阅读说明：本技术 一种嵌入式Au纳米薄膜电极表面等离子体共振免疫分析的方法 (Surface plasma resonance immunoassay method for embedded Au nano-film electrode ) 是由 潘宏程 郭垠 陈雯 于 2019-10-17 设计创作，主要内容包括：本发明公开了一种嵌入式Au纳米薄膜电极表面等离子体共振免疫分析的方法。其方法步骤：采用柠檬酸钠还原法制备的胶体金,并利用自组装的方式将胶体金修饰在ITO电极上,通过简单的退火处理改变该传感器的局与表面等离子共振灵敏度,实现对鼠抗人IgG的灵敏检测。本发明方法制备过程简单,成本低廉。(The invention discloses a method for surface plasma resonance immunoassay of an embedded Au nano film electrode. The method comprises the following steps: the colloidal gold prepared by a sodium citrate reduction method is modified on an ITO electrode in a self-assembly mode, and the local and surface plasmon resonance sensitivity of the sensor is changed through simple annealing treatment, so that the sensitive detection of mouse anti-human IgG is realized. The method has simple preparation process and low cost.)

1. a method for surface plasma resonance immunoassay of an embedded Au nano film electrode is characterized by comprising the following specific steps:

(1) Respectively using analytically pure acetone and analytically pure ethyl for the ITO conductive glass cut in advanceultrasonically cleaning alcohol and secondary water for 3min respectively, and drying in a 50 ℃ oven; placing the dried clean ITO in a medium containing 70% H₂SO₄And 30% H₂O₂Soaking the prepared piranha solution, taking out, washing with deionized water, ultrasonically cleaning, and drying with nitrogen; soaking the dried ITO in 10% KH550 ethanol solution for 2h, taking out, ultrasonically cleaning with ethanol, blow-drying with nitrogen, placing in a thermostat at 120 ℃ for 1 h, and drying for later use;

(2) Synthesizing gold nano sol by a citrate reduction method, diluting 1% chloroauric acid by 100 times, taking 100mL of diluted chloroauric acid aqueous solution with the concentration of 0.01% to a round bottom flask, heating the solution in an oil bath to boil, quickly adding 2mL of 1% trisodium citrate solution as a reducing agent under the stirring condition, continuously heating the solution for 30min, observing that the solution turns from light yellow to gray and then turns to black, finally turning to orange red, stopping heating, cooling to room temperature, and storing the solution in a refrigerator until the solution is used;

(3) Soaking the ITO treated in the step (1) in the gold nano sol prepared in the step (2) for 24 hours; then, placing the Au nano-film electrode in a tubular furnace, introducing nitrogen, after exhausting air, carrying out thermal annealing at 650 ℃ for 5 hours, cooling the electrode to room temperature, and taking out for later use;

(4) Putting the nano-gold ITO electrode prepared in the step (3) into mouse anti-human IgG solutions with different concentrations, placing the nano-gold ITO electrode in a constant-temperature water bath at 37 ℃ for 1 hour, standing the nano-gold ITO electrode in a refrigerator at 4 ℃ overnight, washing the surface of the electrode by PBST washing liquid, and removing unbound coating liquid; then putting the mixture into a sealing solution, wherein the sealing solution is a 5% skimmed milk powder-PBST solution, incubating the mixture for 1 hour at a constant temperature of 37 ℃, and washing the mixture with a PBST washing solution; and obtaining the embedded Au nano film electrode surface plasma resonance immunosensor.

2. When mouse anti-human IgG is not combined on the surface of the embedded Au nano film electrode and the embedded Au nano film electrode is inserted into a solution with RI equal to 1.333, the embedded Au nano film electrode is tested by using an ultraviolet-visible absorption spectrum, and the LSPR peak wavelength W is obtained₀(ii) a When the surface of the embedded Au nano film electrode is combined with different concentrationsAnd inserting the mouse anti-human IgG into a solution with RI equal to 1.333 to obtain the LSPR peak wavelength W, wherein the peak wavelength is W-W₀To analyze the signal, a measurement of mouse anti-human IgG was performed;

The embedded Au nano film electrode prepared by the method can utilize surface plasma resonance to carry out immunoassay, so the invention provides the application of the embedded Au nano film electrode in detecting mouse anti-human IgG.

Technical Field

the invention belongs to the field of nano film materials and the field of electrochemical sensors, and particularly relates to a preparation method of an embedded Au nano film electrode. In addition, the invention also relates to a method for measuring mouse anti-human IgG by using the surface plasma resonance immunoassay method.

Background

The Surface Plasmon Resonance (SPR) phenomenon is a phenomenon in which a resonant wave that changes the transmission of a light wave is formed at the interface between a dielectric and a metal by the light wave. When light is irradiated to the interface under the condition that the light is larger than the total reflection angle, a part of light is reflected, the other part of light is coupled into the plasma, and evanescent waves of the light exist in the surface plasma. When the wave vector of the incident light is parallel to the component of the interface and the wave vector of the surface plasma wave, the surface plasma resonates under the action of light, the light wave loses energy in transmission and macroscopically shows strong absorption of the light wave, and the phenomenon is called plasma resonance. The advantage of SPR sensors is; the anti-interference ability is strong, the measuring accuracy is high, the volume is small, the response speed is fast, the mechanical strength is strong, and the like. Therefore, the SPR technology has wide application prospect in the fields of chemistry, biology, environmental detection and the like.

The Surface Plasmon Resonance (SPR) effect of the gold nanoparticles under the excitation of visible light can generate very large field enhancement on the surface of the gold nanoparticles, and for example, the surface enhanced Raman scattering based on the Surface Plasmon Resonance (SPR) effect can increase the Raman signal of the molecules by several orders of magnitude or even more than ten orders of magnitude. The SPR effect also makes important contributions to the quantitative analysis of biomolecular interactions and the like for metal nano-films. Therefore, gold nanoparticles have been widely used as a functional material in the fields of sensors, electronics, chemistry, medicine, optics, and the like. Bioactive substances such as antibody proteins and the like have weak self-detectable signals and are difficult to quantitatively analyze or detect, and exogenous markers are required to be introduced, so that trace bioactive substances are quantified or traced through strong detectable signals. The existing labeled immunoassay includes enzyme immunoassay, chemiluminescence immunoassay, fluorescence immunoassay and the like. Among the methods, the biomarker technology based on quantum dot fluorescence is most emphasized, but the method has the defects of wide fluorescence spectrum peak, poor signal selectivity, photolysis and photobleaching, and certain limitations.

At present, most of other detection technologies have some defects, the expected effect cannot be completely achieved, and the detection requirement in actual work is met. Therefore, the existing detection technology aiming at ultra-low concentration substances has the defects of high sensitivity, quick response and trace loss, cannot completely achieve the expected effect, and meets the detection requirement in actual work. Therefore, it is very important to develop a surface plasma resonance immunoassay technology with high sensitivity, fast response, low solvent consumption and trace amount without damage.

Disclosure of Invention

in view of the defects of the prior art, the invention aims to provide the material preparation and the application of the surface plasmon resonance immunoassay method of the embedded Au nano film electrode, and provides a method for measuring mouse anti-human IgG by adopting the surface plasmon resonance immunoassay.

In order to achieve the technical effects, the invention adopts the following technical scheme:

Modifying an ITO electrode by using nanogold, and constructing an embedded Au nano film electrode to realize the measurement of mouse anti-human IgG; a method for surface plasma resonance immunoassay of an embedded Au nano film electrode comprises the following steps:

(1) Ultrasonically cleaning ITO conductive glass which is cut in advance for 3min by respectively using analytically pure acetone, analytically pure ethanol and secondary water, and drying in a 50 ℃ drying oven; the dried clean ITO was placed in piranha solution (70% H)₂SO₄：30％H₂O₂) Soaking, taking out, washing with deionized water, ultrasonically cleaning, and drying with nitrogen; and (3) soaking the dried ITO in 10% KH550 ethanol solution for 2h, taking out, ultrasonically cleaning with ethanol, blow-drying with nitrogen, placing in a thermostat at 120 ℃ for 1 h, and drying for later use.

(2) Synthesizing gold nano sol by a citrate reduction method, diluting 1% chloroauric acid by 100 times, taking 100mL of diluted chloroauric acid aqueous solution (0.01%) to a round bottom flask, heating the diluted chloroauric acid aqueous solution to boiling in an oil bath, rapidly adding 2mL of 1% trisodium citrate solution as a reducing agent under the stirring condition, continuously heating for 30min, observing that the solution turns from light yellow to gray, then turns to black, finally turns to orange red, stopping heating, cooling to room temperature, and storing in a refrigerator until the solution is used.

(3) And (3) soaking the ITO treated in the step (1) in the gold nano sol prepared in the step (2) for 24 hours. And then, placing the Au nano film electrode in a tube furnace, introducing nitrogen, after exhausting air, thermally annealing at 650 ℃ for 5 hours, cooling the electrode to room temperature, and taking out for later use.

(4) and (3) putting the nano-gold ITO electrode prepared in the step (3) into mouse anti-human IgG solutions with different concentrations, placing the nano-gold ITO electrode in a constant-temperature water bath at 37 ℃ for 1 hour, standing the nano-gold ITO electrode in a refrigerator at 4 ℃ overnight, washing the surface of the electrode by PBST washing liquid, and removing unbound coating liquid. Then putting the mixture into a sealing solution (5% skimmed milk powder-PBST solution), incubating at the constant temperature of 37 ℃ for 1 hour, and washing with a PBST washing solution. And obtaining the embedded Au nano film electrode surface plasma resonance immunosensor.

When the surface of the embedded Au nano-film electrode is not combined with the mouse anti-human IgG and is inserted into a solution with RI equal to 1.333, the ultraviolet-visible absorption spectrum is used for testing to obtain the LSPR peak wavelength W₀(ii) a When the surface of the embedded Au nano-film electrode is combined with mouse anti-human IgG with different concentrations and is inserted into a solution with RI equal to 1.333, the LSPR peak wavelength W is obtained and is W-W₀To analyze the signal, a measurement of mouse anti-human IgG was performed;

The embedded electrode prepared by the method can detect mouse anti-human IgG, so the invention provides the application of the sensor in detecting the content of the mouse anti-human IgG.

Compared with the prior art, the electrochemical sensor has the following advantages and remarkable progress:

(1) The embedded Au nano film prepared by the method has simple preparation process and high efficiency.

(2) the annealing method is utilized to simply and skillfully improve the surface plasma resonance effect of the embedded Au nano film.

(3) The surface plasma resonance effect is combined with immunoassay to carry out high-sensitivity, fast-response and trace nondestructive detection on bioactive substances, proteins and the like.

The invention provides a material preparation process based on a surface plasma resonance immunoassay method of an embedded Au nano film electrode and a method for detecting mouse anti-human IgG (immunoglobulin G) by utilizing an embedded Au nano film ITO electrode, taking mouse anti-human IgG as a detection object and utilizing ultraviolet-visible absorption spectrum for testing.

Has the advantages that: under the preferred experimental conditions, the concentration of the target mouse anti-human IgG is 1X 10^-3g/L～9×10^{- 3}The peak wavelength of LSPR is linear in the range of g/L (FIG. 3). The linear function relations are respectively: w-1.3288 c +568.63(c is the concentration of IgG), correlation coefficient R²The experimental detection limit reaches 1.457 multiplied by 10 when the product is 0.9869^-4g/L。

Drawings

Fig. 1 Scanning Electron Microscope (SEM) photograph of the embedded Au nano-thin film electrode.

FIG. 2 optimization of different annealing temperatures. In the figure, a-e are respectively the ultraviolet-visible absorption spectra of the embedded Au nano-film which is not annealed and is processed at 450 ℃, 350 ℃, 550 ℃ and 650 ℃.

FIG. 3 is a graph of UV-Vis absorption spectrum as a function of murine anti-human IgG concentration.

Detailed Description