CN111781376A - Rapid detection method of Alzheimer disease biomarker - Google Patents
Rapid detection method of Alzheimer disease biomarker Download PDFInfo
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- CN111781376A CN111781376A CN202010653821.0A CN202010653821A CN111781376A CN 111781376 A CN111781376 A CN 111781376A CN 202010653821 A CN202010653821 A CN 202010653821A CN 111781376 A CN111781376 A CN 111781376A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
- G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Abstract
The invention discloses a rapid detection method of Alzheimer disease biomarkers; due to the heterogeneity of dementia pathology and the complexity of the pathogenesis of alzheimer's disease, simultaneous detection of multiple biomarkers of alzheimer's disease is essential for the early accurate diagnosis of alzheimer's disease; therefore, the detection method is based on a double-resistance sandwich type mode, and can be used for simultaneously and rapidly detecting a plurality of Alzheimer disease biomarkers by adopting a lateral flow test strip technology based on surface enhanced Raman scattering; compared with the prior art, the kit has the characteristics of strong specificity, high sensitivity, low cost, multiple detection and rapid detection, and has wide application prospect in early diagnosis and monitoring of the Alzheimer disease.
Description
Technical Field
The invention relates to a rapid detection method of an Alzheimer disease biomarker, in particular to a detection method of a lateral flow test strip based on surface enhanced Raman scattering. Belongs to the field of instant detection.
Background
Alzheimer's Disease (AD) is the "fourth leading killer" of health in the elderly following cardiovascular disease, cerebrovascular disease and cancer. It is generally accepted that disease modifying treatment of alzheimer's disease is most effective in the early and symptomatic mild stages of the disease. According to recent reports, early diagnosis can reduce the risk of AD by one third, and a high sensitivity biomarker for detecting alzheimer disease is expected to diagnose AD early.
At present, β -amyloid (A β), tau and neurofilament light chain proteins have been demonstrated as possible biomarkers for AD.A single biomarker cannot accurately diagnose AD, particularly for early diagnosis of AD, due to the heterogeneity of the dementia pathology and the complexity of the pathogenesis of AD, while simultaneously detecting multiple biomarkers for AD, such as by analyzing t-tau/A β in combination42、Aβ42/Aβ40And p-tau181/Aβ42The accuracy of diagnosis can be greatly improved, and the AD can be detected 15 years in advance, while the single detection A β has low accuracy and can detect AD. only several years in advance, so that the simultaneous detection of multiple biomarkers of AD is very necessary for early diagnosis of AD.
Currently, positron emission tomography is a clinical detection method for AD biomarkers. But it requires tens of millions of valuable equipment and injection of a dose of radiopharmaceutical into a subject, and is not suitable for large-scale early screening of people without symptoms. Other detection methods aiming at AD biomarkers mainly comprise enzyme-linked immunosorbent assay, electrochemical biosensors and high-sensitivity optical analysis platforms based on surface plasmon resonance. Due to the extremely low content of the AD biomarker, the detection sensitivity of two detection methods of enzyme-linked immunosorbent assay and electrochemical biosensor is not enough and the detection cost is high. However, the high-sensitivity optical analysis platform based on surface plasmon resonance can only detect a single AD biomarker at present, and can not accurately diagnose and predict AD. Therefore, it is necessary to invent a detection method which is low in cost, high in sensitivity, high in specificity and capable of performing multiplex detection.
Surface Enhanced Raman Scattering (SERS) measures the amplified raman scattering signal near or adsorbed to a noble metal nanoparticle or nanostructured surface molecule. SERS has many advantages such as super high sensitivity, can many targets detect and flexible operation. The lateral flow test paper is combined with a lateral flow test paper technology, so that the detection sensitivity can be improved, and the high-specificity multi-target detection can be realized.
Therefore, the invention discloses a lateral flow test strip based on surface enhanced Raman scattering, which is used for carrying out high-sensitivity and high-specificity multiplex detection and rapid detection on AD biomarkers so as to carry out early diagnosis on AD.
Disclosure of Invention
The invention aims to provide a rapid detection method of an Alzheimer disease biomarker, which is based on a lateral flow test strip technology of surface enhanced Raman scattering and has the characteristics of strong specificity, high sensitivity, low cost, multiple detection and rapid detection.
The detection method comprises the following steps:
(1) preparing a lateral flow test strip for detecting the Alzheimer disease biomarker;
(2) dropwise adding a sample solution to be detected onto a sample pad of the test strip;
(3) after a period of time (10 to 30 minutes), raman spectroscopy was performed on the control lines on the test strips using a raman spectrometer:
if the obtained Raman spectrum of the quality control line has characteristic peaks of all used Raman labels, the test result of the test strip is effective, otherwise, a new test strip needs to be replaced or the test strip needs to be prepared again for detection;
if all the characteristic peaks of the used Raman labels appear in the obtained Raman spectrum of the quality control line, performing Raman spectrum analysis on the detection line on the test strip by using a Raman spectrometer, and obtaining the concentration of the corresponding Alzheimer disease biomarker.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a lateral flow test strip for rapidly detecting AD biomarkers is composed of five parts: sample pad, gold label pad, nitrocellulose membrane (NC membrane), absorbent pad and backing. The NC film is fixed in the middle of the back lining, the absorption pad is fixed at the tail end of the back lining, and the sample pad and the gold label pad are fixed at the head end of the back lining. The NC film, gold label pad, sample pad, absorbent pad were affixed to the backing in sequence with the ends of each section overlapping to ensure continuous flow along each section by capillary action after the sample was dropped onto the sample pad.
The AD biomarkers are beta-amyloid, tau protein and neurofilament light chain protein.
The sample pad is saturated with a buffered saline solution containing a surfactant.
And the NC membrane is transversely provided with a detection line and a quality control line, wherein the quality control line is arranged on the right side of the detection line in parallel.
The detection line is one or more straight lines, one or more capture antibodies are fixed on the straight lines, and the capture antibodies are monoclonal antibodies or polyclonal antibodies of beta-amyloid protein, tau protein and neurofilament light chain protein.
And a goat anti-mouse IgG antibody, a rabbit anti-mouse IgG antibody or a goat anti-rabbit 1gG antibody is fixed on the quality control line.
The gold label pad contains one or more SERS nano labels connected with one or more detection antibodies, and the detection antibodies are connected to the SERS nano labels through adsorption or covalent bonding.
The detection antibody is a monoclonal antibody or a polyclonal antibody of beta-amyloid protein, tau protein and neurofilament light chain protein.
The SERS nano-tag refers to a metal nano-particle coded by a Raman tag.
The Raman label is any one of nai Er blue A, crystal violet, rhodamine 6G, methylene blue, p-aminophenol, water-soluble 3H-indocyanine type biological fluorescent labeling dye, methyl blue, 4' -bipyridyl, malachite green isothiocyanate, 4-thiopyridine, p-fluorophenylthiol, p-thioaniline, p-aminophenol, p-thiothiophenol, p-boryl benzoic acid or toluidine blue.
The metal nanoparticles are nanoparticles with a single structure or nanoparticles with a core-shell structure, and the nanoparticles with the single structure are gold nanoparticles, silver nanoparticles or platinum nanoparticles; the nanoparticles of the core-shell structure are gold core silver shell nanoparticles, gold core gold shell nanoparticles, silver core silver shell nanoparticles, silica core gold shell nanoparticles, gold core silica shell nanoparticles, silica core silver shell nanoparticles or silver core silica shell nanoparticles.
Has the advantages that: compared with the prior art, the invention has the following advantages:
1. the sensitivity is high: according to the invention, the SERS nano-tag and the lateral flow test paper technology are combined, and the Raman tag on the detection line is detected by the Raman spectrometer, so that the detection limit can reach fg/mL level, and the sensitivity is high;
2. the specificity is strong: the test strip is prepared on the basis of a double-resistance sandwich type mode, an NC membrane is provided with a detection line and a quality control line, whether the test strip works normally or not can be judged through the quality control line, the result is accurate, and the specificity is strong;
3. multiplex detection: the test strip can be provided with a plurality of detection lines, and each detection line can also be fixed with a plurality of capture antibodies, so that a plurality of AD biomarkers can be detected, and the cost is effectively reduced;
4. and (3) rapid detection: the whole detection process can be completed within half an hour;
5. the cost is low: the lateral flow test strip is low in manufacturing cost.
Drawings
FIG. 1 is a schematic representation of a lateral flow strip for detecting a single AD biomarker;
FIG. 2 is a schematic diagram of a lateral flow test strip for simultaneously detecting multiple AD biomarkers in multiple detection lines;
FIG. 3 is a schematic diagram of a lateral flow test strip for simultaneously detecting multiple AD biomarkers in one detection line;
in the figure: 1. the kit comprises a sample pad, 2, a nitrocellulose membrane, 3, a gold label pad, 4, an SERS nano label, 5, a detection line, 6, a capture antibody, 7, a quality control line, 8, a quality control antibody, 9, an absorption pad, 10 and a backing.
Detailed Description
The invention is further illustrated by the following examples.
Example 1: detection of beta-amyloid
As shown in figure 1, a lateral flow test strip for rapidly detecting AD biomarkers is constructed, and the lateral flow test strip consists of five parts, namely a sample pad 1, a gold-labeled pad 3, a nitrocellulose membrane 2, an absorption pad 9 and a backing 10, wherein the nitrocellulose membrane 2 is fixed at the middle position of the backing 10, the absorption pad 9 is fixed at the tail end of the backing 10, and the sample pad 1 and the gold-labeled pad 3 are fixed at the head end of the backing 10NBA@ Au SERS nanotags 4), β -amyloid monoclonal antibody is fixed on the nitrocellulose membrane 2 to form a detection line 5, and goat anti-mouse IgG antibody is fixed on the nitrocellulose membrane 2 to form a quality control line 7.
When the sample solution is dropped on the sample pad 1, the sample solution flows forward by capillary action, and β -amyloid in the sample solution is mixed with Ag when the sample solution flows through the gold label pad 3NBAThe detection antibody β on the surface of the @ Au SERS nanotag 4 reacts with the amyloid detection antibody to form an antigen-antibody hybrid complex, which flows forward along with the liquid, and when the immune complex reaches the detection line 5, the complex is captured by the β -amyloid capture antibody 6 immobilized on the detection line 5. therefore, a sandwich-type hybrid complex is formed on the detection line 5. the rest of the sample solution is Ag connected with the β -amyloid detection antibodyNBAThe @ Au SERS nanotags 4 continue to flow forward and are captured by goat anti-mouse IgG antibodies on the quality control line 7.
And performing Raman spectrum analysis on the quality control line 7 and the detection line 5 by using a Raman spectrometer. Raman spectrum on quality control line 7The characteristic peak of the Naerlang A shows that the detection result of the lateral flow test strip is effective, and the quantitative detection of β -amyloid can be realized by detecting Ag on the detection line 5NBA@ Au SERS nanotag 4.
Example 2: three detection lines 5 are used for simultaneously detecting beta-amyloid protein, tau protein and neurofilament light chain protein
As shown in figure 2, a lateral flow test strip for rapidly detecting AD biomarkers is constructed, and the lateral flow test strip consists of five parts, namely a sample pad 1, a gold-labeled pad 3, a nitrocellulose membrane 2, an absorption pad 9 and a backing 10, wherein the nitrocellulose membrane 2 is fixed at the middle position of the backing 10, the absorption pad 9 is fixed at the tail end of the backing 10, and the sample pad 1 and the gold-labeled pad 3 are fixed at the head end of the backing 10NBA@ Au SERS nano tag 4), tau protein monoclonal antibody connected AgNBAAg connected with @ Au SERS nano tag 4 and neurofilament light chain protein monoclonal antibodyNBAThe @ Au SERS nanotags 4. the nitrocellulose membrane 2 is provided with three detection lines 5 and a quality control line 7, β -amyloid monoclonal antibody, tau monoclonal antibody and neurofilament light chain protein monoclonal antibody are respectively fixed on the three detection lines 5, and a goat anti-mouse IgG antibody is fixed on the quality control line 7.
When the sample solution is dropped onto the sample pad 1, the sample solution flows forward by capillary action, and when the sample solution flows through the gold pad 3, the corresponding target antigens (β -amyloid, tau, and neurofilament light chain protein) are linked to AgNBAThe corresponding detection antibodies (β -amyloid, tau protein and neurofilament light chain protein detection antibodies) on the surface of the @ Au SERS nanotag 4 react to form three different types of antigen-antibody hybrid complexes.The complex will follow the liquid to flow forward, and when the immune complex reaches the first detection line 5, β -amyloid and β -amyloid detect antibody-linked AgNBAThe complex formed by the @ Au SERS nanotags 4 is captured by β -amyloid capture antibody 6 immobilized on the detection line 5. therefore, a sandwich β -amyloid antigen-antibody hybrid complex is formed on the first detection line 5.
The remaining solution continues to flow forward, and similarly, a sandwich tau protein antigen-antibody hybrid complex is formed on the second detection line 5 and a sandwich neurofilament light chain protein antigen-antibody hybrid complex is formed on the third detection line 5. Finally, the remaining sample solution was combined with three detection antibody-linked AgNBAThe @ Au SERS nanotags 4 continue to flow forward and are captured by goat anti-mouse IgG antibodies on the quality control line 7.
Raman spectroscopy analysis is carried out on the quality control line 7 and the detection line 5 by using a Raman spectrometer, the characteristic peak of Narlan A appears in the Raman spectrum on the quality control line 7, the detection result of the lateral flow test strip is effective, and β -amyloid protein, tau protein and neurofilament light chain protein can be quantitatively detected by measuring Ag on the detection line 5NBA@ Au SERS nanotag 4.
Example 3: one detection line 5 is used for simultaneously detecting beta-amyloid protein, tau protein and neurofilament light chain protein
As shown in figure 3, a lateral flow test strip for rapidly detecting AD biomarkers is constructed, and the lateral flow test strip consists of five parts, namely a sample pad 1, a gold-labeled pad 3, a nitrocellulose membrane 2, an absorption pad 9 and a backing 10, wherein the nitrocellulose membrane 2 is fixed at the middle position of the backing 10, the absorption pad 9 is fixed at the tail end of the backing 10, and the sample pad 1 and the gold-labeled pad 3 are fixed at the head end of the backing 10Silver core gold shell SERS nano-tag 4 (Ag) connected with cloned antibody and wrapped with nailan ANBA@ Au SERS nano-tag 4) and tau protein monoclonal antibody connected silver core gold shell SERS nano-tag 4 (Ag) wrapped with methylene blueMB@ Au SERS nano tag 4) and neurofilament light chain protein monoclonal antibody connected silver core gold shell SERS nano tag 4 (Ag) wrapped with rhodamine 6GR6G@ Au SERS nanotags 4), the nitrocellulose membrane 2 is provided with a detection line 5 and a quality control line 7, the detection line 5 is fixedly provided with β -amyloid monoclonal antibody, tau monoclonal antibody and neurofilament light chain protein monoclonal antibody, and the quality control line 7 is fixedly provided with goat anti-mouse IgG antibody.
When the sample solution is dropped onto the sample pad 1, the sample solution flows forward by capillary action. As the sample solution flows through the gold pad 3, the corresponding target antigens (β -amyloid, tau, and neurofilament light chain protein) react with the corresponding detection antibodies (β -amyloid, tau, and neurofilament light chain protein detection antibodies) immobilized on the SRES nanotags, forming three immune complexes, and continue to flow forward along the strip. When these immune complexes reach the detection line 5, the three complexes are captured by three capture antibodies 6 (beta-amyloid, tau protein and neurofilament light chain protein capture antibodies) pre-immobilized on the detection line 5, respectively, thereby forming three double-anti sandwich-type immune complexes on the detection line 5. The remaining solution continued to flow forward and was captured by goat anti-mouse IgG antibody on quality control line 7.
Raman spectroscopy analysis is carried out on the quality control line 7 and the detection line 5 by using a Raman spectrometer, characteristic peaks of nai' ERlan A, methylene blue and rhodamine 6G appearing in the Raman spectrum on the quality control line 7 indicate that the detection result of the lateral flow test strip is effective, and β -amyloid, tau protein and neurofilament light chain protein can be quantitatively detected by measuring Ag on the detection line 5NBA@Au、AgMB@ Au and AgR6G@ Au SERS nanotag 4.
The above examples are only preferred embodiments of the present invention, and are not intended to limit the present invention, and it will be apparent to those skilled in the art that several modifications and improvements can be made to the present invention, and these modifications and improvements are also considered to be within the scope of the present invention.
Claims (10)
1. A rapid detection method of Alzheimer's disease biomarkers, which is characterized by comprising the following steps:
(1) preparing a lateral flow test strip for detecting the Alzheimer disease biomarker;
(2) dropwise adding a sample solution to be detected onto a sample pad of the test strip;
(3) and (3) after standing for 10-30 minutes, performing Raman spectrum analysis on the quality control line on the test strip by using a Raman spectrometer:
if the obtained Raman spectrum of the quality control line has characteristic peaks of all used Raman labels, the test result of the test strip is effective, otherwise, the test strip needs to be replaced with a new test strip for detection again;
if all the characteristic peaks of the used Raman labels appear in the obtained Raman spectrum of the quality control line, performing Raman spectrum analysis on the detection line on the test strip by using a Raman spectrometer, and obtaining the concentration of the corresponding Alzheimer disease biomarker.
2. The method for rapidly detecting the biomarker of alzheimer's disease according to claim 1, wherein the biomarker comprises: the bottom of the lateral flow test strip is a backing, and a sample pad, a gold label pad, a nitrocellulose membrane and an absorption pad are sequentially arranged on the backing from left to right; the nitrocellulose membrane is provided with a detection line and a quality control line which are arranged in parallel, and the quality control line is arranged on the right side of the detection line.
3. The method for rapidly detecting the Alzheimer's disease biomarker according to claim 2, wherein the sample pad is pretreated by the following steps: the sample pad is placed in a buffered saline solution containing a surfactant for soaking.
4. The method for rapidly detecting the biomarker of alzheimer's disease according to claim 2, wherein the biomarker comprises: the detection line is one or more straight lines, and one or more capture antibodies are fixed on the straight lines.
5. The method for rapidly detecting the biomarker of Alzheimer's disease according to claim 4, wherein the biomarker comprises: the capture antibody is a monoclonal antibody or a polyclonal antibody of beta-amyloid protein, tau protein and neurofilament light chain protein.
6. The method for rapidly detecting the biomarker of alzheimer's disease according to claim 2, wherein the biomarker comprises: and a goat anti-mouse IgG antibody, a rabbit anti-mouse IgG antibody or a goat anti-rabbit 1gG antibody is fixed on the quality control line.
7. The method for rapidly detecting the biomarker of alzheimer's disease according to claim 2, wherein the biomarker comprises: the gold label pad contains one or more SERS nano labels connected with one or more detection antibodies, and the detection antibodies are connected to the SERS nano labels through adsorption or covalent bonding.
8. The method for rapidly detecting the biomarker of alzheimer's disease according to claim 7, wherein the biomarker comprises: the detection antibody is a monoclonal antibody or a polyclonal antibody of beta-amyloid protein, tau protein and neurofilament light chain protein.
9. The method for rapidly detecting the biomarker of alzheimer's disease according to claim 7, wherein the biomarker comprises: the SERS nano-tag is a metal nano-particle coded by a Raman tag;
the Raman label is any one of nai Er blue A, crystal violet, rhodamine 6G, methylene blue, p-aminophenol, water-soluble 3H-indocyanine type biological fluorescent labeling dye, methyl blue, 4' -bipyridyl, malachite green isothiocyanate, 4-thiopyridine, p-fluorophenylthiol, p-thioaniline, p-aminophenol, p-thiothiophenol, p-hydrophobic benzoic acid or toluidine blue;
the metal nanoparticles are any one of gold nanoparticles, silver nanoparticles, platinum nanoparticles, gold-core silver-shell nanoparticles, gold-core gold-shell nanoparticles, silver-core silver-shell nanoparticles, silica-core gold-shell nanoparticles, gold-core silica-shell nanoparticles, silica-core silver-shell nanoparticles or silver-core silica-shell nanoparticles.
10. The method for rapidly detecting the biomarker of alzheimer's disease according to claim 1, wherein the biomarker comprises: the Alzheimer disease biomarkers are beta-amyloid protein, tau protein and neurofilament light chain protein.
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