Application of nano-gold directly bonded with luminol in immunoassay

Abstract

An application of luminol directly bonded gold nanoparticles in immunoassay is provided. An immunoassay probe with luminol directly bonded gold nanoparticles is composed of antibody labeled with luminol directly bonded gold nanoparticles, wherein the luminol directly bonded gold nanoparticles are prepared by a one-pot method through the reduction of chlorauric acid by luminol. A chemiluminescence immunoassay based on the luminol directly bonded gold nanoparticles immunoassay probe and a kit for performing the assay are also provided. The chemiluminescence immunoassay can be used for determining various analytes in samples. The probe and the assay can be applied to clinical diagnosis and treatment, pharmaceutical analysis, food safety assessment and environmental monitoring.

Description

technical field

[0001] The invention relates to the application of nano-gold directly bonded with luminol, in particular to its application in immunoassay. Background technique

[0002] Chemiluminescence is the emission of light during a chemical reaction. As an optical detection method, it does not require a light source, has the advantages of high sensitivity, wide linear range, low background noise, simple instrument, and low price, and has been widely used in immunoassays (Wu, A.H.B.Clin.Chim.Acta 2006 , 369, 119; Zhan, W.; Bard, A.J.Anal.Chem.2007, 79, 459; Yin, X.B.; Qi, B.; Sun; X.P.; Yang; X.R.; Wang; , 3525; Jie, G.F.; Huang, H.P.; Sun, X.l.; Zhu, J.J. Biosens. Bioelectron.2008, 23, 1896; Egashira, N.; Morita, S.; T.Anal.Chem.2008, 80, 4020), has become one of the main clinical analysis methods in many large hospitals. The performance of various chemiluminescent immunoassay methods depends largely on the probes employed. Existing commercial chemiluminescent anal...

Examples

preparation example Construction

[0058] The preparation method of the nano-gold immunoassay probe directly bonded to luminol of the present invention comprises the following steps:

[0059] (1) 100 mL of 0.01% (w / w) HAuCl 4 Heat the solution to boiling, under the conditions of condensing and refluxing and fully stirring, add 1.5-2.0mL solution containing 0.01mol / L luminol and 0.01mol / L sodium hydroxide, keep boiling and continue to stir and reflux for 30min, then remove The heating source continues to stir for more than 15 minutes to obtain a nano-gold solution with a particle diameter of 14-25nm;

[0060] (2) The synthesized nano-gold solution was dialyzed at normal temperature for 2 days, and the ultrapure water dialyzate was changed every 8 hours to obtain the nano-gold directly bonded to luminol;

[0061] (3) Add 0.5 mL of 1.0 mg / mL antibody to the gold colloid solution dialyzed in the above step (2) with 0.1 mol / L sodium hydroxide to adjust the pH to 8.0, and incubate at room temperature for 30 minutes ...

Embodiment 1

[0114] Embodiment 1. Preparation of the chemiluminescent immunoprobe of the present invention

[0115] (1) 100 mL of 0.01% (w / w) HAuCl 4 Heat the solution to boiling, under the conditions of condensing and refluxing and fully stirring, quickly add 1.5ml of a solution containing 0.01mol / L luminol and 0.01mol / L sodium hydroxide, keep boiling and continue to stir and reflux for 30min, then remove the heating Continue to stir for more than 15 minutes to obtain a nano-gold solution with a particle size of 25nm, cool to room temperature, and store at 4°C in the dark;

[0116] (2) The nano-gold solution synthesized in step (1) was dialyzed at normal temperature for 2 days, and the ultrapure water dialysate was changed every 8 hours. Free luminol and its oxidation products could be fully removed by the dialyzing operation, so that the nano-gold Luminol molecules in gold only exist in the form of bonding with the surface of gold nanoparticles, that is, gold nanoparticles directly bond...

Embodiment 2

[0118] Example 2. Immobilization of Sandwich Immune Complexes on Nanogold Modified Electrodes

[0119] Such as figure 2 As shown, the immobilization of the sandwich-type immune complex involved in the chemiluminescent immunoassay method of the present invention on the nano-gold modified electrode comprises the following steps:

[0120] (1) Preparation of nano-gold modified electrode:

[0121] A. Put the polished bare gold electrode into 2mmol / L 1,3'-propanedithiol, soak it at room temperature for 20 hours, take it out and wash it with ethanol and ultrapure water to obtain 1,3'-propanedithiol Dithiol-modified gold electrodes;

[0122] B. Drop 60 μL of streptavidin-linked gold nanoparticles on the 1,3′-propanedithiol-modified electrode prepared in step A, put it in the dark at 4°C for 4 hours, take it out and wash it with ultrapure water Washing to obtain a gold electrode modified by streptavidin / nanogold / 1,3'-propanedithiol;

[0123] (2) Immobilization of the double-antibo...

technical field

[0001] The invention relates to the application of nano-gold directly bonded with luminol, in particular to its application in immunoassay. Background technique

[0002] Chemiluminescence is the emission of light during a chemical reaction. As an optical detection method, it does not require a light source, has the advantages of high sensitivity, wide linear range, low background noise, simple instrument, and low price, and has been widely used in immunoassays (Wu, A.H.B.Clin.Chim.Acta 2006 , 369, 119; Zhan, W.; Bard, A.J.Anal.Chem.2007, 79, 459; Yin, X.B.; Qi, B.; Sun; X.P.; Yang; X.R.; Wang; , 3525; Jie, G.F.; Huang, H.P.; Sun, X.l.; Zhu, J.J. Biosens. Bioelectron.2008, 23, 1896; Egashira, N.; Morita, S.; T.Anal.Chem.2008, 80, 4020), has become one of the main clinical analysis methods in many large hospitals. The performance of various chemiluminescent immunoassay methods depends largely on the probes employed. Existing commercial chemiluminescent anal...

Examples

preparation example Construction

[0058] The preparation method of the nano-gold immunoassay probe directly bonded to luminol of the present invention comprises the following steps:

[0059] (1) 100 mL of 0.01% (w / w) HAuCl 4 Heat the solution to boiling, under the conditions of condensing and refluxing and fully stirring, add 1.5-2.0mL solution containing 0.01mol / L luminol and 0.01mol / L sodium hydroxide, keep boiling and continue to stir and reflux for 30min, then remove The heating source continues to stir for more than 15 minutes to obtain a nano-gold solution with a particle diameter of 14-25nm;

[0060] (2) The synthesized nano-gold solution was dialyzed at normal temperature for 2 days, and the ultrapure water dialyzate was changed every 8 hours to obtain the nano-gold directly bonded to luminol;

[0061] (3) Add 0.5 mL of 1.0 mg / mL antibody to the gold colloid solution dialyzed in the above step (2) with 0.1 mol / L sodium hydroxide to adjust the pH to 8.0, and incubate at room temperature for 30 minutes ...

Embodiment 1

[0114] Embodiment 1. Preparation of the chemiluminescent immunoprobe of the present invention

[0115] (1) 100 mL of 0.01% (w / w) HAuCl 4 Heat the solution to boiling, under the conditions of condensing and refluxing and fully stirring, quickly add 1.5ml of a solution containing 0.01mol / L luminol and 0.01mol / L sodium hydroxide, keep boiling and continue to stir and reflux for 30min, then remove the heating Continue to stir for more than 15 minutes to obtain a nano-gold solution with a particle size of 25nm, cool to room temperature, and store at 4°C in the dark;

[0116] (2) The nano-gold solution synthesized in step (1) was dialyzed at normal temperature for 2 days, and the ultrapure water dialysate was changed every 8 hours. Free luminol and its oxidation products could be fully removed by the dialyzing operation, so that the nano-gold Luminol molecules in gold only exist in the form of bonding with the surface of gold nanoparticles, that is, gold nanoparticles directly bond...

Embodiment 2

[0118] Example 2. Immobilization of Sandwich Immune Complexes on Nanogold Modified Electrodes

[0119] Such as figure 2 As shown, the immobilization of the sandwich-type immune complex involved in the chemiluminescent immunoassay method of the present invention on the nano-gold modified electrode comprises the following steps:

[0120] (1) Preparation of nano-gold modified electrode:

[0121] A. Put the polished bare gold electrode into 2mmol / L 1,3'-propanedithiol, soak it at room temperature for 20 hours, take it out and wash it with ethanol and ultrapure water to obtain 1,3'-propanedithiol Dithiol-modified gold electrodes;

[0122] B. Drop 60 μL of streptavidin-linked gold nanoparticles on the 1,3′-propanedithiol-modified electrode prepared in step A, put it in the dark at 4°C for 4 hours, take it out and wash it with ultrapure water Washing to obtain a gold electrode modified by streptavidin / nanogold / 1,3'-propanedithiol;

[0123] (2) Immobilization of the double-antibo...

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