阅读说明：本技术 一种基于表面增强拉曼技术的生物传感器及制备方法 (Biosensor based on surface enhanced Raman technology and preparation method thereof ) 是由 张茂峰 熊良钟 熊清爵 陈敏 阮志燕 王振 于 2019-09-11 设计创作，主要内容包括：本发明提供了一种基于表面增强拉曼技术的生物传感器及制备方法,该生物传感器为三层夹心结构,包括上层结构：键合拉曼检测分子和检测抗体的金核银壳纳米棒；中层结构：抗原；下层结构：键合捕获抗体的金核银壳纳米棒,该基于表面增强拉曼技术的夹心免疫检测生物传感器通过拉曼检测分子与金核银壳纳米棒的结合,大大增强了检测信号,对于超痕量物质及单分子检测的灵敏度显著提高,最低限可达fg/ml。(The invention provides a biosensor based on a surface enhanced Raman technology and a preparation method thereof, wherein the biosensor is of a three-layer sandwich structure and comprises an upper layer structure: a gold core silver shell nanorod bonded with the Raman detection molecule and the detection antibody; the structure of the middle layer: an antigen; the following layer structure: the sandwich immunoassay biosensor based on the surface enhanced Raman technology greatly enhances detection signals through the combination of Raman detection molecules and the gold-core silver-shell nanorod, obviously improves the sensitivity of detection of ultra-trace substances and single molecules, and can reach fg/ml at the lowest limit.)

1. A sandwich immunoassay biosensor based on surface enhanced raman technology, the biosensor comprising:

the top layer is a gold-core silver-shell nanorod bonded Raman detection molecule, a functional modifier and a detection antibody; and the substrate is a gold-core silver-shell nanorod bonding capture antibody.

2. The biosensor of claim 1, wherein the raman detecting molecule is vitamin K4, and the vitamin K4 molecule introduces a thiol group via diazotization sulfhydrylation.

3. The biosensor of claim 1, wherein the functional modifications comprise glutaraldehyde and cysteamine.

4. The biosensor of claim 1, wherein the biosensor forms a sandwich structure of detection antibody-antigen to be detected-capture antibody during operation.

Technical Field

The invention relates to the field of Raman scattering, in particular to a sandwich immunoassay biosensor based on a surface enhanced Raman technology.

Background

At present, the commonly used immunoassay method is enzyme-linked immunosorbent assay (ELISA), the method detects trace substances in body fluid by the specific combination of an antibody and an enzyme complex and then color development detection, the detection limit of the method can reach pg/ml, but substances with higher sensitivity requirements cannot be detected.

Therefore, in order to reliably and accurately detect ultra-trace or even single-molecule level analytes, Surface Enhanced Raman (SERS) has been gradually developed and widely applied to various fields such as chemical sensing, biological analysis, biosensing, and early cancer diagnosis. Typical SERS immunoassay detection methods are based on sandwich immune complexes capable of capturing antibodies immobilized on a solid substrate. The corresponding antigen is captured from the sample solution and detected by the characteristic raman spectroscopy of the SERS-labeled antibody after the sandwich structure is formed. Quantitative information was obtained from dose-dependent SERS experiments.

In addition, multiple detection of CA19-9, MMP7 and MUC4 in serum samples from pancreatic cancer patients has been demonstrated on a gold substrate-gold nanoparticle sandwich platform, with detection limits as low as 2ng/mL, however, with previous sandwich immunoassay design methods mostly using smooth macroscopic glass, gold, silver films or their bimetallic film substrates, resulting in limited sensitivity.

In order to improve the sensitivity of SERS detection, currently, a substrate material for SERS detection is bonded with Raman detection molecules, wherein the best effect is P-ATP molecules, and the sensitivity is ng/ml-pg/ml.

Disclosure of Invention

The invention provides a sandwich immunoassay biosensor based on a surface enhanced Raman technology, aiming at solving the problem of limited sensitivity of a sandwich type SERS immunoassay method in the prior art, comprising:

the top layer is a gold-core silver-shell nanorod bonded vitamin K molecule, a functional modifier and a detection antibody; and the substrate is a gold-core silver-shell nanorod bonding capture antibody.

Further, the functional modifier comprises glutaraldehyde and cysteamine.

Further, the biosensor can form a sandwich structure of detection antibody-antigen to be detected-capture antibody during working.

The invention provides a sandwich immunoassay biosensor based on a surface enhanced Raman technology, wherein a top layer and a substrate both adopt gold-core silver-shell nanorods as base materials, the gold-core silver-shell nanorods can remarkably improve electromagnetic signals in the surface Raman detection process, on the basis, the gold-core silver-shell nanorod at the top layer is firmly bonded with a diazotized sulfydryl vitamin K4 molecule through sulfydryl, so that the signal is further obviously enhanced, simultaneously, functional modified molecules of glutaraldehyde and cysteine are bonded to firmly fix the detection antibody by the top layer gold core silver shell nanorod and bond the capture antibody by the gold core silver shell nanorod serving as the substrate, the sandwich immunoassay biosensor based on the surface enhanced Raman technology can obviously improve the sensitivity of surface enhanced Raman detection by combining and fixing the antigen to be detected through specificity and integrating the structure.

It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed.

Drawings

Further objects, features and advantages of the present invention will become apparent from the following description of embodiments of the invention, with reference to the accompanying drawings, in which:

FIG. 1 shows a sandwich structure of "detection antibody-antigen to be detected-capture antibody" formed by a biosensor and an antigen to be detected in the present invention;

FIG. 2 shows a Raman spectrum of a SERS immunoassay with an IL-6 concentration of 1fg/mL and a blank sample in example 1;

FIG. 3 shows Raman intensity as a function of KIM1 concentration;

FIG. 4 shows 1145cm^-1The raman intensity of the peak is plotted as a function of KIM1 concentration, and the linear dynamic response range is shown in the inset.

Detailed Description

The objects and functions of the present invention and methods for accomplishing the same will be apparent by reference to the exemplary embodiments. However, the present invention is not limited to the exemplary embodiments disclosed below; it can be implemented in different forms. The nature of the description is merely to assist those skilled in the relevant art in a comprehensive understanding of the specific details of the invention.

Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings. In the drawings, like reference characters designate like or similar parts, or like or similar steps.