CN113544510A - Alzheimer disease diagnosis method using silver nano fissure shells - Google Patents

Alzheimer disease diagnosis method using silver nano fissure shells Download PDF

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CN113544510A
CN113544510A CN202080018727.6A CN202080018727A CN113544510A CN 113544510 A CN113544510 A CN 113544510A CN 202080018727 A CN202080018727 A CN 202080018727A CN 113544510 A CN113544510 A CN 113544510A
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beta
agngs
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金悰浩
梁眞京
黄仁俊
金慧仁
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Nanoyou Co.,Ltd.
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Industry University Cooperation Foundation IUCF HYU
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Abstract

The invention relates to an Alzheimer disease diagnosis method using a silver nano crack shell, which can detect a plurality of Alzheimer disease target biomarkers in a high-sensitivity multiplex manner and diagnose Alzheimer disease in a non-invasive early stage.

Description

Alzheimer disease diagnosis method using silver nano fissure shells
Technical Field
The present invention relates to a method for diagnosing Alzheimer's disease using silver nano fissured shells (Ag Nanogapshell: AgNGS). More particularly, the present invention relates to a diagnostic method for alzheimer's disease using SERS immunoassay based on silver nanoparture shells, and to a diagnostic method that can detect multiple alzheimer's disease target biomarkers with high sensitivity and multiple times and diagnose alzheimer's disease early in a non-invasive manner.
Background
Clinical diagnosis of Alzheimer's Disease (AD) relies on brain imaging techniques such as PET, inquiry, Enzyme-linked immunosorbent assay (ELISA) such as A.beta.40 and A.beta.42 present in cerebrospinal fluid. Recently, diagnostic methods based on diverse nanotechnologies are being developed for the accuracy of AD diagnosis and early diagnosis. In particular, the SERS diagnostic method has attracted considerable attention in terms of sensitivity and multiplex detection, but the SERS diagnostic methods developed according to the prior art have problems that detection is limited to a single target and sensitivity is also extremely low. Therefore, research is continuing to be directed to the development of SERS-based AD diagnostics capable of detecting multiple target biomarkers in blood with high sensitivity.
Previous methods require expensive equipment and are associated with patient distress due to invasive cerebrospinal fluid sampling. In addition, since the degree of progression of the disease cannot be tracked, there is a fatal problem that diagnosis can be made only after the symptoms progress to a considerable extent. In addition, in order to track the degree of onset and progression of AD of a complex mechanism, a technique of simultaneously tracking multiple biomarkers of interest is necessary to improve the accuracy of disease diagnosis and the possibility of early diagnosis, but the prior art has a limit of tracking only a single biomarker. Further, for early diagnosis, a technique for detecting a plurality of biomarkers from a patient sample such as blood by a non-invasive or minimally invasive method is required, but since a very small amount of AD target biomarkers at the pg/mL level are present in a blood sample, a very sensitive diagnostic technique for detecting the same is required, but conventional techniques such as ELISA have a problem that the sensitivity cannot be achieved. Therefore, techniques for non-invasive multiplex detection and early diagnosis of AD with high sensitivity are still required.
Disclosure of Invention
Technical problem
The technical problem to be achieved by the present invention is to solve the above problems and to provide a composition for alzheimer's disease diagnosis comprising a silver nanoslit shell, which can simultaneously detect a plurality of biomarkers for alzheimer's disease diagnosis.
In addition, another object of the present invention is to provide a method for diagnosing alzheimer's disease using the diagnostic composition and a method for identifying and diagnosing alzheimer's disease and mild cognitive impairment.
However, the problems to be achieved by the present invention are not limited to the above-mentioned problems, and other problems not mentioned are understood by those of ordinary skill in the relevant art from the following descriptions.
Technical scheme
In order to solve the above problems, the present invention provides a composition for diagnosing alzheimer's disease comprising a silver nanoslit shell as an active ingredient, wherein 1 or more antibodies selected from the group consisting of an antibody specific to Amyloid β (Amyloid beta: a β)40 and an antibody specific to a β 42 are introduced into the silver nanoslit shell.
The present invention also provides a biosensor for diagnosing alzheimer's disease, comprising a silver nanoslit shell into which an antibody specific to a β 40 or a β 42 is introduced, and a substrate to which the silver nanoslit shell is fixed.
As an embodiment of the invention, the substrate may comprise magnetic beads, and the silver nano-slit shell is fixed by the magnetic beads.
In addition, the present invention provides an alzheimer's disease diagnosis method and an information providing method for alzheimer's disease diagnosis, comprising:
(1) a step of preparing AgNGS-A betA 40 by attaching an antibody specific to Amyloid betA (Amyloid betA: A betA) 40 to the surface of A silver nano-fissure shell (AgNGS); (2) a step of preparing AgNGS-A betA 42 by bonding an antibody specific to A betA 42 to the surface of AgNGS; (3) a step of mixing the AgNGS-AbetA 40 and AgNGS-AbetA 42 and A biological sample in vitro to induce formation of A silver nano-crack shell based Sandwich complex (AgNGS-based Sandwich complex); (4) a step of measuring a raman signal of each complex in the sample and calculating a ratio of a β 40 to a β 42; and (5) a step of determining Alzheimer's disease when the ratio of A β 40 to A β 42 is 2.5 to 999.
As an embodiment of the present invention, the biological sample may be blood or serum, and preferably, may be serum.
The present invention also provides A composition for differential diagnosis of alzheimer's disease and mild cognitive impairment, which comprises, as active ingredients, A silver nanoslit shell (AgNGS- Α β 40) into which an antibody specific to Α β 40 has been introduced and A silver nanoslit shell (AgNGS- Α β 42) into which an antibody specific to Α β 42 has been introduced.
The present invention also provides A biosensor for differential diagnosis of Alzheimer's disease and mild cognitive impairment, wherein AgNGS-A β 40 and AgNGS-A β 42 are immobilized on A substrate.
Further, the present invention provides a method for discriminating and diagnosing alzheimer's disease and mild cognitive impairment and an information providing method for discriminating and diagnosing comprising the steps of:
an information providing method for differential diagnosis of alzheimer's disease and mild cognitive impairment, comprising:
(1) a step of preparing AgNGS-A betA 40 by attaching an antibody specific to Amyloid betA (Amyloid betA: A betA) 40 to the surface of A silver nano-fissure shell (AgNGS); (2) a step of preparing AgNGS-A betA 42 by bonding an antibody specific to A betA 42 to the surface of AgNGS; (3) a step of mixing the AgNGS-AbetA 40 and AgNGS-AbetA 42 and A biological sample in vitro to induce formation of A silver nano-crack shell based Sandwich complex (AgNGS-based Sandwich complex); (4) a step of measuring a raman signal of each complex in the sample and calculating a ratio of a β 40 to a β 42; and (5) when the number is-999 to 1.1, the patient is classified as a normal person, when the number is 1.1 to 2.5, the patient is classified as mild cognitive impairment, and when the number is 2.5 to 999, the patient is classified as Alzheimer's disease.
In addition, the invention provides application of AgNGS-A betA 40 and AgNGS-A betA 42 for differential diagnosis of Alzheimer disease and mild cognitive impairment.
Technical effects
According to the Alzheimer disease diagnosis method utilizing the silver nano fissure shells, the AgNGS nano probe and the magnetic bead which extremely display the inherent specific Surface-enhanced Raman scattering (SERS) signal of the marker can be utilized to simultaneously detect 2 amyloid peptides (Abeta 40 and Abeta 42) by an immunoassay method.
Particularly, the AgNGS-based SERS immunoassay method can realize ultra-high sensitivity detection of less than 1pg/mL aiming at A beta 40 and A beta 42, and can specifically react with each target, so that the method has the advantage of simultaneously detecting a plurality of target biomarkers without interference.
Further, according to the present invention, a β 40 and a β 42 are detected multiply with high sensitivity even under human serum conditions in a complex environment, and thus there is a possibility of non-invasive early diagnosis of AD based on blood, which has an advantage that it can be applied to diagnosis and follow-up of various diseases requiring high sensitivity and multiplex detection capability in addition to AD.
Drawings
Fig. 1 is a schematic diagram of an immunoassay method and a raman signal measurement method using magnetic beads modified with a target-specific antibody and AgNGS.
Fig. 2 is a graph showing the results of detection of 2 kinds (a β ) of biomarkers for Alzheimer's Disease (AD) ((a) is a microarray map image for each concentration detected for a β, (b) is a graph showing AgNGS raman signals for the region shown in (a), (c) is a graph showing signals for each concentration of (a), (d) is a microarray map image for each concentration detected for a β, (e) is a raman signal for AgNGS for the region shown in (d), and (f) is a graph showing signals for each concentration of (d)).
Fig. 3 is a graph showing specificity assays for AgNGS-based SERS immune responses.
FIG. 4 is a graph showing multiplex detection capability in blood samples.
Fig. 5 is a graph showing the results of detection of a β 40 and a β 42 in clinical specimens and the ratio (ratio) thereof.
PREFERRED EMBODIMENTS FOR CARRYING OUT THE INVENTION
The present inventors have conducted intensive studies on a technique for early diagnosis of AD by detecting multiple biomarkers from a non-invasive sample with high sensitivity, and thus have completed the present invention.
Alzheimer's Disease (AD) dementia is a degenerative brain Disease that begins primarily with a decline in memory of recent events and, if the Disease progresses, is accompanied by abnormalities in various cognitive functions. When brain tissue of an alzheimer patient is examined, neuritic plaques and neurofibrillary tangles are observed, and in an image of a brain structure by MRI, global brain atrophy (brain atrophy) due to the disappearance of nerve cells is visible. In the early stage of the disease, hippocampus (hippopus) and entorhinal cortex (entorhinal cortex), which are major brain regions responsible for memory, appear mainly, but gradually spread to the whole brain via the parietal lobe, prefrontal lobe, and the like. In particular, it is known that Alzheimer's disease is highly developed when the gene has the epsilon 4 allele of apoprotein E (APOE), and that amyloid beta protein (A. beta. protein) production is inhibited in addition to epsilon 4 in the APOE epsilon gene.
The present inventors have confirmed that, when a SERS immunoassay method based on a silver nanogap shell (Ag nanogapsell: AgNGS) is used for a non-invasive sample (particularly serum), biomarker detection for diagnosing alzheimer disease can be performed with extremely high sensitivity, and 2 or more biomarkers can be simultaneously examined.
Accordingly, the present invention can provide a composition for diagnosing alzheimer's disease comprising a silver nanoslit shell into which an antibody specific to a β 40 and/or a β 42 is introduced as an active ingredient.
The composition of the present invention may further comprise a pharmacologically or physiologically acceptable carrier, excipient, and diluent in addition to the antibody-introduced silver nanoslit shell, and examples of suitable carriers, excipients, and diluents that may be contained in such a composition include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. When the composition is formulated into a pharmaceutical preparation, it may further contain a conventional filler, an extender, a binder, a disintegrant, a surfactant, an anticoagulant, a lubricant, a wetting agent, a perfume, an emulsifier, a preservative, and the like.
In the invention, the silver nano crack Shell (Ag nano gap Shell: AgNGS) is A nanoprobe (nanoprobe) and comprises silicA particles serving as basic particles and A metal layer containing silver (Ag), wherein the metal layer wraps the silicA particles and is provided with A plurality of cracks (gaps), Raman markers are introduced into the cracks (gaps) in the metal layer to display surface enhanced Raman scattering signals, and specific antibodies to AbetA 40 or AbetA 42 are introduced into the surface of the metal layer to respectively form AgNGS-AbetA 40 or AgNGS-AbetA 42. The antibodies constituting the AgNGS- Α β 40 and AgNGS- Α β 42 have high specificity and bind to Α β 40 or Α β 42 contained in each sample, forming A silver nano-fissured shell-based sandwich complex (AgNGS-based sandwich complex). The present inventors have confirmed that when Surface-enhanced Raman scattering (SERS) signals of the complex are detected, a β 40 and/or a β 42 can be detected with extremely high sensitivity.
On the other hand, Mild Cognitive Impairment (MCI) is a state in which cognitive function is reduced from normal, and is considered to be a precursor of alzheimer's dementia, but it retains the ability to handle daily life and is not yet demented. In the case of patients with mild cognitive impairment, it is known that the probability of developing alzheimer's disease is considerable, but 25 to 30% of patients with mild cognitive impairment return to normal when they receive appropriate treatment at an early stage. In order to prevent alzheimer's disease and treat it as soon as possible after onset, it is necessary to diagnose patients with mild cognitive impairment at an early stage, and for proper treatment, it is preferable to diagnose patients with mild cognitive impairment separately from patients with alzheimer's disease.
Accordingly, the present inventors reacted A serum sample of A normal person or A patient diagnosed with mild cognitive impairment or alzheimer's disease with AgNGS-A β 40 and AgNGS-A β 42 of the present invention to induce complex formation, and calculated the ratio of A β 40 to A β 42 from SERS signal values according to the following formulA 1.
[ formula 1]
Figure BDA0003245668630000061
IA-BThe sum of the intensity values of the entire ranges A to B
As a result, the samples of normal persons, mild cognitive impairment and alzheimer's disease had different values of a β 40/a β 42, and it was found that the smaller the result value, the more normal the cognitive ability, and the larger the result value, the more problematic the cognitive ability and the like. Specifically, it is found that the a β 40/a β 42 value is in the range of 1.1 to 2.5 in the case of mild cognitive impairment, and can be determined as normal when having a value smaller than that, and can be determined as alzheimer's disease when having a value higher than that.
Therefore, the present inventors can quickly and accurately identify and diagnose dementia patients and patients with mild cognitive impairment, and particularly, can prevent dementia by follow-up examination or appropriately treat dementia at a proper time by early diagnosis, since it is possible to provide accurate diagnosis results using a non-invasive sample (serum).
Detailed Description
While the invention is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will herein be described in detail in the summary of the invention. However, the present invention is not limited to the specific embodiments, and all modifications, equivalents, and alternatives included in the spirit and technical scope of the present invention are understood to be included. In explaining the present invention, when it is judged that the detailed description of the related known art may obscure the gist of the present invention, the detailed description is omitted.
[ examples ] A method for producing a compound
Example 1 preparation of AgNGS-A β 40 and AgNGS-A β 42
Silver nanoclosure shells into which an antibody specific to a β 40 or a β 42 was independently introduced were prepared by the following method (see KR 10-1944346).
1-1. silica particle Synthesis and thiol reactive group formation
(1) Tetraethyl orthosilicate was dissolved in 40ml of pure ethanol, and ammonium hydroxide was added to the solution to react at room temperature for 20 hours.
(2) After unreacted materials were removed by a centrifuge, the resulting solution was finally diluted to a concentration of 1 mg/ml.
(3) To 1ml of the synthesized silica particles (1mg/ml), mercaptopropyltriethoxysilane and ammonium hydroxide were added and reacted at ordinary temperature for 12 hours.
(4) And removing unreacted substances by using a centrifugal machine. (MPTS-silica)
1-2, forming silver nano crack shell on the surface of silicon dioxide particles by using mercaptan-silver binding force
(1) Mixing MPTS-silica, PVP and AgNO3After dissolving in 50ml of ethylene glycol, octylamine was added to conduct a reduction reaction. 1 minute after addition of octylamine, Raman labels (4-fluorobenzothiophenol, 4-bromobenzenethiol, 4-chlorobenzenethiol, thiophenol) were added.
(2) And removing unreacted substances by using a centrifugal machine. (AgNGS)
1-3, in order to introduce chemical active groups on the surface of the silver nano fissure shell, utilizing mercaptoundecanoic acid and mercaptohexanol Forming a self-assembled monolayer
(1) After mercaptohexanol and mercaptoundecanoic acid were added to the synthesized AgNGS, the reaction was carried out in ethanol for 1 hour.
(2) And removing unreacted substances by using a centrifugal machine. (SAM-AgNGS)
1-4, introducing selective combination on the surface of silver nano fissure shell Abeta 40 or Abeta 42Antibody of (2)
(1) EDC/NHS (1-ethyl-3- (3-dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide) coupling agent was added to the synthesized SAM-AgNGS, and then the mixture was reacted in PBS solution at pH 6.0100 mM for 30 minutes.
(2) And removing unreacted substances by using a centrifugal machine.
(3) An antibody selectively reactive with A.beta.40 or A.beta.42 (1mg/ml, 10l) was added thereto and stirred, followed by reaction in PBS buffer at pH 7.4100 mM for 2 hours.
(4) After removing unreacted substances by a centrifuge, the solution was dispersed in a 1% bovine serum albumin solution and stored under refrigeration.
Example 2 detection of 2 biomarkers of Alzheimer's Disease (AD) (A β 40, A β 42)
Fig. 2 is a graph showing the results of detection of 2 kinds (a β ) of biomarkers for Alzheimer's Disease (AD) ((a) is a microarray map image for each concentration detected for a β, (b) is a graph showing AgNGS raman signals for the region shown in (a), (c) is a graph showing signals for each concentration of (a), (d) is a microarray map image for each concentration detected for a β, (e) is a raman signal for AgNGS for the region shown in (d), and (f) is a graph showing signals for each concentration of (d)).
As can be confirmed by reference to fig. 2, a β 40, a β 42 can be detected in a concentration-dependent manner by AgNGS-based SERS immunoassay.
Example 3 specificity test for AgNGS-based SERS immunoreaction
Fig. 3 is a graph showing specificity assays for AgNGS-based SERS immune responses.
Fig. 3(a) is a target biomarker specifically reacting only to each target with AgNGS modified with an antibody specific to each target, immunoreacting and showing raman signals. FIG. 3(b) shows that when the concentration of A.beta.42 was fixed at 10ng/mL and the concentration of A.beta.40 was changed to 0-10 ng/mL, the AgNGS-based SERS immunoassay method for a mixed solution of the two biomarkers showed a Raman signal tilt that reflected the change in the concentrations of the two biomarkers. In addition, if referring to fig. 3(c), when a β 40 and a β 42 are present in the ratio of the x-axis, AgNGS-based SERS immunoassay shows a raman signal bias reflecting the concentration change of both biomarkers. From the corresponding results, it can be demonstrated that AgNGS-based SERS immunoassays can react specifically to multiple biomarkers without cross-reaction.
Example 4 confirmation of multiplex detectability in blood samples
FIG. 4 is a graph showing multiplex detection capability in blood samples. If referring to fig. 4, in human serum samples where a β 40 and a β 42 are present at concentrations of 0-1000 ng/mL, when a β 40 and a β 42 are detected by AgNGS-based SERS immunoassay, a raman signal propensity is shown reflecting the change in concentration of both biomarkers. From this, it was found that a β 40 and a β 42 can be specifically detected even in a blood sample having a complicated configuration.
Example 5 diagnosis in clinical samples Using the ratio of A β 40 to A β 42 (ratio)
Serum samples were obtained and used from the boramel hospital for normal persons and patients diagnosed with mild cognitive impairment or alzheimer.
Fig. 5 is a graph showing the detection results of a β 40 and a β 42 in clinical samples. Referring to fig. 5, when a β 40 and a β 42 present in serum of alzheimer's disease patients were measured by AgNGS-based SERS immunoassay, and the concentration ratio of the two biomarkers was calculated, it was found that the ratio was in the range of 1.1 to 2.5 in the case of mild cognitive impairment patient samples, had a value of less than 1.1 in the case of normal persons, and showed a value of more than 2.5 in the case of alzheimer's disease. From the results, it was possible to distinguish and diagnose patients with alzheimer's disease from patients with Mild Cognitive Impairment (MCI) and normal persons (NC).
It will be apparent to those skilled in the art that specific details of the present invention have been described above, and that such detailed description is merely a preferred embodiment, and is not intended to limit the scope of the present invention. The substantial scope of the present invention is, therefore, defined by the appended claims and equivalents thereof.

Claims (9)

1.A composition for diagnosing Alzheimer's disease, comprising a silver nanoslit shell as an active ingredient, wherein 1 or more antibodies selected from the group consisting of an antibody specific to amyloid beta 40 and an antibody specific to A beta 42 are introduced into the silver nanoslit shell.
2. The composition for Alzheimer's disease diagnosis according to claim 1,
the silver nano slit shell is composed of silicon dioxide particles and a metal layer which completely wraps the silicon dioxide particles and has a surface forming a plurality of slits,
the metal layer comprises silver, a Raman label is introduced into the silver cleft, and the antibody is introduced to the surface of the metal layer.
3. An information providing method for Alzheimer's disease diagnosis, comprising:
(1) a step of preparing AgNGS-A betA 40 by joining A specific antibody for amyloid betA 40 to the surface of A silver nano fissure shell;
(2) a step of preparing AgNGS-A betA 42 by bonding an antibody specific to A betA 42 to the surface of AgNGS;
(3) mixing the AgNGS-A betA 40 and AgNGS-A betA 42 with A biological sample in vitro to induce the formation of A sandwich composite body based on A silver nano fissure shell;
(4) a step of measuring a raman signal of each complex in the sample and calculating a ratio of a β 40 to a β 42; and
(5) a step of determining Alzheimer's disease when the ratio of A β 40 to A β 42 is 2.5 to 999.
4. The information providing method for Alzheimer's disease diagnosis according to claim 3,
the biological sample is 1 or more non-invasive samples selected from the group consisting of blood and serum.
5. A method of diagnosing alzheimer's disease comprising:
(1) a step of preparing AgNGS-A betA 40 by joining A specific antibody for amyloid betA 40 to the surface of A silver nano fissure shell;
(2) a step of preparing AgNGS-A betA 42 by bonding an antibody specific to A betA 42 to the surface of AgNGS;
(3) mixing the AgNGS-A betA 40 and AgNGS-A betA 42 with A biological sample in vitro to induce the formation of A sandwich composite body based on A silver nano fissure shell;
(4) a step of measuring a raman signal of each complex in the sample and calculating a ratio of a β 40 to a β 42; and
(5) a step of diagnosing Alzheimer's disease when the ratio of A β 40 to A β 42 is 2.5 to 999.
6. A composition for differential diagnosis of Alzheimer's disease and mild cognitive impairment, which comprises a silver nanoslit shell into which an antibody specific to amyloid beta 40 has been introduced and a silver nanoslit shell into which an antibody specific to Abeta 42 has been introduced as active ingredients.
7. An information providing method for differential diagnosis of alzheimer's disease and mild cognitive impairment, comprising:
(1) a step of preparing AgNGS-A betA 40 by joining A specific antibody for amyloid betA 40 to the surface of A silver nano fissure shell;
(2) a step of preparing AgNGS-A betA 42 by bonding an antibody specific to A betA 42 to the surface of AgNGS;
(3) mixing the AgNGS-A betA 40 and AgNGS-A betA 42 with A biological sample in vitro to induce the formation of A sandwich composite body based on A silver nano fissure shell;
(4) a step of measuring a raman signal of each complex in the sample and calculating a ratio of a β 40 to a β 42; and
(5) a normal person at-999 to 1.1, a mild cognitive impairment at 1.1 to 2.5, and Alzheimer's disease at 2.5 to 999.
8. The information providing method for the differential diagnosis of Alzheimer's disease and Mild cognitive impairment according to claim 7,
the biological sample is 1 or more non-invasive samples selected from the group consisting of blood and serum.
9. A method for differential diagnosis of alzheimer's disease and mild cognitive impairment comprising:
(1) a step of preparing AgNGS-A betA 40 by joining A specific antibody for amyloid betA 40 to the surface of A silver nano fissure shell;
(2) a step of preparing AgNGS-A betA 42 by bonding an antibody specific to A betA 42 to the surface of AgNGS;
(3) mixing the AgNGS-A betA 40 and AgNGS-A betA 42 with A biological sample in vitro to induce the formation of A sandwich composite body based on A silver nano fissure shell;
(4) a step of measuring a raman signal of each complex in the sample and calculating a ratio of a β 40 to a β 42; and
(5) a step of judging the normal person when the number is-999 to 1.1, diagnosing mild cognitive impairment when the number is 1.1 to 2.5, and diagnosing Alzheimer's disease when the number is 2.5 to 999.
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