阅读说明：本技术 一种基于表面增强拉曼散射的金纳米标记的试纸条、制备方法及使用方法 (Gold nano-labeled test strip based on surface-enhanced Raman scattering, preparation method and use method ) 是由 肖瑞 汪崇文 陆路春 于 2019-11-19 设计创作，主要内容包括：本发明公开了一种基于表面增强拉曼散射的金纳米标记的试纸条、制备方法及使用方法,涉及生物检测技术领域。其包括共轭垫,共轭垫上含有金纳米棒和拉曼分子,拉曼分子修饰于金纳米棒的表面,制备共轭垫所用的金纳米棒的局域表面等离子体共振峰(LSPR)可以根据需要自由调整。试纸条具有大散射截面和明显的拉曼散射峰且没有荧光干扰,具有灵敏度高、特异性好的优点。该试纸条使用该方法简单,检测速度快,检测范围广。(The invention discloses a gold nano-label test strip based on surface-enhanced Raman scattering, a preparation method and a use method thereof, and relates to the technical field of biological detection. The gold nanorod-based conjugate pad comprises a gold nanorod and Raman molecules, wherein the Raman molecules are modified on the surface of the gold nanorod, and a local surface plasmon resonance peak (LSPR) of the gold nanorod used for preparing the conjugate pad can be freely adjusted according to needs. The test strip has a large scattering cross section and an obvious Raman scattering peak, has no fluorescence interference, and has the advantages of high sensitivity and good specificity. The test strip is simple in use, high in detection speed and wide in detection range.)

1. A gold nano-labeled test strip based on surface-enhanced Raman scattering is characterized by comprising a conjugate pad, wherein the conjugate pad contains a gold nanorod and Raman molecules, the Raman molecules are modified on the surface of the gold nanorod, and a local surface plasma resonance peak of the gold nanorod used for preparing the conjugate pad can be adaptively adjusted.

2. The test strip of claim 1, wherein the local surface plasmon resonance peak of the gold nanorod is between 690 and 825 nm;

preferably, the local surface plasmon resonance of the gold nanorods is 785 nm.

3. The test strip of claim 1, wherein the Raman molecule is DTNB, MBA, DTTC, or PATP;

preferably, the raman molecule is DTNB.

4. The test strip of claim 1, wherein the surface of the gold nanorod is further modified with bovine serum albumin, and the bovine serum albumin is coupled with the amino-terminal peptide chain of the detection antibody through a carboxyl-terminal peptide chain;

preferably, the detection antibody is a tumor marker detection antibody;

preferably, the detection antibody is a murine anti-human alpha-fetoprotein detection antibody.

5. The test strip of claim 1, further comprising a PVC base plate, an antibody-sensitized nitrocellulose membrane, a sample pad, and absorbent paper, wherein the antibody-sensitized nitrocellulose membrane, a gold-labeled conjugate pad, the sample pad, and the absorbent paper are sequentially adhered to the PVC base plate;

preferably, the antibody coating film is coated with an indicating antibody and a capturing antibody to form a quality control line and a detection line respectively;

preferably, the indication antibody is a Y anti-X antibody, the capture antibody is an X anti-human tumor marker, X is a monoclonal antibody, and Y is a polyclonal antibody;

preferably, said X is from a mouse or rat and said Y is from a sheep, rabbit or horse;

preferably, the capture antibody is a mouse anti-human alpha-fetoprotein capture antibody, the indicator antibody is goat anti-mouse IgG, and the antibody coating membrane is an NC membrane.

6. A method for detecting a tumor marker using the test strip of any one of claims 1 to 5, which is not involved in the treatment or diagnosis of a disease, comprising the steps of: the sample to be tested is placed on the sample pad.

7. The method of claim 6, further comprising recording a surface enhanced Raman scattering signal of the detection line with a Raman spectrometer or visually observing a color of a band of the detection line;

preferably, the excitation wavelength of the Raman spectrometer is 785 nm.

8. A method for preparing the test strip of any one of claims 1 to 5, comprising the steps of: modifying the gold nanorods by using Raman molecules to prepare the gold nanorods modified with the Raman molecules.

9. The preparation method according to claim 8, further comprising preparing a conjugate pad using gold nanorods modified with raman molecules, and then assembling the sensitized nitrocellulose membrane, the conjugate pad, a sample pad, and an absorbent pad in this order on a PVC base plate;

preferably, the method for preparing the conjugate pad comprises the steps of coating bovine serum albumin on the surface of the gold nanorod modified with the Raman molecules to obtain the gold nanorod coated with the bovine serum albumin, coupling the detection antibody with the gold nanorod coated with the bovine serum albumin to obtain the gold nanorod coupled with the antibody, uniformly mixing the gold nanorod coupled with the antibody with the gold-labeled diluent, and dropwise adding the gold-labeled diluent on the gold-labeled conjugate pad to obtain the conjugate pad.

10. The method according to claim 8, wherein the method further comprises preparing gold nanorods, and the preparing gold nanorods comprises adding 0.8ml of chloroauric acid, 0.3-0.35ml of 0.01M silver nitrate, 0.4ml of hydrochloric acid, 0.32ml of ascorbic acid, and 0.1ml of seed solution to 40ml of CTAB solution.

Technical Field

The invention relates to the technical field of biological detection, in particular to a gold nano-label test strip based on surface enhanced Raman scattering, a preparation method and a use method thereof.

Background

Cancer is one of the most life-threatening diseases in the world, and the mortality rate caused by cancer is high because its symptoms usually appear late. Thus, early diagnosis of cancer can guide active treatment and improve patient survival. Among them, the detection of cancer biomarkers has become one of the most promising approaches for early diagnosis of cancer. A key indicator of tumorigenesis and therapeutic response is the change in cancer biomarker concentration in human biological specimens such as serum, blood, urine, or saliva. The current methods for detecting the tumor biomarkers mainly comprise a chemiluminescence immunoassay, an enzyme-linked immunosorbent assay (ELISA), an immunoblotting method, an electrochemical immunoassay method and the like, but the methods have the defects of complex operation, long detection time, high cost and the like. The above-mentioned drawbacks prevent the wide application of the existing marker detection methods in point-of-care detection. The establishment of a sensitive and convenient detection method is extremely necessary for the diagnosis of tumor markers.

The Lateral Flow Immunoassay (LFIA) has the advantages of simple operation, high analysis speed, low cost and the like, but the traditional immunoassay takes colloidal gold as a label, depends on a colorimetric signal, and has the defects of low sensitivity, limited qualitative and semi-quantitative properties.

Surface Enhanced Raman Scattering (SERS) is a highly sensitive and effective detection tool that can address the limitations of conventional immune lateral flow assays. However, the local surface plasmon resonance peak (LSPR) of the existing SERS-based gold or silver nanoparticles is between 400 and 600nm and does not match the commonly used 785nm excitation wavelength, and thus, is not ideal in the SERS signal enhancement performance.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a gold nano-label test strip based on surface enhanced Raman scattering, a preparation method and a use method thereof so as to solve the technical problems.

The invention is realized by the following steps:

a gold nano-labeled test strip based on surface-enhanced Raman scattering comprises a conjugate pad, wherein the conjugate pad contains a gold nanorod and Raman molecules, the Raman molecules are modified on the surface of the gold nanorod, and a local surface plasmon resonance peak (LSPR) of the gold nanorod used for preparing the conjugate pad can be adaptively adjusted.

In a preferred embodiment of the application of the invention, bovine serum albumin is further modified on the surface of the gold nanorod, and is coupled with the amino-terminal peptide chain of the detection antibody through the carboxyl-terminal peptide chain;

preferably, the detection antibody is a tumor marker detection antibody;

preferably, the detection antibody is a murine anti-human alpha-fetoprotein detection antibody.

The test strip comprises a conjugate pad, wherein the conjugate pad contains a gold nanorod, and the surface of the gold nanorod is modified with Raman molecules and bovine serum albumin. The Raman molecule is used as a Raman reporter molecule and is used for feeding back signals of maximum surface enhanced Raman scattering under excitation light. Bovine Serum Albumin (BSA) has the function of enhancing the stability of the gold nanorods, and prevents the gold nanorods from mutually aggregating to influence the performance of the gold nanorods. The inventor proves that the gold nanorods modified with BSA can keep activity under the environment of higher salt ion strength and impurities. Bovine serum albumin is modified on the surface of the gold nanorods to enhance the high stability of the gold nanorods in a biological sample and prevent the gold nanorods from mutually aggregating to influence the performance of the gold nanorods.

The detection antibody is coupled with the carboxyl of BSA through an amino group, the detection antibody can realize the specific combination of an antigen (tumor marker), and the antigen can also be specifically combined with an antibody on a nitrocellulose membrane, so that the qualitative and quantitative detection of the gold nanorod composite for capturing the antigen is realized.

The invention creatively discovers that the adjustment of the local surface plasma resonance peak of the gold nanorod used for preparing the conjugate pad can be realized by controlling the content of silver nitrate. Therefore, the requirement of conventional 785nm excitation wavelength can be met, the surface enhanced Raman scattering signal is increased more obviously, and the detection of low-content biomarkers is facilitated.

The test strip provided by the invention realizes the interception of the antigen tumor marker by coating the tumor marker detection antibody on the conjugate pad, and the detection antibody is preferably a mouse anti-human alpha-fetoprotein detection antibody.

In a preferred embodiment of the present invention, the local surface plasmon resonance peak of the gold nanorod is 690-825 nm;

preferably, the local surface plasmon resonance peak of the gold nanorods is 785 nm.

When the content of silver nitrate is 0.35ml, the local surface plasmon resonance peak (LSPR) of the gold nanorod is 785 nm. If the content of silver nitrate is more than or less than 0.35ml, the local surface plasmon resonance peak (LSPR) of the gold nanorod deviates from 785nm, so that the Surface Enhanced Raman Scattering (SERS) signal is not obviously increased.

In a preferred embodiment of the present invention, the raman molecule is DTNB, MBA, DTTC or PATP;

preferably, the raman molecule is DTNB.

DTNB (5, 5' -dithiobis (2-nitrobenzoic acid)) is preferred as the Raman molecule in this example, and thiol-containing compounds in antibodies and antigens can react with DTNB to break the disulfide bond of DTNB to produce 2-nitro-5-thiobenzoic acid (NTB-), and if ionized in water at neutral or basic pH, can form NTB ^2-A divalent anion. Such NTB ^2-The ions appear yellow. The detection can be quantitative under visible light or ultraviolet spectrum.

In a preferred embodiment of the present invention, the nitrocellulose membrane (NC membrane) is coated with an indicator antibody and a capture antibody to form a quality control line and a detection line, respectively;

preferably, the indication antibody is a Y anti-X antibody, the capture antibody is an X anti-human tumor marker, X is a monoclonal antibody, and Y is a polyclonal antibody;

preferably, X is from a mouse or rat and Y is from a sheep, rabbit or horse;

preferably, the capture antibody is a murine anti-human alpha-fetoprotein capture antibody, the indicator antibody is a goat anti-murine IgG, (the antibody envelope is NC membrane).

When a target antigen is dripped on the sample pad, the antigen passes through the conjugate pad under the action of capillary force, is specifically combined with a detection antibody on the conjugate pad, continuously moves towards the direction of absorbent paper along the nitrocellulose membrane, passes through the detection line (T line), is specifically combined with the antigen combined with the gold nanorods and a capture antibody on the detection line, and is specifically combined with the remaining antigen combined with the gold nanorods and an indication antibody, so that the detection line and the quality control line are provided with strips. The indicator antibody must be paired with the same type of detector antibody, and the host species of the indicator antibody should be different from that of the detector antibody.

The method for detecting the tumor marker by using the test strip does not relate to the treatment and diagnosis of diseases, and comprises the following steps: the sample to be tested is placed on the sample pad.

When a sample to be detected does not contain a target antigen (tumor marker), the sample enters the conjugate pad under the action of capillary force, the nanorod (AuNRs @ BSA @ Antibody) on the conjugate pad enters a detection line region under the action of capillary force, and the AuNRs @ BSA @ Antibody does not contain the antigen and cannot be specifically recognized with a capture Antibody on the Antibody coating film, so that no strip is generated in the detection line, and the AuNRs @ BSA @ Antibody is specifically combined with an indication Antibody, so that a quality control line is generated with a strip.

In a preferred embodiment of the present invention, the method further comprises recording the surface enhanced raman scattering signal of the detection line with a raman spectrometer or visually observing the color of the strip of the detection line;

preferably, the excitation wavelength of the Raman spectrometer is 785 nm.

For quantitative analysis, Surface Enhanced Raman Scattering (SERS) signal detection is required.

A preparation method of a test strip comprises the following steps: modifying the gold nanorods by using Raman molecules to prepare the gold nanorods (AuNRs-Raman molecules) modified with the Raman molecules.

The preparation method also comprises the steps of preparing a conjugate pad by using the gold nanorods (AuNRs-Raman molecules) modified with Raman molecules, and then sequentially assembling the sensitized nitrocellulose membrane, the conjugate pad, the sample pad and the absorption pad on a PVC (polyvinyl chloride) bottom plate;

preferably, the method for preparing the conjugate pad comprises the steps of coating bovine serum albumin on the surface of a gold nanorod (AuNRs-Raman molecule) modified with a Raman molecule to obtain a gold nanorod (AuNRs @ BSA) coated with the bovine serum albumin, coupling a detection Antibody and the gold nanorod coated with the bovine serum albumin to obtain a gold nanorod (AuNRs @ BSA @ Antibody) coupled with the detection Antibody, and dripping the gold nanorod coupled with the detection Antibody on the gold nanorod with a gold-labeled diluent to obtain the conjugate pad.

In a preferred embodiment of the present invention, the preparation method further comprises preparing gold nanorods, wherein the preparing of the gold nanorods comprises adding 0.8ml of chloroauric acid, 0.3-0.35ml of 0.01M silver nitrate, 0.4ml of hydrochloric acid, 0.32ml of ascorbic acid and 0.11ml of seed solution to 40ml of CTAB solution of gold-containing nano material. According to multiple experiments, the silver nitrate with the best intensity of the Raman signal of the gold nanorod is added in an amount of 0.35ml of 0.01M silver nitrate added in each 40ml of CTAB solution, and the length-diameter ratio of the gold nanorod is the best at the moment. If the addition amount of silver nitrate is larger than or smaller than the above amount, the prepared gold nanorods are too long or too short in size, so that the local surface plasmon resonance peak of the gold nanorods used for preparing the conjugate pad is deviated, and thus the gold nanorods cannot be matched with a given excitation wavelength, the resonance enhancement effect is further reduced, and the detection sensitivity is reduced.

The invention has the following beneficial effects:

the invention provides a gold nano-labeled test strip based on surface-enhanced Raman scattering, which comprises a conjugate pad, wherein the conjugate pad contains a gold nanorod and Raman molecules, a stable hot spot near the tip of the nanorod and the localization of a plasma peak of the stable hot spot enable the nanorod to have a strong Raman scattering enhancement effect, and the content of silver nitrate affects the local surface plasma resonance peak of the gold nanorod, so that the resonance peak is adjusted according to the requirement of excitation wavelength. Therefore, the local surface plasma resonance peak (LSPR) of the gold nanorod in the excitation wavelength range is matched with the excitation wavelength, the maximum plasma coupling effect is generated, and the resonance enhancement effect is further improved. Loss of biological sample can also be reduced at the plasma resonance peak. The test strip prepared by the preparation method has a large scattering cross section, an obvious Raman scattering peak, no fluorescence interference, high sensitivity and good specificity. The test strip is simple in use, high in detection speed and wide in detection range.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a schematic diagram (b) of the preparation process (a) of gold nanorods and the detection of test strips;

FIG. 2 is TEM pictures of AuNRs under different synthesis conditions, and ultraviolet-visible spectra and SERS intensity graphs of gold nanorods under different length-diameter ratios;

FIG. 3 is a graph showing the results of a SERS marker assay performed with BSA coated gold nanorods;

FIG. 4 is a graph of the analytical performance of the SERS-LFIA test strip;

FIG. 5 is an ultraviolet-visible spectrum of a comparative example using gold nanospheres as the substrate.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.