KR102006426B1 - Multi-detection Biosensor of Alzheimer's Disease Based on Rayleigh Scattering with Colorimetric Assay and Multi-detecting Method Using The Same - Google Patents

Abstract

The present invention relates to a nano plasmonic sensor based on Alzheimer's disease markers present in the blood, beta amyloid 40, beta amyloid 42, tau protein-recognized gold plasmon nanoparticles, and the Rayleigh scattering phenomenon of the sensor Alzheimer's multiple detection method using colorimetric analysis. The present invention is a simple diagnostic method using the blood is capable of simultaneous multiple detection of several onset markers and has the advantage of improved sensitivity of the diagnosis using chaotropic solvent.

Description

Multi-detection Biosensor of Alzheimer's Disease Based on Rayleigh Scattering with Colorimetric Assay and Multi-detecting Method Using The Same}

The present invention relates to Alzheimer's diagnostic biosensor using Rayleigh scattering phenomenon and colorimetric analysis of gold nanoparticles and multiple detection method using the biosensor, and more specifically beta amyloid 40, an Alzheimer's disease marker in blood, It relates to a beta amyloid 42, a nano plasmonic sensor that recognizes tau protein and a multi-detection method of Alzheimer's using the sensor.

Alzheimer's disease is a histological feature of having amyloid plaque and neurofibrillar tangle in the hippocampus and cortex, and worsening cognitive impairment that affects the patient's memory. It is a degenerative disease that weakens. Alzheimer's disease can only slow the progression of the disease if the disease progresses above a certain level, so early diagnosis is very important. Until now, diagnosis methods such as brain imaging, cognitive function test and cerebrospinal fluid test are available, but these methods can be applied only after the symptoms of Alzheimer's disease can be applied. In addition, it requires a large diagnostic cost economically, accompanied by a surgical method is very high risk. Therefore, the development of an easy method for early diagnosis of Alzheimer's disease is emerging, and as a part of this, there is an urgent need for the development of a highly sensitive diagnostic method for detecting a small amount of pathogenic biomarker present in the blood.

At present, the most importantly pointed causative agents in the pathogenesis of Alzheimer's disease include beta amyloid 40, beta amyloid 42 peptide, and tau protein.

Beta amyloid peptides are produced by cleaving amyloid proproteins by beta secretase and gamma secretase. Therefore, the activity of these enzymes increases the production of beta amyloid peptide is closely related to Alzheimer's dementia disease. However, the alpha-secretase cleaves the beta-amyloid peptide at its intermediate portion, and the activity of beta-amyloid peptide is increased, thereby reducing the production of beta-amyloid peptide. Alpha secre- tase predominates in normal subjects and interferes with amyloid beta cleavage by beta-secretase. When these beta amyloid peptides accumulate in the brain, they produce large amounts of active oxygen, causing oxidative damage and causing death of nerve cells. In addition, beta amyloid peptide causes excessive accumulation of intracellular calcium, resulting in excitotoxicity and cell death. Neuronal cell death caused by the beta amyloid peptide causes cell death in memory of the hippocampus, a memory-related site, and memory loss, a representative symptom of dementia. The major plaque component of beta amylase, which is produced by cleaving sequentially by secretase, is known as an isoform of beta amyloid 42 peptide that is involved in the formation of neurotoxic oligomers and plaques in the pathogenesis of Alzheimer's disease.

Recently, in addition to beta amyloid, it has been reported that the tau protein has an abnormal structure and forms an inclusion body, which causes degenerative brain disease. The tau protein is used to stabilize microtubules such as railroad connecting bars. Excessive accumulation of tau protein as a necessary substance is known to disrupt the network of normal neurons as microtubules collapse. The cause of accumulation of tau protein is the excessive attachment of phosphorus to tau protein to form a weight due to phosphorylation.The disease caused by the accumulation of these tau proteins and the aggregation of nerve cells is called tauopathy It is also indicated as the cause of various degenerative brain diseases.

Recently, the technology of biosensor is focused on the development of non-label measurement technology that does not use a labeling material, and typically, the method using surface plasmon resonance phenomenon occurring on the metal surface (RQ DUAN et al., Neoplasma, 59 (3): 348, 2012; Wei Zhou et al., International Journal of Nanomedicine, 6: 381, 2011), A method for measuring the change of refractive index of light due to the bending phenomenon of three-dimensional microstructure Cantilever (Balle, MK. Et al., Ultramicroscopy, 82; 1, 2000), a method for measuring the resonance frequency change of a quartz crystal with a change in the mass of biomolecules (Marx, KA., Biomacromolecules, 4; 1099, 2003), Method for measuring electric field effects based on (Yuqing, M. et al., Biotechnol. Adv., 21; 527, 2003) and electrochemical measurement methods (Hanahan, G. et al., J. Environ. Monit., 6, 657, 2004; Sang Hee Han et al., Analytica Chimica Acta, 665: 79, 2010).

Among them, localized surface plasmon resonance (LSPR) method can detect the reaction quantitatively without complicated pre-purification and labeling process, and thus, various studies on the interaction between biomolecules immobilized on the surface have been varied. Has been used. In the LSPR method, when light having various wavelengths is applied to a material having a local surface, such as metal nanoparticles, unlike the bulk metal, polarization occurs on the surface of the metal nanoparticles, and has a unique property of important electric field strength. These LSPR optical properties are sensitive to changes in dielectric constant (refractive index) occurring near nanoparticles. This change in dielectric constant (refractive index) can be used to detect adsorption between biomolecules. Based on LSPR's optical properties to measure biomaterial concentration, thickness, and binding kinetics data for specific biological analytes, including antigen / antibody, ligand / receptor, protein / protein reaction, and DNA hybridization over the past decades Various biomolecular interaction assays have been performed.

Accordingly, the present inventors have made intensive efforts to develop a new multi-detection platform for early diagnosis of Alzheimer's disease. The present invention has been completed by confirming that the plasmonic sensor can be detected using the plasmonic sensor.

Republic of Korea Patent No. 10-1003124

Disclosure of Invention An object of the present invention is to provide a method for diagnosing Alzheimer's disease using a Rayleigh scattering phenomenon and a colorimetric analysis method of a plasmonic sensor based on gold nanoparticles to which an antibody is immobilized, and a biosensor for diagnosing Alzheimer's disease.

In order to achieve the above object, the present invention, (A) is isolated from the sample to the plasmonic sensor based on gold nanoparticles immobilized with an antibody or aptamer to a target protein that specifically increases or decreases expression in Alzheimer's patients Contacting the sample to induce binding of the target protein with the antibody or aptamer; And (B) measuring light scattering spectra according to the binding, and determining whether Alzheimer's disease occurs through analysis of the maximum wavelength mobility (Δλ max) obtained therefrom.

The present invention also provides an Alzheimer's diagnostic biosensor in which an antibody or aptamer of at least one target protein selected from the group consisting of beta amyloid 40 peptide, beta amyloid 42 peptide and tau protein is immobilized on gold nanoparticles.

The method for diagnosing Alzheimer's disease according to the present invention allows the diagnosis of Alzheimer's disease using blood readily obtainable from the human body, thereby enabling an objective analysis without requiring complicated surgical procedures for diagnosing Alzheimer's disease. The application of gold nanoparticles allows multiple detection markers to be identified on a single platform, enabling multiple detections, and cao to compensate for the reduced detection of disease markers in the blood with other proteins. Pretreatment with tropic solvents has the advantage of improving diagnostic sensitivity.

1 shows an HR-TEM image of 50 nm size circular gold nanoparticles prepared from Example 1-1.
2 shows HR-TEM images of rod-shaped gold nanoparticles having an aspect ratio of 1.73 prepared from Examples 1-2.
3 shows an HR-TEM image of rod-shaped gold nanoparticles having an aspect ratio of 3.59 prepared from Examples 1-3.
4 shows UV-Vis spectra of 50 nm size circular gold nanoparticles prepared from Example 1-1.
5 shows the UV-Vis spectra of rod-shaped gold nanoparticles having an aspect ratio of 1.73 prepared from Examples 1-2.
6 shows the UV-Vis spectra of rod-shaped gold nanoparticles having an aspect ratio of 3.59 prepared from Examples 1-3.
FIG. 7 shows a dark field image of 50 nm size circular gold nanoparticles prepared from Example 1-1 exposed at 1000 × magnification.
FIG. 8 shows a dark field image of rod-shaped gold nanoparticles with an aspect ratio of 1.73 prepared from Examples 1-2 exposed at 1000 × magnification.
FIG. 9 shows a dark field image of rod-shaped gold nanoparticles with an aspect ratio of 3.59 prepared from Examples 1-3 exposed at 1000 × magnification.
FIG. 10 shows an HR-TEM image of a mixture of 50 nm circular, rod-shaped aspect ratio 1.73, rod-shaped gold nanoparticles with aspect ratio 3.59.
FIG. 11 shows UV-Vis spectra of a mixture of 50 nm circular, rod-shaped aspect ratio 1.73, rod-shaped gold nanoparticles having an aspect ratio of 3.59.
FIG. 12 shows a dark field image of a mixture of 50 nm circular, rod-shaped aspect ratio 1.73, rod-shaped gold nanoparticles with aspect ratio 3.59. FIG.
FIG. 13 shows localized surface plasmonic resonance spectra of a mixture of 50 nm circular, rod-shaped aspect ratio 1.73, rod-shaped gold nanoparticles having aspect ratio 3.59. FIG.
FIG. 14 shows the UV-Vis change spectra that appear in each step of binding PEG and antibodies to circular gold nanoparticles.
FIG. 15 shows the LSPR light scattering change spectrum for each stage of binding PEG and antibody to circular gold nanoparticles.
16 shows LSPR maximal scattering wavelength shift according to antigen-specific binding in the nanoplasmonic biosensor of the present invention.
17 is a graph showing LSPR peak scattering wavelength shift and sensitivity according to beta amyloid 40 concentration in the nanoplasmonic biosensor according to the present invention.
18 is a graph showing LSPR peak scattering wavelength shift and sensitivity according to beta amyloid 40 concentration in the presence of chaotropic solvent in the nanoplasmonic biosensor according to the present invention.
19 shows the detection amount of beta amyloid 42 according to the chaotropic solvent concentration.
20 shows the detection amount of tau protein according to the chaotropic solvent concentration.
Figure 21 shows the detection amount of beta amyloid 40 according to the chaotropic solvent concentration.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

The present invention relates to an Alzheimer's diagnostic nanoplasmonic biosensor capable of detecting Alzheimer's disease markers at the same time.

The present invention focuses on the fact that as the cerebrospinal fluid of 500 ml per day flows into the blood, the mechanism of Alzheimer's disease occurring in the brain is sufficiently reflected in the blood, and thus a nanoplasmonic biosensor capable of simultaneously detecting the Alzheimer's disease marker in the blood sample. I wanted to develop.

In the present invention, the antibody or aptamer binding to beta amyloid 40 peptide, beta amyloid 42 peptide and tau protein, which are markers for the development of Alzheimer's disease, is immobilized on rod-shaped gold nanoparticles having a circular, aspect ratio of 1.73 and aspect ratio of 3.59, respectively. Different optical and visual characteristics were used to identify Alzheimer's onset markers. Gold nanoparticles of different shapes and sizes enable colorimetric analysis through surface resonance plasmonic spectra and dark field images showing different maximum wavelengths. At this time, the antibody or aptamer of the onset marker was linked to the heterofunctional polyethylene glycol (SH-PEG-COOH) by treating the gold nanoparticles. As the gold nanoparticles bind to the onset markers, resonance occurs in a larger wavelength region, and the presence or absence of the onset markers is confirmed by changing the spectrum of the nanoparticles bound to antibodies or aptamers of specific onset markers. It was. On the other hand, by quantifying the onset markers by standardizing the maximum wavelength mobility generated by applying the artificially synthesized markers to the sensor, the nanoplasmonic biosensor of the present invention is very high in the Alzheimer's pathogens It was confirmed that it can be detected with sensitivity and accuracy.

Therefore, in one aspect, the present invention provides a sample isolated from a specimen in a plasmonic sensor based on gold nanoparticles in which an antibody or aptamer is immobilized to a target protein that specifically increases or decreases expression in an Alzheimer's patient. Contacting to induce binding of the target protein with the antibody or aptamer; And (B) measuring light scattering spectra according to the binding, and determining whether Alzheimer's disease is detected through analysis of the maximum wavelength mobility (Δλ max) obtained therefrom.

In the present invention, it is particularly preferable that the sample is blood in view of the fact that all the body fluid discharged from the human body can be utilized and cerebrospinal fluid flows into the blood.

In the present invention, the target protein may be any one or more selected from the group consisting of beta amyloid 40 peptide, beta amyloid 42 peptide, and tau protein, but is not limited thereto.

In the present invention, the gold nanoparticles may be different in shape and / or size depending on the type of antibody or aptamer immobilized. That is, by preparing and using gold nanoparticles having different shapes and / or sizes as many as the number of antibodies or aptamers respectively binding to a plurality of target proteins, it is possible to simultaneously detect various target proteins and thereby improve the accuracy of Alzheimer's diagnosis. .

In the present invention, the binding of the target protein with the antibody or aptamer may be induced in a reaction sample containing a chaotropic solvent to compensate for the decrease in detection due to the binding of the onset marker present in the blood to another protein. Can be. In this case, since the chaotropic solvent has a property of weakening non-covalent bonds between molecules by interfering with the arrangement of water molecules present in the solvent, it acts to increase the sensitivity of the Alzheimer's disease marker when added to the blood.

In the present invention, the binding between the target protein and the antibody or aptamer may be induced in a reaction sample containing a chaotropic solvent.

In the present invention, the blood may further comprise a pretreatment step of reacting a target protein present in the blood with a solution containing a chaotropic solvent in a step before binding to the antibody or aptamer.

In the present invention, the chaotropic solvent any one selected from the group consisting of guanidine hydrocholoride, guanidine thiocyanate, potassium thiocyanate and trichloroacetate (sodium trichloroacetate) It may be more than, but is not limited thereto.

In the present invention, the chaotropic solvent for the detection of beta amyloid 42 is preferably 5-7 M guanidine hydrochloride or 2-5 M guanidine thiocyanate, but is not limited thereto.

In the present invention, the chaotropic solvent for detecting the tau protein is preferably 5-7 M guanidine hydrochloride or 4-6 M guanidine thiocyanate, but is not limited thereto.

In the present invention, the chaotropic solvent for the detection of beta amyloid 40 is preferably 1.5-3.5 M potassium thiocyanate hydrochloride or 5-7 M guanidine hydrochloride, but is not limited thereto.

In the present invention, the mobility of the maximum wavelength may be represented by the concentration of the onset factor coupled to the surface of the plasmonic sensor.

On the other hand, the present invention provides an Alzheimer's diagnostic biosensor in which an antibody or aptamer of at least one target protein selected from the group consisting of beta amyloid 40 peptide, beta amyloid 42 peptide and tau protein is fixed to gold nanoparticles.

In the present invention, the gold nanoparticles may be different from each other in shape and / or size depending on the type of antibody or aptamer immobilized.

In the present invention, the average particle size of the gold nanoparticles is preferably 10 ~ 150nm, more preferably 40 ~ 130. On the other hand, the circular gold nanoparticles are preferably 40 to 60 nm, more preferably about 50 nm, and when the gold nanoparticles are rod-shaped, the aspect ratio is preferably 1 to 5, and more preferably 1.6 to 3.7. desirable.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these examples are for illustrative purposes only and that the scope of the present invention is not construed as being limited by these examples.

Example 1 Gold Nanoparticles Fabrication

In order to fabricate the nanoplasmonic biosensor based on the surface plasmon resonance phenomenon of the present invention, a circular gold nanoparticle having a size of about 50 nm, a rod of 1.73 aspect ratio and a rod of 3.59 aspect ratio is known in the art. synthesis), and the material used for the synthesis was purchased from Sigma Aldrich (USA).

1-1. Fabrication of 50nm Circular Gold Nanoparticles

10 ml of 0.1 M HAuCl ₄ and 9.9 ml of sterile water were mixed in a 20 ml vial, and heated at 200 ° C. with stirring at 1000˜1500 rpm. 1 ml of 0.04M Sodium citrate was added to the boiled gold aqueous solution, and when the aqueous solution turned yellow was maintained for 5 minutes, the mixture was stirred at 1150 rpm at room temperature. Filtration was performed using a 0.2 μl filter to remove aggregated product from the reaction solution, and the size and shape were high resolution transmission electron microscopy (HR-TEM, jEOL JEM-3011) and UV / VIS spectroscopy. It was measured using a photometer (UV / VIS 3600, Shimadzu).

1-2. Fabrication of rod-shaped gold nanoparticles with an aspect ratio of 1.73

The gold nanoparticle seed solution was mixed with 5 ml of 0.5 mM HAuCl _{4 and} 5 ml of 0.2 M CTAB, followed by 0.006 M NaBH ₄ 1 mL was added and the Au (III) -CTAB aqueous solution was stirred at 1200 rpm. Yellow to yellow brown color was used. The seed solution was used at room temperature for 30 minutes before use.

1.4 g of CTAB and 0.2468 g of NaOL were dissolved in sterile water at 50 ° C., the solution was cooled to 30 ° C., 2.4 ml of 4 mM AgNO ₃ was added, and the mixture was left for 15 minutes without stirring. After 50 ml of 1 mM HAuCl ₄ was added and stirred at 700 rpm for 90 minutes, the color of the aqueous solution was lost (colorless). 0.3 ml of 37 wt.% (12.1 M) HCl was added and stirred at 400 rpm for 15 minutes to adjust the pH, 0.25 ml of 0.064M ascorbic acid was added thereto, vigorously stirred for 30 seconds, 0.04 ml of seed solution was added, and the mixture was stirred for 30 seconds. After vigorous stirring, the mixture was grown to 30 DEG C without stirring for 12 hours. At this time, the color turns purple. After centrifugation at 7000 rpm for 30 minutes to separate the product and remove the supernatant, the size and shape of the high resolution transmission electron microscopy (HR-TEM, jEOL JEM-3011) and UV / VIS spectrophotometer (UV / VIS 3600, Shimadzu).

1-3. Rod-shaped gold nanoparticles with an aspect ratio of 3.59

The gold nanoparticle seed solution was mixed with 5 ml of 0.5 mM HAuCl _{4 and} 5 ml of 0.2 M CTAB, followed by 0.006 M NaBH ₄ 1 mL was added and the Au (III) -CTAB aqueous solution was stirred at 1200 rpm. Yellow to yellow brown color was used. The seed solution was used at room temperature for 30 minutes before use.

The growth solution was dissolved 1.8g of CTAB and 0.2468g of NaOL in 50 ℃ sterilized water, the solution was lowered to 30 ℃ temperature, 4mM AgNO ₃ 4.8ml was added and left for 15 minutes without stirring. After 50 ml of 1 mM HAuCl ₄ was added and stirred at 700 rpm for 90 minutes, the color of the aqueous solution was lost (colorless). 37 wt.% (12.1M) of HCl was added thereto and stirred at 400 rpm for 15 minutes to adjust the pH, 0.25 ml of 0.064M ascorbic acid was added and stirred vigorously for 30 seconds, 0.04 ml of seed solution was added, and vigorously for 30 seconds. After stirring, the mixture was grown to 30 DEG C without stirring for 12 hours. At this time, the color turns purple. After centrifugation at 7000 rpm for 30 minutes to separate the product and remove the supernatant, the size and shape of the high resolution transmission electron microscopy (HR-TEM, jEOL JEM-3011) and UV / VIS spectrophotometer (UV / VIS 3600, Shimadzu).

As a result, it was confirmed that circular (Fig. 1), bar (Fig. 2), and bar (Fig. 3) gold nanoparticles having an aspect ratio of 1.73 and an aspect ratio of 3.59 were prepared, respectively. It was confirmed that different optical and visual properties are induced in the particles (FIGS. 4 to 9). Meanwhile, the circular and rod-shaped gold nanoparticles are mixed (FIG. 10) and observed to show surface resonance plasmonic spectra (FIG. 11) and dark field images showing different maximum wavelengths even when the respective gold nanoparticles are mixed. Through colorimetric (Fig. 12) was confirmed to be observed.

Example 2 Substrate Fabrication

The glass substrate was surface-treated with a silane compound containing an amine or an alkyl end group as a support of the nano plasmonic sensor. The silane compound was (3-Aminopropyl) triethoxysilane, and the amine group was attached to the substrate by immersing the glass substrate in a 5% (3-Aminopropyl) triethoxysilane solution for 15 minutes.

Example  3: antibodies to circular gold nanoparticles Combined Nanoplasmonic  Bio sensor manufacturing board manufacturing

Heterofunctional polyethylene glycol (SH-PEG-COOH) was treated with rod-shaped gold nanoparticles and bound to antibodies against the beta amyloid 40 Alzheimer's disease invention marker. Heterofunctional polyethylene glycol (SH-PEG-COOH) was used at a molecular weight of 2000 and Laysan Bio. It was purchased from Ltd. and used. Heterofunctional polyethylene glycol (SH-PEG-COOH) acted as a stabilizer and provided a functional group that could bind gold nanoparticles to glass substrates and bind to antibodies. 2 mg of SH-PEG-COOH having a molecular weight of 2000 was dissolved in 1 ml of sterile water to prepare 1 mM. Thereafter, 20 μl of the prepared solution was added to 1 ml of the 50 nm circular gold nanoparticle aqueous solution synthesized in Example 1-1, stirred for 24 hours, and centrifuged at 7000 rpm for 15 minutes to separate the surface-treated gold nanoparticles. The separated pellets were resuspended in sterile water at an optical density of 2, and 1ul of 0.7M 1-Ethyl-3- (3-dimethylaminopropyl) carbodi-imide / N-hydrosuccimide (EDC / NHS) based on 100ul of aqueous gold solution. Mix well and incubate for 15 minutes. Meanwhile, 50ul of beta amyloid 40 antibody to be attached to the surface of gold nanoparticles was diluted in PBS at a concentration of 50ug / ml. After 15 minutes, the antibody dilution was added to PEG-treated gold solution and pipetted, and reacted without stirring for 4 hours. After 4 hours, the mixture was centrifuged at 5000 rpm for 10 minutes and redispersed in 100ul of sterile water.

Gold nanoparticle aqueous solution containing the circular gold nanoparticle aqueous solution to which the antibody was bound was diluted to an optical density of 0.05, and dropped by 10 µl onto a glass substrate. Then, after about 50 seconds, the gold nanoparticles aqueous solution was removed again.

Looking at the change in the wavelength of the UV-Vis spectrum appearing at each step of binding the antibody to the circular gold nanoparticles, the plasmonic spectrum of the circular 50nm gold nanoparticles prepared in Example 1-1 (Fig. 14, 50nm bare AuNPs) is 530.5 Although there was a peak at nm, the Rayleigh light scattering spectrum (Fig. 14, PEGylated 50 nm AuNPs) of the same single nanoparticle after surface treatment with a molecular weight of 2000 heterofunctional PEG 1 mM showed a peak at 533 nm and a 50 μl / ml antibody 50ul. After attaching to the surface of gold nanoparticles and reacting for 4 hours (FIG. 14, 50 ug / ml antibody binding), a peak was found at 537.2 nm. That is, it was confirmed that UV-Vis maximum wavelength shift of 2.5 nm after PEG attachment and 4.2 nm after antibody attachment occurred compared to the circular gold nanoparticles.

On the other hand, in the case of LSPR light scattering change spectrum which appears in each step of binding the antibody to the circular gold nanoparticles, the molecular weight compared to the plasmonic spectrum of the circular 50nm gold nanoparticles prepared in Example 1-1 (FIG. 15, 50nm bare AuNPs) Rayleigh light scattering spectrum (FIG. 15, After SAM) of the same single nanoparticle after surface treatment with 2000 heterofunctional PEG 1 mM and 50ul of antibody at 50 ug / ml concentration were attached to the surface of gold nanoparticles (FIG. 15, After ab binding) LSPR peak wavelength shifts of 17.3 nm and 31 nm occurred at, respectively.

Example  4: Antibodies to rod gold nanoparticles Combined Nanoplasmonic  Bio sensor manufacture

Heterofunctional polyethylene glycol (SH-PEG-COOH) was treated with rod-shaped gold nanoparticles and bound to antibodies against beta amyloid 42, two marker markers of Alzheimer's disease invention of tau protein. Heterofunctional polyethylene glycol (SH-PEG-COOH) was used at a molecular weight of 2000 and 3400, respectively, Laysan Bio. It was purchased from Ltd. and used. Heterofunctional polyethylene glycol (SH-PEG-COOH) acted as a stabilizer, and instead of CTAB on the surface of rod-shaped gold nanoparticles with aspect ratios 1.73 and 3.59, gold nanoparticles adhere well to the glass substrate and can bind to antibodies. To provide functional groups. To 1946.5 µl of sterile water, 53.5 µl of rod-like gold nanoparticles having an aspect ratio of 3.59 of 300 µg Au / ml and 50 mg of molecular weight 3400 SH-PEG-COOH were added. In the case of rod-shaped gold nanoparticles having an aspect ratio of 1.72, 37.05 µl and a molecular weight of 3400 SH-PEG-COOH 40 mg were added to 1962.95 µl of sterile water at a concentration of 300 µg Au / ml. The solution was stirred for 4 days at room temperature at 200 rpm and then centrifuged at 5000 rpm for 4 minutes. Centrifugation removed SH-PEG-COOH that was not bound to gold and was resuspended in 1 ml of sterile water. Afterwards, EDC and NHS were used to connect gold nanoparticles with antibodies. Gold nanoparticles aqueous solution containing rod-shaped gold nanoparticles aqueous solution was diluted to an optical density of 0.1 and dropped to 10 μl on a glass substrate. Thereafter, after about 5 minutes, the aqueous gold nanoparticles were removed again.

Example 5 Nanoplasmonic Biosensor Effect Verification

As gold nanoparticles bind to biomolecules, resonance occurs in a larger wavelength region, and the spectral changes of the nanoparticles in which the beta amyloid 40, the beta amyloid 42, and the tau protein are bound to the antibody are observed. The presence or absence of a pathogen was confirmed.

That is, it was confirmed that the antigen-specific binding occurs without cross-talk in the biosensors prepared in Examples 3 and 4, wherein all antigens were added at 100 nM, and PBS was used as a diluting solution.

In the case of the 50 nm circular gold nanoparticle biosensor manufactured by Example 3, only 10 nM beta amyloid 40 was reacted by the beta amyloid 40 antibody immobilized on the surface, resulting in a maximum Rayleigh light scattering wavelength shift of about 10 nm, and 100 nM beta amyloid 42 and For tau protein, only 1.4 nm and 1.1 nm of migration occurred, respectively. Meanwhile, in the aspect ratio 1.73 bar gold nanoparticle biosensor manufactured by Example 4, beta amyloid 42 was fixed to the surface of the particle, so that the maximum scattering wavelength shift of 17 nm occurred for 100 nM beta amyloid 42, but the same concentration was achieved. For beta amyloid 40 and tau protein, only 1.95 nm and 1.93 nm of migration occurred. In the aspect ratio 3.53 rod-type gold nanoparticle biosensor manufactured by Example 4, the maximum scattering wavelength of 19 nm was shifted to 100 nM tau protein by immobilizing the tau protein antibody, but 2.45 nm to the same concentration of beta amyloid. It was confirmed that only a shift of 2.5 nm occurs (FIG. 16).

In addition, it was confirmed that the beta amyloid 40 can be quantified by identifying the maximum wavelength mobility generated by applying a certain amount of the sensor. To this end, beta amyloid 40 in undiluted plasma was spiked at each concentration from 5pM to 50nM and reacted with a gold nanoparticle sensor immobilized on a glass substrate.

Maximum Wavelength (LSPR Δλ max (nm)) Shift and LOD Values According to Beta Amyloid 40 Concentration Conc. Δλ _max (nm) (w / o pretreatment) Δλ _max (nm) (w / pretreatment) 5 pM 2.5013 2.92791 50 pM 2.8746 3.04 500 pM 5.1664 6.0662 5 nM 7.7586 8.8575 50 nM 8.1795 8.906656033 LOD (pM) 8.45pM 5.67pM

The LOD (detection limit) is calculated according to the following equation from three standard deviations of the peak-to-peak wavelength shift back from the baseline (without amyloid beta 40) through repeated experiments:

, 여기서 δ는 대조군의 표준 편차, slope는 센서의 감도이다. LOD =

Where δ is the standard deviation of the control and slope is the sensitivity of the sensor.

Only the biosensor according to the present invention can be used as a sensor having a minimum detection concentration of 8.45pM, it was confirmed that the chaotropic solvent of Example 8 can have a lower minimum detection concentration of 5.67pM. That is, when the chaotropic solvent was used, more maximal wavelength shift occurred, and thus the sensitivity was about 3pM higher than when the solvent was not used (Fig. 17 and Fig. 18).

Example 6 Enzyme Immune Response Analysis of Beta Amyloid 42 According to Chaotropic Solvents

Ammonium bicarbonate (ABC) was dissolved in water to prepare a 25 mM aqueous solution, and guanidine hydrocholoride, guanidine thiocyanate, potassium thiocyanate and 25 mM hydrogen carbonate prepared above. Stock solution was prepared by dissolving in ammonium (ABC) aqueous solution at 20M.

Chaotropic Solvent Concentration for Beta-amyloid 42 Enzyme Immune Response Analysis blood 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl Dilution solution 50 μl 47.5 μl 45 μl 40 쨉 l 35 μl 30 μl 25 μl 20 쨉 l 10 μl Guanidine hydrochloride (20M)
Guanidine thiocyanate (20M)
Potassium Thiocyanate (20M) - 2.5 μl 5 μl 10 μl 15 μl 20 쨉 l 25 μl 30 μl 40 쨉 l Final concentration (M) 0 0.5 One 2 3 4 5 6 8

The concentration of the chaotropic solvent was analyzed as described above in the enzyme immunoassay kit (SensoLyte ^™ , Catalog # AS-55552) of beta amyloid 42. As a sample, blood collected from humans was used, and experiments were performed using plasma from which agglomerates were removed from blood.

Briefly describing the experimental method, human beta amyloid 42 standard (Component B) was reconstituted with 1 ml of peptide standard reconstitution buffer (Component G). That is, the peptide was hydrated for 10 minutes, upside down and gently mixed, and then 100 μl of the reconstituted standard was dispensed per vial and stored at −80 ° C. until use.

After aligning and labeling the strips (Component A) according to the standard and the number of wells used for the samples, the diluted standard and the samples were each duplicated (duplicate) and the experiment was performed. Sequential dilution of human beta amyloid 42 standard was prepared immediately before the experiment, which proceeded as described in Table 3 below.

The detection antibody (Component H) was diluted 200-fold in the detection antibody dilution buffer (Component I). 50 μl of the antibody solution was dispensed into each well, and 100 μl of diluted beta amyloid 42 standard in each well was dispensed with blanks. Meanwhile, 100 μl of the diluted sample was added to each well as shown in Table 2, and 50 μl of the antibody solution was added to each well. The adhesive plate cover (Component J) was covered and reacted by blocking the overnight light at 4 ° C. The 10X washing buffer (Component D) was diluted with distilled water to prepare a 1X washing buffer, and the solution of the plate reacted overnight was removed, and then washed 6 to 7 times using 350 μl of 1X washing buffer in each well. Allow enough 20 seconds for the wash buffer to work between each wash before the wash solution is removed. Plates were wiped using paper towels for accurate optical readings. 100 μl of TMB color substrate solution (Component E) was added to each well and the plate was tapped lightly and reacted for about 5-15 minutes at room temperature until a blue color gradient appeared along the well. 50 μl of termination solution (Component F) was added to each well and the plate was lightly tapped to turn blue to yellow. Absorbance (OD) was measured at 450 nm using a microplate absorbance reader within 20 minutes after addition of the termination solution.

As a result, it was confirmed that the detection amount of beta amyloid 42 was higher in 6M of guanidine hydrochloride and 3-4M of guanidine thiocyanate (FIG. 19).

Example 7 Analysis of Enzyme Immune Response of Tau Proteins According to Chaotropic Solvents

Ammonium bicarbonate (ABC) was dissolved in water to prepare a 25 mM aqueous solution, and guanidine hydrocholoride, guanidine thiocyanate, potassium thiocyanate and 25 mM hydrogen carbonate prepared above. Stock solution was prepared by dissolving in ammonium (ABC) aqueous solution at 20M.

Concentration of Chaotropic Solvent (Guanidine Hydrochloride) for Analysis of Tau Protein Enzyme Immune Response blood 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl Dilution solution 50 μl 47.5 μl 45 μl 40 쨉 l 35 μl 30 μl 25 μl 20 쨉 l 10 μl Guanidine hydrochloride (20M) - 2.5 μl 5 μl 10 μl 15 μl 20 쨉 l 25 μl 30 μl 40 쨉 l Final concentration (M) 0 0.5 One 2 3 4 5 6 8

Concentration of Chaotropic Solvent (Guanidine Thiocyanate) for Analysis of Tau Protein Enzyme Immune Response blood 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl Dilution solution 50 μl 47.5 μl 45 μl 40 쨉 l 35 μl 30 μl 25 μl 20 쨉 l 10 μl Guanidine thiocyanate (20M) - 2.5 μl 5 μl 10 μl 15 μl 20 쨉 l 25 μl 30 μl 40 쨉 l Final concentration (M) 0 0.5 One 2 3 4 5 6 8

Concentration of Chaotropic Solvent (Potassium Thiocyanate Hydrochloride) for Analysis of Tau Protein Enzyme Immune Response blood 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl Dilution solution 50 μl 47.5 μl 45 μl 40 쨉 l 35 μl 30 μl 25 μl 20 쨉 l 10 μl Potassium Thiocyanate (20M) - 2.5 μl 5 μl 10 μl 15 μl 20 쨉 l 25 μl 30 μl 40 쨉 l Final concentration (M) 0 0.5 One 2 3 4 5 6 8

The concentration of chaotropic solvent was analyzed as described above according to the instructions of the enzyme immunoassay kit (invitrogen, Catalog nos. KHB0041, KHB0042) of the tau protein. As a sample, blood collected from humans was used, and experiments were performed using plasma from which agglomerates were removed from blood.

Briefly describing the experimental method, 100 μl of standard dilution buffer was added to the zero well, except for the color source blank. 100 μl of standard was added to the titration wells for the standard curve and 50 μl of sample and 50 μl of standard dilution buffer were added to the wells for sample analysis. After the plate was covered with a cover and reacted at room temperature for 2 hours, the solution was thoroughly removed by suction and washed 4 times with 1 × washing buffer. 100 μl of human FGF-b Biotin Conjugate solution was then added to each well, except for the colorant blank, and mixed by gently tapping the side of the plate. After the plate was covered with a cover and reacted at room temperature for 1 hour, the solution was thoroughly removed by suction and washed 4 times with 1 × wash buffer. 100 μl of Stre ptavidin-HRP was added to each well, except for the chromogen blank, and the plate was covered with gerber and reacted at room temperature for 30 minutes. The solution in each well was thoroughly aspirated off and washed four times with 1 × wash buffer. 100 μl of Stabilized Chromogen was added to each well so that each substrate solution turned blue. The reaction was carried out for 30 minutes at room temperature in dark conditions. 100 μL of the termination solution was added to each well and the sides of the plate were tapped lightly for mixing so that the solution of each well changed from blue to yellow.

As a result, it was confirmed that tau protein was detected high when guanidine hydrochloric acid 6M or guanidinethiocinan acid salt 4M was used (FIG. 20).

Example 8 Analysis of Enzyme Immune Response of Amyloid Beta 40 by Chaotropic Solvents

Ammonium bicarbonate (ABC) was dissolved in water to prepare a 25 mM aqueous solution, and guanidine hydrocholoride, guanidine thiocyanate, potassium thiocyanate and 25 mM hydrogen carbonate prepared above. Stock solution was prepared by dissolving in ammonium (ABC) aqueous solution at 20M.

Concentration of Chaotropic Solvent (Guanidine Hydrochloride) for Enzyme Immune Response Analysis of Amyloid Beta40 blood 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl Dilution solution 50 μl 47.5 μl 45 μl 40 쨉 l 35 μl 30 μl 25 μl 20 쨉 l 10 μl Guanidine hydrochloride (20M) - 2.5 μl 5 μl 10 μl 15 μl 20 쨉 l 25 μl 30 μl 40 쨉 l Final concentration (M) 0 0.5 One 2 3 4 5 6 8

Concentration of Chaotropic Solvent (Guanidine Thiocyanate) for Enzyme Immune Response Analysis of Amyloid Beta40 blood 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl Dilution solution 50 μl 47.5 μl 45 μl 40 쨉 l 35 μl 30 μl 25 μl 20 쨉 l 10 μl Guanidine thiocyanate (20M) - 2.5 μl 5 μl 10 μl 15 μl 20 쨉 l 25 μl 30 μl 40 쨉 l Final concentration (M) 0 0.5 One 2 3 4 5 6 8

Concentration of Chaotropic Solvent (Potassium Thiocyanate Hydrochloric Acid) for Enzyme Immune Response Analysis of Amyloid Beta40 blood 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl 50 μl Dilution solution 50 μl 47.5 μl 45 μl 40 쨉 l 35 μl 30 μl 25 μl 20 쨉 l 10 μl Potassium Thiocyanate (20M) - 2.5 μl 5 μl 10 μl 15 μl 20 쨉 l 25 μl 30 μl 40 쨉 l Final concentration (M) 0 0.5 One 2 3 4 5 6 8

The concentration of the chaotropic solvent was analyzed as described above using the enzyme immunoassay kit (Cusabio, Catalog no. CSB-E08299h) of amyloid beta 40. As a sample, blood collected from humans was used, and experiments were performed using plasma from which agglomerates were removed from blood.

As a result of confirming the detection amount of beta amyloid 40 according to the concentration of chaotropic solvent, the detection amount of beta amyloid 40 was increased in all cases of using chaotropic solvent. When 6M was used, it could be confirmed that the detection amount of beta amyloid 40 was increased (FIG. 21).

Example  9: Chaotropic  Alzheimer's invention according to solvent Of cover factor Polyacrylamide  Analysis using gel electrophoresis

CAST GEL determined acrylamide content according to the molecular weight of the Alzheimer's disease marker. Beta amyloid 40 and beta amyloid 42 were prepared as 15% Tris-glycine polyacrylamide gel with reference to the molecular weight of about 4kDa and tau protein, depending on the type of isoform, about 36.8 ~ 45.9kDa. 15% Tris-glycine polyacrylamide gel was prepared by mixing 2.3 ml of sterile water, 5.0 ml of 30% acrylamide mix, 2.5 ml of 1.5M Tris buffer (pH 8.8), 0.1 ml of 10% ammonium persulfate, and 0.004 ml of TEMED. The components of the stacking gel were prepared by mixing 2.7 ml of sterile water, 0.67 ml of 30% acrylamide mix, 0.5 ml of 1.0M Tris buffer (pH 6.8), 0.04 ml of 10% ammonium persulfate, and 0.004 ml of TEMED. The gel solution was added to the gel caster in turn, cast, and the sample was diluted with 10X Tris-glycine buffer and the protein weight standard marker was prepared. At this time, if dilution of the sample is necessary, it was prepared by diluting with 1X Tris-glycine buffer. After gel preparation, the standard marker was placed on the first line, the samples were set on the other line, and loaded at 20V for about 10 hours. After that, the stacking gel portion was removed from the polyacrylamide gel, and the loaded protein was stained by dipping in 50 ml of the brilliant blue G solution diluted 10-fold. At this time, the dye was shaken at 50 rpm on an orbital shaker for 13 hours, and rinsed with sterile water until the band was visible after dyeing.

Meanwhile, 25mM aqueous solution was prepared by dissolving ammonium bicarbonate (ABC) in water, and guanidine hydrocholoride, guanidine thiocyanate, and potassium thiocyanate 25mM prepared above. A stock solution was prepared by dissolving at 20 M in aqueous ammonium carbonate (ABC) solution. Samples were prepared using this and electrophoresed.

blood 10 μl 5 μl 5 μl 5 μl 5 μl 5 μl 5 μl 5 μl 5 μl Dilution solution
(Tris-glycine buffer) - 4.75 μl 4.5 μl 4 μl 3.5 μl 3 μl 2.5 μl 2 μl 1 μl Chaotropic Solvent (20M) - 0.25 μl 0.5 μl 1 μl 1.5 μl 2 μl 2.5 μl 3 μl 4 μl Final concentration 0 0.5 One 2 3 4 5 6 8

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (11)

(A) A sample isolated from a sample is contacted with a nanoplasmonic sensor based on gold nanoparticles to which an antibody or aptamer is immobilized to a target protein that specifically increases or decreases expression in an Alzheimer's patient. Inducing binding to the antibody or aptamer; And
A method for diagnosing Alzheimer's disease, comprising: measuring light scattering spectra according to the binding, and determining whether Alzheimer's disease occurs through analysis of the maximum wavelength mobility (Δλ) obtained therefrom,
The target protein Alzheimer's diagnostic method, characterized in that further comprising a pre-treatment step of reacting with a solution containing a chaotropic solvent in the step before binding to the antibody or aptamer. The method of claim 1,
And said sample is blood. The method of claim 1,
The target protein is any one or more selected from the group consisting of beta amyloid 40 peptide, beta amyloid 42 peptide and tau protein. The method of claim 1,
The gold nanoparticles are different in shape and / or size depending on the type of antibody or aptamer immobilized. delete The method of claim 1,
The chaotropic solvent is guanidine hydrocholoride (guanidine hydrocholoride), guanidine thiocyanate (guanidine thiocyanate) and potassium thiocyanate (potassium thiocyanate) characterized in that any one or more selected from the group consisting of. The method according to claim 6,
Chaotropic solvent for the detection of beta amyloid 42 is characterized in that 5 to 7M guanidine hydrochloride or 2 to 5M guanidine thiocyanate. The method according to claim 6,
Chaotropic solvent for the detection of tau protein is characterized in that the 5 to 7M guanidine hydrochloride or 4 to 6M guanidine thiocin ansulfate. The method according to claim 6,
Chaotropic solvent for the detection of beta amyloid 40 is characterized in that the 1.5-3.5M potassium thiocyanate hydrochloric acid or 5-7M guanidine hydrochloric acid. Chaotropic solvent for target protein pretreatment; And gold nanoparticles to which an antibody or aptamer of at least one target protein selected from the group consisting of beta amyloid 40 peptide, beta amyloid 42 peptide and tau protein is immobilized. The method of claim 10,
The gold nanoparticles are biosensor, characterized in that the shape and / or size is different from each other according to the type of the antibody or aptamer is fixed.

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