Homogeneous phase multi-component immunoassay method based on surface plasma coupling effect
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A surface plasma and coupling effect technology, applied in the field of protein quantification and immunoassay technology, can solve the problems of many elution steps, time-consuming and laborious, and high difficulty in operation, and achieve the effects of good specificity, enhanced Raman signal, and high sensitivity
Active Publication Date: 2013-02-06
HUNAN UNIV
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Problems solved by technology
The current immunoassay technology uses the principle of antigen-antibody immune sandwich reaction. The traditional detection method is mainly based on solid-phase reaction. First, the capture antibody is fixed, and then the antigen and detection antibody are combined sequentially. There are many elution steps, which are time-consuming and labor-intensive.
In addition, some new immunoassay techniques have been established, such as magnetic separation analysis technology, microfluidic immunoassay technology, nanotube technology based on field effect transistors, etc. These analytical methods are sensitive, fast and specific, but require the help of complex The instrument is difficult to operate, and the experimenter needs to master certain techniques
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Embodiment 1
[0028] Embodiment 1: detection of human interferon-γ
[0029] The specific operation steps are:
[0030] (1) Preparation of gold nanorod probes and gold nanosphere probes
[0031]Preparation method of gold nanorod probe: (1) Take 1 mL of 50 mM TCEP solution and add it to human interferon-γ antibody solution (polyclonal antibody, 0.1 mg / mL, 150 mL), mix well, and place in 37 °C Incubate in a shaker for 30 minutes; (2) Take 4 mL of gold nanorod (length-to-diameter ratio ~2.7) solution and centrifuge at 8000 rpm for 10 minutes to remove excess cetyltrimethylammonium bromide (CTAB) solution. The nanorods were redispersed in 5 mL of sterilized water; 280 mL of 15 mM Brij 56 solution was added to the gold nanorod solution, mixed thoroughly, and incubated in a shaker at 37°C for 30 minutes to replace the CTAB on the surface of the gold nanorods ; take it out, centrifuge at 8000 rpm for 10min, remove the upper layer solution, redisperse the gold nanorods in 2 mL sterilized water; (3...
Embodiment 2
[0037] Example 2: Multicomponent detection of cytokines
[0038] A test sample containing three cytokines (interferon-γ, interleukin-2, tumor necrosis factor-α) was selected as a model system. For the three cytokines to be tested, three gold nanorod probes Ⅰ, Ⅱ, Ⅲ and three gold nanosphere probes Ⅰ, Ⅱ, Ⅲ, gold nanorod probe Ⅰ and gold nanosphere probe Ⅰ were prepared respectively. Can specifically bind human interferon-γ, gold nanorod probe II and gold nanosphere probe II can specifically bind human interleukin-2, gold nanorod probe III and gold nanosphere probe III can specifically bind tumor necrosis factor-alpha.
[0039] The specific operation steps are:
[0040] (1) Preparation of gold nanorod probes and gold nanosphere probes
[0041] The preparation method of gold nanorod probe Ⅰ modified with Raman dye DTNB and human interferon-γ antibody: (1) Take 1 mL of 50 mM TCEP solution and add it to human interferon-γ antibody solution (polyclonal antibody, 0.1 mg / mL, 150 m...
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Abstract
The invention discloses a homogeneous phase multi-component immunoassay method based on a surface plasma coupling effect. A gold nanosphere probe (Raman reactive dyes and capture antibodies are embellished on the surface of the gold nanosphere probe) and a gold nanorod probe (Raman reactive dyes and detection antibodies are embellished on the surface of the gold nanorod probe) are respectively prepared; the Raman reactive dyes carry sulfydryl self and are free of fluorescent background; and the antibodies are subjected to TCEP (Tris(2-carboxyethyl) phosphine) reduction treatment. The antigen of an object to be tested has immune sandwich reaction with the antibodies so as to generate an immune complex, so that the distances between nano particles are shortened, the surface plasma coupling effect is generated, the Raman signals of the surface embellishing dyes on the surfaces of the nano particles are significantly enhanced, and the SERS (Surface Enhanced Raman Scattering) quantitative analysis of the antigen is realized by using the coupling enhancing effect. The method is simple and rapid, the detection process needs only one step of operation without repeated elution or separating samples, the synchronous quantitative analysis to a plurality of components in the samples is realized with high sensitivity and good uniqueness, and thus the method is expected to become a new method in proteomics research and drug therapeutic effect evaluation.
Description
technical field [0001] The invention belongs to a homogeneous multi-component immunoassay method based on the surface plasmon coupling effect, specifically refers to the biological functionalization and application of nanomaterials, surface-enhanced Raman resonance (SERS) and coupling enhancement effects, protein quantification and immunoassay analytical skills. Background technique [0002] Quantitative detection of protein molecular markers is of great significance to proteomics research. As an analytical index, it can sensitively reflect the efficacy of therapeutic drugs in disease models. The current immunoassay technology uses the principle of antigen-antibody immune sandwich reaction. The traditional detection method is mainly based on solid-phase reaction. First, the capture antibody is fixed, and then the antigen and detection antibody are combined sequentially. There are many elution steps, which are time-consuming and labor-intensive. In addition, some new immunoa...
Claims
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