in sequence; 2) PANI-HCNT/Ab1/SA/Ab2Preparation of @ SICNPs modified electrode: dissolving 5 mg of Polyaniline (PANI) powder in 1 mL of N, N-Dimethylformamide (DMF), dissolving 5 mg of Helical Carbon Nanotube (HCNT) powder in 1 mL of N, N-Dimethylformamide (DMF), mixing 5 mg/mL of PANI solution and 5 mg/mL of HCNT solution according to the volume ratio of 5:1, dripping 3 mu L of PANI-HCNT mixed solution on the surface of a clean glassy carbon electrode, drying in an oven, cooling to room temperature, dripping 3 mu L of 2.5 wt.% of glutaraldehyde aqueous solution on the electrode, activating for 40 min at 4 ℃, soaking the obtained modified electrode in 10 ng/mL of sialic acid primary antibody (Ab) for 40 min at 10 ng/mL1) In solution, incubate for 50 min at 4 ℃ and then remove excess Ab with phosphate buffered pH7.41Finally, immersing the electrode into BSA aqueous solution with the concentration of 1.0 wt.% for reaction for 1 h, sealing the non-specific active sites on the surface of the electrode, and flushing out the surface residual liquid to obtain the PANI-HCNT/Ab1Modifying the electrode; the electrodes were immersed in 5 μ Ι _ of Sialic Acid (SA) standard solutions of different concentrations and incubated in a refrigerator for 40 min; washing the surface of the electrode with a phosphoric acid buffer solution with pH7.4, and naturally drying at room temperature to obtain PANI-HCNT/Ab1a/SA modified electrode; finally, 3. mu.L Ab was taken2@ SICNPs solution was droplet coated on PANI-HCNT/Ab1Modification of electrode surface by SA, incubation for 50 min at 4 deg.C, washing with pH7.4 phosphoric acid buffer solution to remove residual liquid, and naturally drying at room temperature to obtain PANI-HCNT/Ab1/SA/Ab2@ SICNPs modified electrodes.
The invention relates to preparation and application of a sialic acid immunosensor based on silver iodide nanoparticle photothermal effect induction in-situ signal amplification, which is characterized by comprising the following steps of: 1) the measurement is carried out by adopting a three-electrode system and using PANI-HCNT/Ab1/SA/Ab2The @ SICNPs modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is an auxiliary electrode, sialic acid is detected on an electrochemical workstation, the initial potential is set to be-0.5V, and the final potential is set to be 0.6V; 2) in a phosphate buffer solution at pH7.4, 1X 10 pairs of Differential Pulse Voltammetry (DPV)-4Measuring the electrochemical behavior of a series of sialic acid standard solutions with different concentrations in ng/mL-1 ng/mL on a modified electrode, and drawing a working curve by recording an electrochemical signal generated after 808nm laser irradiation; and replacing the sialic acid standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
The invention has the following remarkable advantages:
(1) the photo-thermal material silver iodide nanoparticle labeled antibody is used as a signal probe, can be used as an electrochemical signal source, and can realize signal amplification by utilizing the self photo-thermal effect. Compared with the traditional electrochemical immunosensor, the sensor adopts a signal probe with the dual functions of a signal source and a photothermal effect, the electrode pollution is avoided due to the excellent affinity of the signal probe, the phenomenon of signal instability caused by signal source leakage does not exist in the detection process, and the analysis performance of the sensor is obviously improved.
(2) The chitosan-modified silver iodide nanoparticles have better film-forming property, are beneficial to immobilization of signal probes, and have the advantages of difficult leakage of signal sources, good biocompatibility and the like.
(3) The polyaniline-spiral carbon nanotube composite is used as a sensor substrate, the spiral carbon nanotube shows good conductivity which is beneficial to electron transmission, and in addition, the unique spiral three-dimensional structure has a larger specific surface area, so that the loading capacity of polyaniline on a sensor interface is improved, and more active sites can be provided for the fixation of an antibody.
Drawings
FIG. 1 is a schematic diagram of the preparation process of the sialic acid sandwich type electrochemical immunosensor of the present invention.
FIG. 2A shows different concentrations of 1X 10-4And (a-h) an electrochemical response diagram of a sialic acid standard solution sensing electrode of ng/mL-1 ng/mL.
FIG. 2B is a graph of the electrochemical response of the sensing electrode as a linear function of sialic acid standard solution concentration.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples.
Example 1
Preparation of a sialic acid immunosensor based on silver iodide nanoparticle photothermal effect induced in-situ signal amplification (as shown in figure 1):
(1) pretreating a glassy carbon electrode: firstly, mechanically grinding and polishing a glassy carbon electrode on chamois paved with alumina powder, washing residual powder on the surface by using secondary water, then moving the chamois into an ultrasonic water bath for cleaning until the chamois is cleaned, and finally, thoroughly washing the chamois by using ethanol, dilute acid and water in sequence;
(2) under dark conditions, 10 mL of 0.1M AgNO was added to 10 mL of a 0.5 wt.% Chitosan (CS) solution3Stirring the solution for 30 min to obtain a mixed solution; slowly adding 10 mL of 0.15M KI solution into the mixed solution, stirring for 3h, centrifuging the obtained SICNPs compound at the rotating speed of 6000 rpm for 20min, washing with secondary water and ethanol, drying at 60 ℃ for 12h, and dispersing the obtained product with 50 mL of secondary water to obtain 2 mM SICNPs stock solution;
(3) 50 μ L of sialic acid secondary antibody (Ab)2) Mixing the solution with 50 μ L of 2.5 wt.% glutaraldehyde aqueous solution for 40 min, adding 300 μ L of 2 mM stock SICNPs, and stirring at 4 deg.CIncubate for 2h under stirring, add 50. mu.L of 1.0 wt.% BSA solution in water to react for 1 h, block Ab2Surface non-specific active sites, centrifuging the mixed solution at 6000 rpm for 20min, washing with secondary water and ethanol, and removing supernatant to obtain Ab2@ SICNPs complex, dispersed in phosphate buffer pH7.4, and then 20. mu.L of BSA aqueous solution was added thereto, and the mixture was left at 4 ℃ for further use.
(4) Dripping 3 μ L PANI-HCNT mixed solution on the surface of a clean glassy carbon electrode, drying in an oven, cooling to room temperature, dripping 3 μ L2.5 wt.% glutaraldehyde aqueous solution on the electrode, activating at 4 deg.C for 40 min, soaking the obtained modified electrode in 10 ng/mL sialic acid primary antibody (Ab)1) In solution, incubate for 50 min at 4 ℃ and then remove excess Ab with phosphate buffered pH7.41Finally, immersing the electrode into BSA aqueous solution with the concentration of 1.0 wt.% for reaction for 1 h, sealing the non-specific active sites on the surface of the electrode, and flushing out the surface residual liquid to obtain the PANI-HCNT/Ab1Modifying the electrode;
(5) take 3. mu.L Ab2@ SICNPs solution is dripped on the PANI-HCNT/Ab prepared in the step (4)1Modifying the electrode surface, incubating at 4 deg.C for 50 min, washing with pH7.4 phosphoric acid buffer solution to remove residual solution, and naturally drying at room temperature to obtain PANI-HCNT/Ab1/SA/Ab2@ SICNPs modified electrodes.
Example 2
Preparation of Helical Carbon Nanotubes (HCNT) used in example 1:
quartz plate (50 mm × 30 mm × 2 mm) as substrate, ultrasonic cleaning in ethanol for 30 min, washing with secondary water, and passing through 0.1M ferric nitrate (Fe (NO)3)·9H2O) drying at room temperature after the aqueous solution treatment; then placing the quartz plate in a horizontal quartz tube furnace, heating to 800 ℃ and keeping the temperature at H2After reacting for 90 min in the atmosphere, the obtained sample is placed in N2And slowly cooling to room temperature in the atmosphere to obtain the helical carbon nanotube.
Preparation of chitosan-modified silver iodide nanoparticles (SICNPs) used in example 1:
under dark conditions, 10 mL of 0.1M AgNO was added to 10 mL of a 0.5 wt.% Chitosan (CS) solution3Stirring the solution for 30 min to obtain a mixed solution; and slowly adding 10 mL of 0.15M KI solution into the mixed solution, stirring for 3h, centrifuging the obtained SICNPs compound at the rotating speed of 6000 rpm for 20min, washing with secondary water and ethanol, drying at 60 ℃ for 12h, and dispersing the obtained product with 50 mL of secondary water to obtain a 2 mM SICNPs stock solution.
Chitosan-modified silver iodide nanoparticles (SICNPs) labeled secondary antibodies (Ab) used in example 12@ SICNPs) preparation of the solution:
50 μ L of 1 ng/mL sialic acid secondary antibody (Ab)2) Mixing the solution with 50 μ L of 2.5 wt.% glutaraldehyde aqueous solution for 40 min, adding 300 μ L of 2 mM stock solution of SICNPs, incubating at 4 deg.C for 2h under mild stirring, adding 50 μ L of 1.0 wt.% BSA aqueous solution, reacting for 1 h, blocking Ab2Surface non-specific active sites, centrifuging the mixed solution at 6000 rpm for 20min, washing with secondary water and ethanol, and removing supernatant to obtain Ab2@ SICNPs complex, dispersed in phosphate buffer pH7.4, and then 20. mu.L of BSA aqueous solution was added thereto, and the mixture was left at 4 ℃ for further use.
Example 3
An application of a sialic acid immunosensor based on silver iodide nanoparticle photothermal effect induction in-situ signal amplification comprises the following steps:
(1) the assay was performed using a three-electrode system with the PANI-HCNT/Ab prepared in example 11/SA/Ab2The @ SICNPs modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is an auxiliary electrode, SA is detected on an electrochemical workstation, the initial potential is set to be-0.5V, and the final potential is set to be 0.6V;
(2) in phosphate buffer solution of pH7.4, 1X 10 pairs of Differential Pulse Voltammetry (DPV)-4Measuring the electrochemical behavior of a series of sialic acid standard solutions with different concentrations in ng/mL-1 ng/mL on a modified electrode, and drawing a working curve by recording an electrochemical signal generated after 808nm laser irradiation;
(3) and replacing the sialic acid standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
Claims (5)
1. A preparation method of a sialic acid immunosensor based on silver iodide nanoparticle photothermal effect induction in-situ signal amplification is characterized by comprising the following steps: (1) pretreatment of the glassy carbon electrode GCE: firstly, mechanically grinding and polishing a glassy carbon electrode on chamois paved with alumina powder, washing residual powder on the surface by using secondary water, then moving the chamois into an ultrasonic water bath for cleaning until the chamois is cleaned, and finally, thoroughly washing the chamois by using ethanol, dilute acid and water in sequence;
（2）PANI-HCNT/Ab1/SA/Ab2preparation of @ SICNPs modified electrode: dissolving 5 mg polyaniline PANI powder in 1 mL of N, N-dimethylformamide DMF, dissolving 5 mg of helical carbon nanotube HCNT powder in 1 mL of N, N-dimethylformamide DMF, mixing 5 mg/mL of PANI solution with 5 mg/mL of HCNT solution according to the volume ratio of 5:1, dripping 3 mu L of PANI-HCNT mixed solution on the surface of a clean glassy carbon electrode, drying in an oven, cooling to room temperature, dripping 3 mu L of 2.5 wt.% of glutaraldehyde water solution on the electrode, activating at 4 ℃ for 40 min, soaking the obtained modified electrode in 10 ng/mL of sialic acid-anti-Ab1In solution, incubate for 50 min at 4 ℃ and then remove excess Ab with phosphate buffered pH7.41Finally, immersing the electrode into BSA aqueous solution with the concentration of 1.0 wt.% for reaction for 1 h, sealing the non-specific active sites on the surface of the electrode, and flushing out the surface residual liquid to obtain the PANI-HCNT/Ab1Modifying the electrode; the electrode was immersed in 5 μ L of sialic acid SA standard solutions of different concentrations and incubated in a refrigerator for 40 min; washing the surface of the electrode with a phosphoric acid buffer solution with pH7.4, and naturally drying at room temperature to obtain PANI-HCNT/Ab1a/SA modified electrode; finally, 3. mu.L of chitosan-modified silver iodide nanoparticle is taken to mark secondary antibody Ab2@ SICNPs solution was droplet coated on PANI-HCNT/Ab1Modification of electrode surface by SA, incubation for 50 min at 4 deg.C, washing with pH7.4 phosphoric acid buffer solution to remove residual liquid, and naturally drying at room temperature to obtain PANI-HCNT/Ab1/SA/Ab2@ SICNPs modifying electrodesA pole;
(3) detection of sialic acid: the measurement is carried out by adopting a three-electrode system and using PANI-HCNT/Ab1/SA/Ab2The @ SICNPs modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is an auxiliary electrode, sialic acid is detected on an electrochemical workstation, the initial potential is set to be-0.5V, the final potential is set to be 0.6V, and 1 × 10 is subjected to differential pulse voltammetry in phosphoric acid buffer solution with the pH value of 7.4-4Measuring the electrochemical behavior of a series of sialic acid standard solutions with different concentrations in ng/mL-1 ng/mL on a modified electrode, and drawing a working curve by recording an electrochemical signal generated after 808nm laser irradiation; and replacing the sialic acid standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
2. The method of claim 1, wherein the helical carbon nanotube HCNT is prepared by: using quartz plate 50 mm × 30 mm × 2 mm as substrate, ultrasonic cleaning in ethanol for 30 min, washing with secondary water, and passing through 0.1M ferric nitrate Fe (NO)3)·9H2Drying at room temperature after the O aqueous solution treatment; then placing the quartz plate in a horizontal quartz tube furnace, heating to 800 ℃ and keeping the temperature at H2After reacting for 90 min in the atmosphere, the obtained sample is placed in N2And slowly cooling to room temperature in the atmosphere to obtain the helical carbon nanotube.
3. The method of claim 1, wherein Ab is2@ SICNPs solution was prepared by the following method: 1) preparing chitosan modified silver iodide nanoparticles SICNPs: under dark conditions, 10 mL of 0.1M AgNO was added to 10 mL of 0.5 wt.% chitosan CS solution3Stirring the solution for 30 min to obtain a mixed solution; slowly adding 10 mL of 0.15M KI solution into the mixed solution, stirring for 3h, centrifuging the obtained SICNPs compound at the rotating speed of 6000 rpm for 20min, washing with secondary water and ethanol, drying at 60 ℃ for 12h, and dispersing the obtained product with 50 mL of secondary water to obtain 2 mM SICNPs stock solution; 2) chitosan-modified silver iodide nanoparticle labelDouble antibody Ab2Preparation of @ SICNPs solution: 50 μ L of 1 ng/mL sialic acid DiantiAb2Mixing the solution with 50 μ L of 2.5 wt.% glutaraldehyde aqueous solution for 40 min, adding 300 μ L of 2 mM stock solution of SICNPs, incubating at 4 deg.C for 2h under mild stirring, adding 50 μ L of 1.0 wt.% BSA aqueous solution, reacting for 1 h, blocking Ab2Surface non-specific active sites, centrifuging the mixed solution at 6000 rpm for 20min, washing with secondary water and ethanol, and removing supernatant to obtain Ab2@ SICNPs complex, dispersed in phosphate buffer pH7.4, and then 20. mu.L of BSA aqueous solution was added thereto, and the mixture was left at 4 ℃ for further use.
4. A sensor for detecting sialic acid based on silver iodide nanoparticle photothermal effect induced in-situ signal amplification comprises a working electrode, a platinum wire electrode as a counter electrode and Ag/AgCl as a reference electrode, and is characterized in that the working electrode adopts PANI-HCNT/Ab1/SA/Ab2@ SICNPs modified electrode, the PANI-HCNT/Ab1/SA/Ab2The @ SICNPs modified electrode is prepared by the following method: 1) polishing of glassy carbon electrode: firstly, mechanically grinding and polishing a glassy carbon electrode on chamois paved with alumina powder, washing residual powder on the surface by using secondary water, then moving the chamois into an ultrasonic water bath for cleaning until the chamois is cleaned, and finally, thoroughly washing the chamois by using ethanol, dilute acid and water in sequence; 2) PANI-HCNT/Ab1/SA/Ab2Preparation of @ SICNPs modified electrode: dissolving 5 mg polyaniline PANI powder in 1 mL of N, N-dimethylformamide DMF, dissolving 5 mg of helical carbon nanotube HCNT powder in 1 mL of N, N-dimethylformamide DMF, mixing 5 mg/mL of PANI solution with 5 mg/mL of HCNT solution according to the volume ratio of 5:1, dripping 3 mu L of PANI-HCNT mixed solution on the surface of a clean glassy carbon electrode, drying in an oven, cooling to room temperature, dripping 3 mu L of 2.5 wt.% of glutaraldehyde water solution on the electrode, activating at 4 ℃ for 40 min, soaking the obtained modified electrode in 10 ng/mL of sialic acid-anti-Ab1In solution, incubate for 50 min at 4 ℃ and then remove excess Ab with phosphate buffered pH7.41Finally, the electrode is immersed into BSA aqueous solution with the concentration of 1.0 wt.% for reaction for 1 h, and non-electrode surfaces are blockedSpecific active sites are washed away to obtain PANI-HCNT/Ab after surface residual liquid is removed1Modifying the electrode; immersing the electrode in 5 μ L of sialic acid SA standard solutions with different concentrations, and incubating in a refrigerator for 40 min; washing the surface of the electrode with a phosphoric acid buffer solution with pH7.4, and naturally drying at room temperature to obtain PANI-HCNT/Ab1a/SA modified electrode; finally, 3. mu.L of chitosan-modified silver iodide nanoparticle is taken to mark secondary antibody Ab2@ SICNPs solution was droplet coated on PANI-HCNT/Ab1Modification of electrode surface by SA, incubation for 50 min at 4 deg.C, washing with pH7.4 phosphoric acid buffer solution to remove residual liquid, and naturally drying at room temperature to obtain PANI-HCNT/Ab1/SA/Ab2@ SICNPs modified electrodes.
5. Use of a sensor according to claim 4 for detecting sialic acid, characterized in that the steps are as follows: 1) the measurement is carried out by adopting a three-electrode system and using PANI-HCNT/Ab1/SA/Ab2The @ SICNPs modified electrode is a working electrode, Ag/AgCl is a reference electrode, a platinum wire electrode is an auxiliary electrode, SA is detected on an electrochemical workstation, the initial potential is set to be-0.5V, and the final potential is set to be 0.6V; 2) in phosphate buffer solution of pH7.4, 1X 10 was measured by differential pulse voltammetry-4Measuring the electrochemical behavior of a series of sialic acid standard solutions with different concentrations in ng/mL-1 ng/mL on a modified electrode, and drawing a working curve by recording an electrochemical signal generated after 808nm laser irradiation; and replacing the sialic acid standard solution with the sample solution to be detected for detection, and checking the detection result through a working curve.
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| CN102476046A (en) * | 2010-11-24 | 2012-05-30 | 沈阳建筑大学 | Polyaniline/carbon nano tube adsorbent with multi-layer pore structure and preparation method thereof |
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| CN102476046A (en) * | 2010-11-24 | 2012-05-30 | 沈阳建筑大学 | Polyaniline/carbon nano tube adsorbent with multi-layer pore structure and preparation method thereof |
| CN109813773A (en) * | 2019-03-04 | 2019-05-28 | 福建师范大学 | Preparation and application of a sialic acid biosensor based on photoelectric and temperature dual-signal responses based on polymer dots |
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