KR20170073032A - Biological material immobilized-highly sensitive sandwich immunoassay biochip for diagnosing hepatocellular carcinoma with qdot nanoprobe and sandwich immunoassay using the same - Google Patents

Abstract

The present invention relates to a sandwich immunoassay biochip for the diagnosis of liver cancer using a biomaterial immobilized quantum dot nanoprobe and an immunoassay using the same. The sandwich immunoassay biochip according to the present invention has an advantage of being able to accurately and quickly detect not only the adsorption of biomolecules such as proteins but also the presence of trace amounts of various disease proteins including liver cancer biomarkers. In addition, the polymer material used in the sandwich immunoassay biochip of the present invention may be used for controlling release of a functional substance in a drug delivery system or the like.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a sandwich immunoassay biochip for high-sensitivity diagnosis of liver cancer using a biomaterial-immobilized quantum dot nanoprobe, and an immunoassay method using the same. BACKGROUND ART [0002] Biomaterials,

The present invention relates to a sandwich immunoassay biochip for the diagnosis of liver cancer using a biomaterial immobilized quantum dot nanoprobe and an immunoassay using the same. According to the present invention, liver cancer can be detected accurately and promptly even when a small amount of liver cancer biomarker exists.

In order to effectively diagnose cancer, biosensors have been actively studied as a technology for detecting the presence or absence of disease by detecting target substances from body fluid samples [1,2]. In addition to the rapid development of biotechnology using biochips, Lab-on-a-chip, which performs all steps such as dilution, mixing, reaction, separation, and quantification of samples based on MEMS technology on one chip, Has been developed. It is expected that the development of such a technology will enable the development of portable high-sensitivity cancer diagnostic devices that can directly diagnose very small amounts of biological samples or extremely low concentrations of biological samples without pretreatment [3].

Liver cancer is one of the most frequent malignant tumors and is one of the second most dead cancer in close China. Therefore, it is important to detect very low concentrations of tumor biomarkers in the blood for early diagnosis of these cancers. One of the most widely used proteins for hepatoma biomarker is alpha fetoprotein (AFP), which is produced in the fetal liver when the fetus is born and the concentration of the protein is rapidly reduced at the same time as the birth of the fetus. . However, in 70% of patients with liver cancer, the levels of AFP remain high in the blood.

Studies on biosensors that have been performed to diagnose liver cancer have been studied mainly by detecting biomarker α-fetoprotein (AFP) and CEA biomarkers related to liver cancer using electrochemical principles. Have been developed in a manner that uses enzymes necessary for the production of electrical signals in response to the presence of a substance. For accurate and rapid diagnosis of diseases, the most important issue is the technology to easily detect and quantify cancer-related biomarkers that are dissolved in very low concentrations in the blood. When a microfluidic device is used, it requires a small sample and a reagent. However, it is very important to stably and efficiently immobilize an antibody specific to a cancer-related biomarker to a microfluidic device. In addition, we need a surface treatment technique that can minimize non-specific adsorption of proteins that we do not want.

There are several problems in the existing studies. Among them, there is a limitation that the used samples are tested with human serum or TrisHCl + BSA in the state where various biomaterials existing in blood are excluded. However, this method can lead to stability problems by immobilizing proteins through noncovalent bonding such as hydrophobic interaction or charge interaction in the binding of the substrate and the capturing antibody to the PDMS surface. However, this method can lead to stability problems by immobilizing proteins through noncovalent bonding such as hydrophobic interaction or charge interaction in the binding of the substrate and the capturing antibody to the PDMS surface.

Non-patent literature

[1] Tothill, Biosensors for cancer markers diagnosis, Seminars in Cell & Developmental Biology, 2009, 20, 55-62

[2] Rasooly et al., Development of biosensors for cancer clinical testing, Biosensors and Bioelectronics, 2006, 21, 1851-1858

[3] Soper et al., Point-of-care biosensor systems for cancer diagnostics / prognostics, Biosensors and Bioelectronics, 2006, 21, 1932-1942

Accordingly, the present inventors have made intensive efforts to develop a biochip for accurate diagnosis of hepatocellular carcinoma (HCC). As a result, the present inventors have confirmed that the detection limit of 0.1 ng / mL is shown when Qdot nanoprobe is used.

As used herein, the term "biochip" refers to a biochip, an optical biochip, and an electrochemical biochip used for enzymatic analysis and immunoassay, The biochip is a biochip used in an immunoassay for detecting an antigen through specific binding of an antigen-antibody, and in particular, the biochip of the present invention is capable of detecting an antigen present in a trace in a sample rapidly and accurately, The present invention relates to a sandwich immunoassay biochip for detecting a specific binding between an antibody and an antigen.

As used herein, the term "quantum dot (Qdot)" is a nano-sized semiconductor material. When the size is smaller than a certain size, the electron movement characteristics in the bulk semiconductor material are more restricted, Means a material exhibiting a quantum confinement effect with a different emission wavelength.

According to a first embodiment of the present invention,

A hepatoma biomarker that binds to the Fc portion of a capturing antibody bound to a functional group of a functionalized surface-modified plastic support; And

A sandwich immunoassay biochip for diagnosing high sensitivity of liver cancer comprising a detection antibody bound to Quantum dots silica nanoprobe in another antigenic determinant not associated with the capture antibody of the liver cancer biomarker is disclosed. An exemplary embodiment of the sandwich immunoassay biochip according to the present invention is shown in FIG.

As used herein, the term " Hepatocellular carcinoma (HCC) "refers to a primary malignant tumor originating in the liver tissue itself occurring in a patient having a risk factor such as abuse, viral hepatitis, and hepatic liver disease. HCC does not have fibrous stroma and hemorrhage and cell necrosis occur, resulting in vascular infiltration into the portal vein system and, in severe cases, can lead to liver rupture and intraperitoneal blood exudation.

As used herein, the term "hepatoma biomarker" refers to hepatocellular carcinoma (AFP) or hepatocellular carcinoma antigen (CEA) But are not limited thereto.

As used herein, the term "antibody" is intended to include all forms of binding specifically to the liver cancer biomarkers of the invention. Therefore, in addition to a monoclonal antibody or polyclonal antibody, a multispecific antibody (i.e., an antibody recognizing two or more antigens or two or more epitopes, for example, referred to as a specific antibody) Fragments of antibodies, recombinant antibodies, chemically modified antibodies that have the ability to specifically bind to the marker. Examples of fragments of the antibody include Fab, F (ab ') 2, scFv (an antibody in which a heavy or light chain Fv is linked by a suitable linker), Fv, Fab / c Is meant to include an antibody fragment obtained by treating with a protein cleaving enzyme, for example, papain or pepsin. The globulin type of the antibody may be any one of IgG, IgM, IgA, IgE, and IgD as long as it can specifically bind to liver cancer biomarkers of the present invention.

In the sandwich immunoassay biochip of the present invention, the functional group may be an amine group. For example, the functional group may be N-acryloxy succinimide (NAS).

In the sandwich immunoassay bio-chip of the present invention, the plastic support may be a polymer selected from the group consisting of cycloolefin copolymer (COC), polymethyl methacrylate (PMMA), polystyrene, polyethylene, acrylonitrile butadiene styrene ), Or polycarbonate. ≪ RTI ID = 0.0 >

In the sandwich immunoassay biochip of the present invention, the surface of the plastic support may be modified with a benzyl group (-CH2-C6H5) and polyethylene glycol.

In the sandwich immunoassay biochip of the present invention, the liver cancer biomarker includes, but is not limited to, AFP (alpha-fetoprotein) or CEA (cancer embryo antigen).

In the sandwich immunoassay biochip of the present invention, the quantum dot is a II-VI compound semiconductor nanocrystal such as CdS, CdSe, CdTe, ZnS, ZnSe, ZnTe, HgS, HgSe, , III-V compound semiconductor nanocrystals such as GaAs, InP, and InAs, but are not limited thereto.

In the sandwich immunoassay biochip of the present invention, the quantum dot may have an emission peak at a wavelength of 520 to 570 nm and a wavelength of 620 to 670 nm. This includes cases where the quantum dots are green and / or red luminescence. For example, light is absorbed from the blue LED chip or the blue light-emitting phosphor to emit green and / or red light. As another example, the quantum dot may have an emission peak at a wavelength of 570 to 620 nm. This absorbs light from the blue LED chip or the blue light-emitting phosphor to emit yellow light.

In the sandwich immunoassay biochip of the present invention, the nanoprobe may be a mesoporous silica bead.

In the sandwich immunoassay biochip of the present invention, the biochip may have a detection limit of 0.1 ng / mL.

According to a second embodiment of the present invention,

(A) contacting a sample to a biochip according to the first embodiment of the present invention;

(B) specifically binding a liver cancer biomarker in a sample to a capture antibody in the bio-chip;

(C) binding a detection antibody bound to a quantum dot nanoprobe to another antigenic determinant not bound to the capture antibody of the liver cancer biomarker; And

(D) measuring a change in luminescence of the quantum dot.

As used herein, the term "sample" includes, but is not limited to, tissue, cells, serum, plasma, saliva, cerebrospinal fluid or urine containing a target substance to be detected.

In the sandwich immunoassay of the present invention, the immunoassay may have a detection limit of 0.1 ng / mL.

1 shows an exemplary embodiment of a sandwich immunoassay biochip according to the present invention.
Fig. 2 shows fluorescence microscopic results after binding (left) and before binding (right) of Qdot on silica nanobeads according to Example 2. Fig.
FIG. 3 shows the BCA assay results of the Qdot nano-probe in which the secondary antibody according to Example 2 is immobilized.
FIG. 4 shows a sandwich immunoassay result using the Qdot nano-probe according to Example 1. FIG.

Unless otherwise defined herein, 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 pertains in more detail, with reference to the accompanying drawings, I want to explain. It is to be understood, however, that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

< Example >

Example  1. Surface modification of plastic substrate and immobilization of antibody

Benzyl methacrylate, poly (ethylene glycol) methyl ether methacrylate and N-acryloxy succinamide (NAS 99%) were mixed in a volume ratio of 35:25:40 to 2,2'-azobisisobutyronitrile (AIBN ) At 70 DEG C, and then dipped in a cycloolefin copolymer (COC) for 1 hour to modify the COC surface.

Rabbit-IgG was bound onto the surface-modified COC using a micro-contact printing method and quenched with PBS. Then, to confirm that the Rabbit-IgG was immobilized, Rhodamine-conjugated anti-rabbit IgG was sprayed onto the COC surface.

Example  2. Secondary antibody  Immobilized Qdot Nano-prop  Produce

First, Qdot containing a carboxyl group was added to a silica nanobead having an amine group, and the reaction was carried out for 10 hours using an EDC solution. The unbound Qdot was removed using a centrifuge and the anti-AFP was immobilized as a detection antibody. Then, the unbound antibody was removed using a centrifuge to complete the Qdot nano-probe having the secondary antibody immobilized thereon. In order to confirm whether the Qdot was immobilized on the silica nano-beads well, it was observed with a fluorescence microscope, and the results are shown in Fig. The BCA assay was performed to confirm whether the secondary antibody was immobilized on the Qdot nano-probe, and the results are shown in Fig.

< Experimental Example >

Experimental Example  1. Liver cancer Marker  Sandwich immunoassay for diagnosis

AFP as an analyte on the plastic support according to Example 1 was diluted 1/10 with respect to 100 ng / ml, treated for each concentration, washed several times with PBS for 1 hr. Then, the Qdot nano-probe immobilized with the secondary antibody according to Example 2 was treated on the plastic substrate for 1 hour, reacted for 1 hour, and washed several times. Quantitative analysis using Image J revealed a concentration-dependent signal from 100 ng / mL to 0.1 ng / mL, indicating a detection limit of 0.1 ng / mL (FIG. 4).

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. The disclosed embodiments are therefore to be considered in all respects from an illustrative point of view, not of limitation. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of the present invention should be construed as being included in the present invention.

Claims (2)

A hepatoma biomarker that binds to the Fc portion of a capturing antibody bound to a functional group of a functionalized surface-modified plastic support; And
And a detection antibody bound to a quantum dots silica nanoprobe in another antigenic determinant not associated with the capture antibody of the liver cancer biomarker, wherein the detection antibody is included in the sandwich immunoassay biochip for high sensitivity diagnosis of liver cancer.

(A) contacting the sample with the biochip of claim 1;
(B) specifically binding a liver cancer biomarker in a sample to a capture antibody in the bio-chip;
(C) binding a detection antibody bound to a quantum dot nanoprobe to another antigenic determinant not bound to the capture antibody of the liver cancer biomarker; And
(D) measuring a change in the emission of the quantum dot.

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