KR102200143B1 - Bio-probe for detecting target protein, manufacturing method thereof, analyzing apparatus having the same and analyzing method thereof - Google Patents

Abstract

Disclosed are a bioprobe for detection of a target protein designed to increase the sensitivity and speed of colorimetric analysis by signal amplification so as to detect an initial amplified protein biomarker, a method for manufacturing the same, an analysis device having the same, and a method thereof. The bioprobe for detection of a target protein includes gold nanoparticles having an average diameter of 15 nm; C-reactive protein (CRP) antibody bound to the gold nanoparticles; And a plurality of 6×histacks bonded to the gold nanoparticles and having high affinity for labeled nickel tagged with an optical reporter. The designed 6X heat-stack-gold nanoparticle-anti-CRP probe is eco-friendly because it is synthesized through an environmentally friendly procedure using only water. In addition, the 6X Histack nanoparticle probe is designed with simple detection (3 steps), so it has simplicity. In addition, the 6X histack-gold nanoparticle-anti-CRP probe is manufactured within 2 hours, so it has rapidity. In addition, the sensitivity of the 6X histack-gold nanoparticle-anti-CRP probe has been demonstrated in the detection of CRP protein requiring an LOD of 27.7 pg/ml and has sensitivity.

Description

A bioprobe for detecting a target protein, a method for manufacturing the same, an analysis device having the same, and a method therefor

The present invention relates to a bioprobe for detection of a target protein, a manufacturing method thereof, an analysis device having the same, and a method thereof, and more particularly, a target protein designed to increase the sensitivity and speed of a colorimetric assay by signal amplification. It relates to a bioprobe for detection, a manufacturing method thereof, an analysis device having the same, and a method thereof.

A biomarker is a kind of biomolecule that exists in a biological or medical specimen, and diagnoses the condition of a disease by qualitatively and/or quantitatively detecting changes in their structure or concentration, and the therapeutic effect of drugs and other diseases. It plays the role of a marker that allows you to comprehensively judge the relevance of For early diagnosis of disease, a technology that quantitatively analyzes biomarkers appearing in the early stages of onset is essential. This is because the technology currently used for disease monitoring is not adequately responding to the technical demand for early diagnosis of disease due to limitations such as sensitivity, quarantine speed, and cost.

In order to quantitatively analyze biomarkers, methods using an enzyme-linked immunosorbent assay (ELISA) method, western blotting method, and mass spectrometry are commonly used. ELISA is a standard method for protein detection. C-reactive protein (CRP) is also detected by ELISA. C-reactive protein (CRP) is a protein consisting of five circular substructures. It is present in the serum and produced in the liver, and an increase in blood can be observed in infection or inflammatory reaction, and is known to increase rapidly in cardiovascular disease (CVD).

Since ELISA requires several steps of 10 or more, the detection time takes from 3 to 6 hours. ELISA is expensive because it requires 2-3 sets of antibodies. The limit of detection (LOD) of CRP ELISA is 200 pg/ml.

Magnetic nanoparticles, quantum dots, and carbon dot-based probes were introduced for protein detection as an amplified signal with LOD in the range of 100 to 200 pg/ml, but it takes a long time (12-24Hrs) and requires a high temperature (180-300oC). And is a toxic reagent.

Other techniques for detecting proteins include Matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF), and High-Performance Liquid Chromatography (HPLC). . The sensitivity of this technique is 200 pg/ml. However, detectors employing such detection techniques are expensive and heavy, and require skilled personnel and a protocol that takes a lot of time (8-24 hours).

In particular, there is High-Performance Liquid Chromatography (HPLC). Although this method has high sensitivity, it has a disadvantage in that the detection process is complicated because it has to undergo processes such as extraction, washing of cartridges, and immuno-affinity columns, and analysis, which requires an expert and requires a long time for analysis and high cost.

Korean Patent Registration No. 10-1088885 Republic of Korea Patent Publication No. 10-2017-0017266 Korean Patent Registration No. 10-1612094 Korean Patent Registration No. 10-1928620

Accordingly, the technical problem of the present invention is focused on this point, and an object of the present invention is a bioprobe for detection of a target protein designed to increase the sensitivity and speed of colorimetric analysis by signal amplification so that the initial amplified protein biomarker can be detected. Is to provide.

Another object of the present invention is to provide a method for producing a bioprobe for detecting the target protein.

Another object of the present invention is to provide an analysis device using the bioprobe for detecting a target protein.

Another object of the present invention is to provide an analysis method using the bioprobe for detecting a target protein.

In order to realize the object of the present invention as described above, a bioprobe for detecting a target protein according to an embodiment includes gold nanoparticles having an average diameter of 15 nm; C-reactive protein (CRP) antibody bound to the gold nanoparticles; And a plurality of 6×histacks bonded to the gold nanoparticles and having high affinity for labeled nickel tagged with an optical reporter.

In one embodiment, the gold nanoparticles may be covered with citrate.

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In one embodiment, the antibody may include a monoclonal antibody.

In order to realize another object of the present invention, a method of manufacturing a bioprobe for detecting a target protein according to an embodiment includes: (i) CRP-antibody-gold nanoparticle conjugate comprising gold nanoparticles having an average diameter of 15 nm Generating a; And (ii) loading the CRP monoclonal antibody-gold nanoparticle conjugate with 6X histack to obtain a 6X histack-gold nanoparticle-anti-CRP probe.

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In one embodiment, step (i) is, (i-1) a mixed solution of a gold nanoparticle solution (AuNP) containing gold nanoparticles having an average diameter of 15 nm and an anti-CRP monoclonal antibody (Anti-CRP mAb) Agitating; (i-2) centrifuging the product obtained in step (i-1); And (i-3) removing the supernatant from the resultant obtained in step (i-2) to obtain a CRP-antibody-gold nanoparticle conjugate.

In one embodiment, the anti-CRP monoclonal antibody and the gold nanoparticle solution may be mixed at a concentration of 10:1.

In one embodiment, the concentration of the anti-CRP monoclonal antibody may be 30 nM, and the concentration of the gold nanoparticle solution may be 3 nM.

In one embodiment, the mixed solution may be stirred for 20 minutes at 500 rpm at room temperature.

In one embodiment, the centrifugation of step (i-2) may take place at 10,000 rpm for 25 minutes.

In one embodiment, step (ii), (ii-1) adding 6X heat stack; (ii-2) stirring the mixture obtained in step (ii-1); (ii-3) centrifuging the product obtained in step (ii-2); And (ii-4) removing the supernatant from the resultant obtained in step (ii-3) to obtain a 6X histack-gold nanoparticle-anti-CRP probe.

In one embodiment, the concentration of 6X heat stack added in step (ii-1) may be approximately 150 nM.

In one embodiment, the agitation of step (ii-2) may be performed at about 180 rpm for about 15 minutes.

In one embodiment, the centrifugation of step (ii-3) may be performed at approximately 4° C. for 20 to 25 minutes at approximately 10,000 rpm.

According to such a bioprobe for detection of a target protein, its manufacturing method, an analysis device having the same, and the method, the designed 6X Heat Stack-Gold Nanoparticle-Anti-CRP probe is synthesized through an environmentally friendly procedure using only water, so it has eco-friendliness have. In addition, the 6X Histack nanoparticle probe is designed with simple detection (3 steps), so it has simplicity. In addition, the 6X histack-gold nanoparticle-anti-CRP probe is manufactured within 2 hours, so it has rapidity. In addition, the sensitivity of the 6X histack-gold nanoparticle-anti-CRP probe has been demonstrated in the detection of CRP protein requiring a limited of detection (LOD) of 27.7 pg/ml, and has sensitivity.

1 is a flowchart illustrating a method of manufacturing a bioprobe for detection of a target protein according to an embodiment of the present invention.
FIG. 2 is a diagram schematically illustrating a synthesis process of a 6X histack-gold nanoparticle-anti-CRP probe, which is a bioprobe for detection of a target protein according to the present invention.
3 is a view for explaining CRP detection based on a 6X histack-gold nanoparticle-anti-CRP probe, which is a bioprobe for detection of a target protein according to the present invention.
4 is a view for explaining a C-reactive protein (CRP) detection process using a 6X histack-gold nanoparticle-anti-CRP probe, a bioprobe for detection of a target protein according to the present invention.
5 is a flowchart illustrating a method of detecting C-reactive protein (CRP) using a 6X histack-gold nanoparticle-anti-CRP probe according to the present invention.
Figure 6a is a table for explaining the detection of C-reactive protein (CRP) according to the present invention, Figure 6b is a graph showing the CRP concentration versus absorbance in the table of Figure 6a.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. In the present invention, various modifications may be made and various forms may be applied, and specific embodiments will be illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific form disclosed, it should be understood to include all changes, equivalents, and substitutes included in the spirit and scope of the present invention.

In describing each drawing, similar reference numerals have been used for similar elements. In the accompanying drawings, the dimensions of the structures are shown to be enlarged compared to the actual size for clarity of the present invention.

Terms such as first and second may be used to describe various components, but the components should not be limited by the terms. These terms are used only for the purpose of distinguishing one component from another component. For example, without departing from the scope of the present invention, a first component may be referred to as a second component, and similarly, a second component may be referred to as a first component. Singular expressions include plural expressions unless the context clearly indicates otherwise.

In the present application, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof, does not preclude in advance the possibility of being added.

In addition, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in this application. Does not.

1 is a flowchart illustrating a method of manufacturing a bioprobe for detection of a target protein according to an embodiment of the present invention. FIG. 2 is a diagram schematically illustrating a synthesis process of a 6X histack-gold nanoparticle-anti-CRP probe, which is a bioprobe for detection of a target protein according to the present invention.

1 and 2, a mixed solution is formed by mixing approximately 30 nM of anti-CRP monoclonal antibody (Anti-CRP mAb) and approximately 3 nM of gold nanoparticle solution (AuNP) (step S110). Here, the gold nanoparticles are covered with citrate and have an average diameter of 15 nm. Gold nanoparticles were synthesized through the Turkevich method.

Then, the mixed solution of anti-CRP monoclonal antibody (Anti-CRP mAb) and gold nanoparticle solution (AuNP) is stirred at room temperature at about 500 rpm for about 20 minutes (step S120).

Then, centrifugation is performed at 10,000 rpm for approximately 25 minutes (step S130).

Subsequently, a supernatant floating in water is removed (step S140). At this time, the pallet dissolved in water.

CRP monoclonal antibody-gold nanoparticles are loaded with 6X Hisstack by the following procedure.

Approximately 150 nM of 6X heat stack is added to the gold nanoparticle solution (step S150). 6X His-tag (or polyhistidine-tag) is an amino acid motif consisting of at least 6 or more histidine (His) residues in a protein, usually at the N-terminus or C-terminus of a protein. It is also known as Hexa histidine-tag, His-tag, His6 tag, etc. 6X Histack can be used to detect proteins. It is also possible by gel staining (SDS-PAGE) using a tag antibody or, instead, a fluorescent probe with metal ions, which is useful in subcellular localization, ELISA, Western blot or immunological assays. The 6X Heat Stack can be successfully used to immobilize proteins on a specific surface, such as a microtiter plate coated with nickel or cobalt or a protein array. .

The mixture obtained in step S150 is subjected to a light stirring treatment at about 180 rpm for about 15 minutes (step S160).

Then, centrifugation treatment is performed at about 4° C. for about 20 minutes at 10,000 rpm (step S170).

Subsequently, the supernatant liquid floating in water is removed (step S180). At this time, the pallet dissolved in water.

Subsequently, a functionalized 6X histack-gold nanoparticle-anti-CRP probe is obtained and stored at approximately 4°C (step S190).

3 is a view for explaining CRP detection based on a 6X histack-gold nanoparticle-anti-CRP probe, which is a bioprobe for detection of a target protein according to the present invention.

Referring to FIG. 3, a CRP-coated substrate is prepared by coating CRP on a substrate. The prepared CRP-coated substrate was injected with a 6X histack-gold nanoparticle-anti-CRP probe prepared by the manufacturing method described in FIGS. 1 and 2 to construct a 6X histack-gold nanoparticle-anti-CRP-CRP complex do.

Subsequently, a plant protein horseradish peroxidase (HRP) was administered to construct a 6X histack-gold nanoparticle-nickel-HRP probe-CRP-anti-CRP complex.

Subsequently, when a fluorescent material is administered as a substrate, the 6X histack-gold nanoparticle-probe-CRP-anti-CRP complex emits yellow light.

4 is a view for explaining a C-reactive protein (CRP) detection process using a 6X histack-gold nanoparticle-anti-CRP probe, which is a bioprobe for detection of a target protein according to the present invention.

Referring to FIG. 4, a 6X histack-gold nanoparticle-anti-CRP probe, which is synthesized and stored, is administered to a plate coated with CRP. A plurality of wells divided into a matrix type are formed in the CRP-coated well plate, and CRPs are coated on each of the wells.

Then, a trimethyl boron (TMB) substrate for color development test is administered. MB (3,3',5,5'-tetramethylbenzidine) is a chromosome that exhibits blue color when oxidized as a result of oxygen free radicals produced through hydrogen peroxide hydrolysis using HRP. TMB is a chromagenic substrate most commonly used to detect HRP and is released in various forms. The TMB substrate solution rapidly detects HRP activity and generates a blue color (maximum wavelength of 370 nm or 652 nm) that turns yellow (maximum wavelength 450 nm) when sulfuric acid or phosphoric acid stop solution is added. The reaction is usually completed between 5 and 30 minutes after addition to the HRP-binding.

Then, after adding a stop solution, the reaction result value is read through a microplate reader, and graphical anaylsis is performed.

5 is a flowchart illustrating a method of detecting C-reactive protein (CRP) using a 6X histack-gold nanoparticle-anti-CRP probe according to the present invention.

Referring to FIG. 5, a coating buffer such as sodium bicarbonate (NaHCO3) (ie, a buffer solution) is coated on the CRP arranged on the well plate (step S210).

Then, it is cultured overnight (step S212).

Then, the well plate is blocked with 200ul of Bovine Serum Albumin (BSA) having a concentration of 3% (step S214).

Then, the well plate is washed with 250ul of PBST (phosphate buffered saline solution containing 0.05% Tween™ 20) solution having a concentration of 2% (step S216). Here, cleaning may be performed about 6 times.

Then, 100ul of the 6X histack-gold nanoparticle-anti-CRP probe according to the present invention is distributed to the wells of the well plate (step S218).

Then, incubate for about 90 minutes at room temperature (step S220).

Subsequently, the well plate is washed with 200ul of PBST having a concentration of 0.2% (step S222). Here, cleaning may be performed about 6 times.

Then, 100 ul of 6X histack-nickel-HRP having a concentration of 1 ug/ml is performed (step S224).

Then, the well plate is cultured in the dark for about 15 minutes (step S226).

Then, the well plate is washed with 200ul of PBST having a concentration of 0.2% (step S228). Here, cleaning may be performed about 6 times.

Then, the TMB substrate is treated for approximately 10 minutes (step S230).

Then, 0.2M HSO4 is added as a stop solution (step S232).

Then, the microplate reader reads the well plate at 450 nm of the reference light (step S234).

Figure 6a is a table for explaining the detection of C-reactive protein (CRP) according to the present invention, Figure 6b is a graph showing the CRP concentration versus absorbance in the table of Figure 6a.

6A and 6B, when the CRP concentration was 0 pg/ml, the absorbance for the reference light 450 nm was measured to be 0.0785. When the CRP concentration was 50 pg/ml, the absorbance for the reference light 450 nm was measured to be 0.1026. When the CRP concentration was 100 pg/ml, the absorbance for the reference light 450 nm was measured to be 0.1247. When the CRP concentration was 500 pg/ml, the absorbance for the reference light 450 nm was measured to be 0.2408. When the CRP concentration was 2000 pg/ml, the absorbance for the reference light 450 nm was measured to be 0.3201.

Hereinafter, a sensitivity comparison between a commercial ELISA-based assay method for C-reactive protein detection and a 6X histack-gold nanoparticle-anti-CRP probe-based immunoassay method according to the present invention will be described with reference to Table 1 below.

[Table 1]

Referring to Table 1, comparing the analysis method using a 6X histack-gold nanoparticle-anti-CRP probe according to the present invention with a commercial ELISA analysis method, a 6X histack-gold nanoparticle-anti-CRP probe according to the present invention The analysis method used can reduce the overall cost and complexity by developing a direct analysis method without using a capture antibody and a secondary antibody.

In addition, when the performance of colorimetric analysis for C-reactive protein is compared with a commercial kit using the same antibody, it can be seen that the assay sensitivity according to the present invention is 27.7 pg/ml or more.

Hereinafter, a comparison between the 6X His-stack gold nano probe according to the present invention and other nanoparticle-based probes will be described with reference to Table 2 below.

[Table 2]

Referring to Table 2, the 6X histack-gold nanoparticle-anti-CRP probe according to the present invention was designed through a green synthesis procedure using water at a relatively low temperature.

Compared to other nano-probe synthesis (ie, magnetic particle-based probe, quantum dot-based probe, carbon dot-based probe), the design procedure of the probe is fast and simple within 2 hours.

As a result of confirming the double conjugation of the peptide (6XHistack) and the antibody on the first commercially available same nanoparticle (34.29pg/ml), the protocol developed according to the present invention improves signal sensitivity.

Hereinafter, a comparison of the 6X histack-gold nanoparticle-anti-CRP probe and other gold nanoparticles according to the present invention will be described with reference to Table 3 below.

[Table 3]

Referring to Table 3, the conjugation time of an aptamer and an antibody in other gold nanoparticle-based technologies is 12 to 16 hours, whereas 6X hisstack in the 6X histack-gold nanoparticle-anti-CRP probe according to the present invention The conjugation time of the antibody and the antibody is within 2 hours. Here, the aptamer refers to a single-stranded nucleic acid (DNA, RNA, or modified nucleic acid) having a stable tertiary structure by itself as a substance capable of specifically binding to a target substance to be detected in a sample. Specifically, the presence of the target material in the sample can be confirmed. The preparation of an aptamer is synthesized by determining the sequence of an oligonucleotide having a high binding ability and selective to the target substance to be identified according to a general aptamer preparation method, and then linking the 5'or 3'end of the oligonucleotide. In order to be able to bind to the functional group of -SH, -COOH, -OH or -NH2 can be made by transforming.

In addition, in the case of LOD obtained by solution analysis through a microplate reader, the other gold nanoparticle-based technology is 10 pg/ml based on paper substrate, whereas the 6X histack-gold nanoparticle-anti-CRP probe according to the present invention is solution based 27.7 pg. /ml.

In addition, other gold nanoparticle-based technologies are buffer-based, which is applied to other gold nano-probe designs, so that the green binding process is impossible, whereas the 6X His-stack-gold nanoparticle-anti-CRP probe according to the present invention is based on water, so the green binding process This is possible. Therefore, since the buffer is not used, the cost can be reduced and it can be said to be eco-friendly.

As described above, according to the present invention, the 6X histack-gold nanoparticle-anti-CRP probe was successfully designed with a simple and fast synthesis protocol. The validity of the 6X histack-gold nanoparticle-anti-CRP probe designed according to the present invention was verified in CRP protein detection.

The 6X histack-gold nanoparticle-anti-CRP probe designed according to the present invention can be applied to the detection of other clinically important protein biomarkers.

Although the above has been described with reference to examples, it is understood that those skilled in the art may variously modify and change the present invention without departing from the spirit and scope of the present invention described in the following claims. You can understand.

Claims (12)

Gold nanoparticles having an average diameter of 15 nm;
C-reactive protein (CRP) antibody bound to the gold nanoparticles; And
A bioprobe for detection of a target protein, characterized in that it comprises a plurality of 6X hist stacks having high affinity for labeled nickel bound to the gold nanoparticles and tagged with an optical reporter. delete The bioprobe for detecting a target protein according to claim 1, wherein the gold nanoparticles are covered with citrate. The bioprobe for detection of a target protein according to claim 1, wherein the antibody comprises a monoclonal antibody. delete delete (i) generating a CRP-antibody-gold nanoparticle conjugate comprising gold nanoparticles having an average diameter of 15 nm; And
(ii) Preparation of a bioprobe for detection of a target protein comprising the step of obtaining a 6X histack-gold nanoparticle-anti-CRP probe by loading 6X histack into the CRP monoclonal antibody-gold nanoparticle conjugate Way. The method of claim 7, wherein step (i),
(i-1) stirring a mixed solution of a gold nanoparticle solution (AuNP) containing gold nanoparticles having an average diameter of 15 nm and an anti-CRP monoclonal antibody (Anti-CRP mAb);
(i-2) centrifuging the product obtained in step (i-1); And
(i-3) A method for producing a bioprobe for detection of a target protein, comprising the step of obtaining a CRP-antibody-gold nanoparticle conjugate by removing the supernatant from the result obtained in step (i-2). The method of claim 8, wherein the anti-CRP monoclonal antibody and the gold nanoparticle solution are mixed at a concentration of 10:1. The method of claim 7, wherein step (ii),
(ii-1) adding 6X heat stack;
(ii-2) stirring the mixture obtained in step (ii-1);
(ii-3) centrifuging the product obtained in step (ii-2); And
(ii-4) Removing the supernatant from the result obtained in step (ii-3) to obtain a 6X histack-gold nanoparticles-anti-CRP probe of a bioprobe for detection of a target protein, characterized in that it comprises Manufacturing method. The bioprobe for detecting a target protein according to any one of claims 1 to 6; And
And a measuring device capable of detecting a signal emitted from the bioprobe for detecting the target protein. (a) contacting the bioprobe for detection of a target protein according to any one of claims 1 to 6 with a sample to be analyzed; And
(b) an analysis method comprising the step of detecting a signal emitted from the bioprobe for detection of a target protein that has passed through step (a).

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