WO2022139194A1 - Quantum-dot probe for immunosensor and fabrication method therefor - Google Patents

Abstract

The present invention relates to a quantum-dot probe for an immunosensor and a fabrication method therefor and, more specifically, to a method for fabricating a quantum-dot probe for an immunosensor into an accumulated form of many quantum-dots by a cluster formation reaction, and a quantum-dot probe for an immunosensor, fabricated thereby. The quantum-dot probe according to the present invention has many quantum-dots accumulated in one cluster, exhibiting improved fluorescence performance and also has many biomolecules conjugated in one cluster, enabling binding specificity for a target material.

Description

Quantum dot probe for immune sensor and manufacturing method thereof

This application claims priority to Republic of Korea Patent Application No. 10-2020-0183254 filed on December 24, 2020, and the entire specification is a reference to the present application.

The present invention relates to a quantum dot probe for an immune sensor and a method for manufacturing the same, and more particularly, to a method for manufacturing a quantum dot probe for an immune sensor in a form in which several quantum dots are accumulated by a cluster formation reaction, and an immune sensor manufactured thereby It relates to a quantum dot probe for use.

A biosensor is a system that forms a detection reaction of a specific substance using biological factors such as a bond between substances when obtaining information from a measurement object and converts it into a recognizable signal such as color, fluorescence, or electrical signal. By lowering the limit, it enables early diagnosis of diseases, etc., and is being used as a drug delivery agent through a biosensor that targets a specific antigen.

A biosensor is basically an external analyte, a receptor that accepts a substance for the first time, a converter that converts the received information into easily identifiable information such as an electrical signal, and an electronic device that directly measures and displays electrical signals. Depending on the substance to be analyzed, there are glucose sensors and urea sensors. Depending on the type of receptor, it is divided into enzyme sensors, immune sensors, and microorganism sensors. Depending on the conversion method, electrochemical biosensors, thermal biosensors, and optical biosensors are classified as Among them, the immune sensor utilizes the specificity of antigen-antibody interaction. That is, the principle of specific binding is used in which the antibody reacts only with the specific structure of the antigen. The immunosensor analysis method using the strong specific binding force between the antigen and the antibody has a very low detection limit compared to other methods, and thus has characteristics suitable for the measurement of physiologically active substances present in low concentrations.

In addition, if we look briefly at the recent trend of biosensor development, the development of small biosensors that can be analyzed with a small amount of biological samples is the mainstream. The immune sensor is a more useful method for detecting a small amount of antigen, antibody, etc. contained in such a small amount of biological sample. In addition, due to the recent miniaturization of biosensors, the absolute amount of biological samples participating in the detection of biomolecules is extremely limited, so the need for developing an immune sensor capable of detecting small amounts of biological samples with high sensitivity is increasing. is rising

The present invention provides a method for manufacturing a quantum dot probe for an immune sensor using a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots.

In addition, the present invention provides a quantum dot probe for an immune sensor prepared by performing a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots.

In addition, the present invention provides an immunosensor in which the quantum dot probe is immobilized on the surface.

In addition, the present invention provides an immunodetection method using the immune sensor.

However, the technical problem to be achieved by the present invention is not limited to the above-mentioned problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

The present invention, activating the quantum dots (step a); and mixing biomolecules with the quantum dots in which step a has been performed, and performing a conjugation reaction between the quantum dots and biomolecules and a cluster formation reaction of the quantum dots (step b). do.

The surface of the quantum dots may be modified with carboxylic acid.

The quantum dots may have a core-shell structure including a core and a shell covering the core.

The core is CdSe, CdS, ZnS, ZnSe, CdTe, CdSeTe, CdZnS, PbSe, AgInZnS, ZnTe, CdSeS, PbS, PbTe, HgS, HgSe, HgTe, GaN, GaP, GaAs, InP, InZnP, InGaP, InGaN, InGaP, and at least one compound selected from among ZnO, wherein the shell is CdSe, ZnSe, ZnS, ZnTe, CdTe, PbS, SrSe, CdO, CdS, ZnO, InP, InS, GaP, GaN, GaO, InZnP, InGaP , InGaN, InZnSCdSe, and may include at least one compound selected from HgSe.

In step a, the quantum dots may be reacted with 1-ethyl-3 (3-dimethylamino) propyl carbodiimide (EDC) and N-hydroxysuccininide (NHS).

In step b, the quantum dots and biomolecules may be functionalized with an azide group and an alkyne group, respectively.

The alkyne group may be characterized as a cyclooctyne group. characterized in that the alkyne group is OCT (octane), ALO (aryl-less octyne), MOFO (monofluorinated cyclooctyne), DIFO (difluorinated cyclooctyne), DIBO (dibenzocyclooctyne), BARAC (biarylazacyclooctynone), DIBAC (Dibenzoazacyclooctyne) or DIMAC (diazazacyclooctyne) can be done with

The biomolecule may be any one or more selected from the group consisting of antibodies, proteins, recombinant proteins, peptides, amino acids, sugars, glycoproteins, vitamins and nucleic acids.

The antibody may be one or more selected from the group consisting of IgM, IgG, IgA, IgE, and IgD, preferably IgG.

In addition, the present invention provides a quantum dot probe for an immune sensor prepared by performing a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots.

The quantum dots and biomolecules are functionalized with an azide group and an alkyne group, respectively, and may be conjugated by a cyclization reaction of azide and alkyne.

In addition, the present invention is an immunosensor in which a detector that specifically binds to a substance to be detected is immobilized on the surface of a quantum dot, wherein the detector is manufactured by performing a conjugation reaction between quantum dots and biomolecules and a cluster formation reaction of quantum dots. It provides an immune sensor, characterized in that the quantum dot probe for the immune sensor.

In addition, the present invention provides an immunodetection method using the immune sensor.

The immunodetection method may include a step of detecting the specific binding of an antigen-antibody by reacting a quantum dot probe for an immune sensor prepared by performing a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots with a substance to be detected. can

The present invention comprises the steps of activating quantum dots (step a); and mixing biomolecules with the quantum dots in which step a has been performed, and performing a conjugation reaction between the quantum dots and biomolecules and a cluster formation reaction of the quantum dots (step b). do.

The present invention also provides a quantum dot probe for an immune sensor prepared by performing a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots.

In addition, the present invention provides an immunosensor in which a detector specifically binding to a substance to be detected is immobilized on the surface. Here, the detector may be a quantum dot probe for an immune sensor prepared by performing a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots.

The present invention also provides an immunodetection method using the immune sensor.

The immune sensor refers to a type of biosensor using the property of forming an antigen-antibody complex. This is based on converting and displaying a certain change into an electrical signal by forming an antigen-antibody complex, and may include both a non-labeled immune sensor and a labeled immune sensor according to the difference in measurement method.

Quantum dots are attractive materials in the field of biosensors because of their excellent photostability and continuous monitoring in real time. Recently, research has been focused on developing sensors for some target analytes in which quantum dots are used as electron donors for fluorescence resonance energy transfer (FRET) between quantum dots and acceptor molecules.

Recently, in the field of biosensors, a technology for binding proteins, peptides, antigens, antibodies, etc. to the surface of a semiconductor quantum dot is in the spotlight. Quantum dots have an advantage in that they have a narrow light emitting area and that the light emission area can be easily controlled. However, there are disadvantages in that the manufacturing process is complicated, the manufacturing cost is high, and the quantum dot manufacturing environment is different from the stabilization conditions of the biomaterial.

According to an embodiment of the present invention, the quantum dots surface-modified with a carboxyl (carboxylic) functional group are free of EDC (1-ethyl-3 (3-dimethylamino) propyl carbodiimide) / NHS (N-hydroxysuccininide) -Activation (pre-activation) may be performed to react with an amino group of a biomolecule (see FIG. 2 ). Since the rate of the reaction between the NHS on the quantum dot and the amine group of the biomolecule is pH dependent, the reaction rate is lowered to pH 7.2 and the mixing ratio of the biomolecule and the quantum dot is adjusted to 1:5, -Can be made to form a quantum dot cluster complex. In such a cluster complex (quantum dot probe), fluorescence performance is improved because several quantum dots are accumulated for one cluster, and a number of biomolecules are also conjugated to improve binding specificity with a target material.

However, in the method by the EDC/NHS, the material stability of EDC/NHS is rather semi-stable, and it is difficult to measure the degree after NHS pre-activation. can be difficult

This problem can be solved by introducing a bioorthogonal reaction (see FIG. 3). The biological orthogonal reaction used in this method applies a binding reaction between azide and cyclooctyne, and this reaction shows an almost uniform binding reaction rate in a wide pH environment, and It is characterized by very high water condition) stability.

Universal bioconjugation utilizes natural functional groups such as amine groups, carboxyl groups, or thiol groups in biochemicals, but as the functional groups are complexly present in biochemicals, non-selective reactions occur. As a result, not only the conjugation with the conjugate, but also the conjugation between biochemicals and the conjugation of the biochemical substances may also react concomitantly to cause aggregation (FIG. 1A). In order to induce an effective bioconjugation reaction while suppressing the side reactions of aggregation, very precise reaction control is required. According to the present invention, a non-selective side reaction is a biological orthogonal reaction introducing a non-natural functional group, specifically, a selective reaction through an azide-alkyl reaction. There is an advantage that can be avoided ( FIG. 1B ).

In the quantum dot probe according to the present invention, since several quantum dots are accumulated in one cluster, fluorescence performance is improved, and since many biomolecules are also conjugated, binding specificity with a target material can be improved. In addition, according to the present invention, an azide group and an alkyne group are introduced into quantum dots and biomolecules, respectively, to induce a bio-conjugation reaction in a bioorthogonal reaction, thereby controlling side reactions of aggregation and conjugation efficiency between quantum dots and biomolecules. can improve

1 is a schematic diagram for a catalytic reaction (A) and a biological orthogonal reaction (B).

2 and 3 are schematic diagrams of a manufacturing process of a quantum dot probe according to an embodiment of the present invention.

4 is a flowchart of a manufacturing process of a quantum dot probe according to an embodiment of the present invention.

5 shows the results of comparing the reactivity of the normal quantum dot conjugate and the cluster quantum dot conjugate to the serum sample of 210 pg/mL BNP in blood.

[Example 1]

Preparation of Clustered Quantum Dot Conjugates

Quantum dots (core: 　CdSe, shell: ZnS) of 6 nm size surface-modified with carboxylic acid were colloidalized in 100 μl of 0.1 M MES buffer at pH 6.0 at a concentration of 8 μM, and 50 mg/m in distilled water. After 10ul of EDC (1-ethyl-3(3-dimethylamino)propyl carbodiimide) solution dissolved in ml and 10ul of NHS (N-hydroxysuccininide) dissolved in 50mg concentration in distilled water were injected, reacted at room temperature for 30 minutes to activate, Sepadex G-25 was used for ion exchange with 0.1M PBS (phosphate buffer saline) at pH 7.4. After injecting mouse anti-BNP IgG antibody to the quantum dots colloidal in 1.0M PBS with pH 7.4 so that the final concentration is 2 mg/ml or more, 100ul of 1.0M biocarbonate buffer solution with pH 9.0 is injected Then, the cluster formation reaction was started at room temperature, and the absorbance was measured at 430 nm during the cluster formation reaction. stopped The cluster reaction solution was centrifuged at 30,000xg for 60 minutes. After centrifugation, the supernatant was discarded leaving only the pellet, and then the pellet was dispersed in 100ul of 0.1M PBS with 1% (w/v) BSA pH 7.4, and 2ul of the sprayed solution was applied to a glass fiber pad with a width of 4mm X length of 6mm. After instillation on the , it was dried to make a conjugate-pad. On the nitrocellulose-membrane, mouse anti-BNP antibody and goat anti-mouse IgG IgG antibody were dissolved at 1 mg/ml in 0.1M PBS solution of pH 7.4, respectively. The solution was dispensed with each nozzle to create a capture line. A test strip is made by arranging a 4 mm wide X 12 mm long polyester fiber salad pad and a 4 mm wide X 8 mm long cellulose inter-pad on an adhesive backing-card measuring 4 mm width X 60 mm length, and making a cassette. assembled to the housing.

[Comparative Example 1]

Preparation of general quantum dot conjugate

Quantum dots (core: 　CdSe, shell: ZnS) having a surface-modified surface of carboxylic acid were ion-exchanged with 0.1M PBS (phosphate buffer saline) at pH 7.4 using Sepadex G-25. . A mouse anti-BNP IgG antibody was injected into the quantum dots of 1.0M PBS with a pH of 7.4 so that the final concentration was 2 mg/ml or more. After centrifugation, the supernatant was discarded leaving only the pellet, and then the pellet was dispersed in 100ul of 0.1M PBS with 1% (w/v) BSA pH 7.4, and 2ul of the sprayed solution was applied to a glass fiber pad with a width of 4mm X length of 6mm. After instillation on the surface, it was dried to make a conjugate-pad. On a nitrocellulose-membrane, mouse anti-BNP antibody and goat anti-mouse IgG IgG antibody were dissolved at 1 mg/ml in 0.1M PBS solution of pH 7.4, respectively. The solution is dispensed into each nozzle to create a capture line. A test strip is made by arranging a 4mm wide X 60mm long adhesive backing-card made of polyester fiber with a width of 4mm X 12mm long and an inter-pad made of a cellulose material with a width of 4mm X 8mm long, and placed on the cassette housing. assembled.

[Experimental Example 1]

Confirmation of reactivity to serum samples using cluster quantum dot conjugates or general quantum dot conjugates

The reactivity of 210 pg/mL BNP to the serum sample was confirmed using the quantum dot conjugate prepared by the method described in the complex prepared by the method described in Example 1 and Comparative Example 1. After dripping 50ul of BNP-positive plasma sample on the sample-pad of the strip, 100ul of 0.1M PBS solution of pH 7.0 was dripped and developed for 15 minutes, then UV-LED was irradiated to the cartridge and a 650nm LP light-filter was installed. A band of fluorescence caused by quantum dots was detected by photographing for 5 ms with a camera. As a result, it was confirmed that detection using the cluster quantum dot conjugate of the present invention exhibited better reactivity ( FIG. 5 ).

In the quantum dot probe according to the present invention, since several quantum dots are accumulated in one cluster, fluorescence performance is improved, and since many biomolecules are also conjugated, binding specificity with a target material can be improved. In addition, according to the present invention, an azide group and an alkyne group are introduced into quantum dots and biomolecules, respectively, to induce a bio-conjugation reaction in a bioorthogonal reaction, thereby controlling side reactions of aggregation and conjugation efficiency between quantum dots and biomolecules. can be improved, so it has great industrial applicability.

Claims (13)

1. activating the quantum dots (step a); and

   mixing biomolecules with the quantum dots in which step a has been performed, and performing a conjugation reaction between the quantum dots and biomolecules and a cluster formation reaction of the quantum dots (step b);

   A method of manufacturing a quantum dot probe for an immune sensor comprising a.
2. The method according to claim 1,

   The quantum dot is a method for producing a quantum dot probe for an immune sensor, characterized in that the surface is modified with carboxylic acid.
3. The method according to claim 1,

   The quantum dot is a method of manufacturing a quantum dot probe for an immune sensor, characterized in that it has a core-shell structure comprising a core and a shell covering the core.
4. 4. The method according to claim 3,

   The core is CdSe, CdS, ZnS, ZnSe, CdTe, CdSeTe, CdZnS, PbSe, AgInZnS, ZnTe, CdSeS, PbS, PbTe, HgS, HgSe, HgTe, GaN, GaP, GaAs, InP, InZnP, InGaP, InGaN, InGaP, and at least one compound selected from ZnO,

   The shell includes at least one compound selected from CdSe, ZnSe, ZnS, ZnTe, CdTe, PbS, SrSe, CdO, CdS, ZnO, InP, InS, GaP, GaN, GaO, InZnP, InGaP, InGaN, InZnSCdSe, and HgSe. A method of manufacturing a quantum dot probe for an immune sensor.
5. The method according to claim 1,

   The step a is a method of manufacturing a quantum dot probe for an immune sensor, characterized in that the quantum dots are reacted with EDC (1-ethyl-3 (3-dimethylamino) propyl carbodiimide) and NHS (N-hydroxysuccininide).
6. The method according to claim 1,

   In step b, the quantum dots and biomolecules are each functionalized with an azide group and an alkyne group.
7. 7. The method of claim 6,

   The alkyne group is OCT (octane), ALO (aryl-less octyne), MOFO (monofluorinated cyclooctyne), DIFO (difluorinated cyclooctyne), DIBO (dibenzocyclooctyne), BARAC (biarylazacyclooctynone), DIBAC (Dibenzoazacyclooctyne) or DIBAC (Dibenzoazacyclooctyne) Dimethoxyazacyclooctyne), characterized in that the group, a method for producing a quantum dot probe for an immune sensor.
8. The method according to claim 1,

   The biomolecule is at least one selected from the group consisting of antibodies, proteins, recombinant proteins, peptides, amino acids, sugars, glycoproteins, vitamins and nucleic acids. manufacturing method.
9. A quantum dot probe for an immune sensor manufactured by performing a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots.
10. 10. The method of claim 9,

    The quantum dots and biomolecules are functionalized with an azide group and an alkyne group, respectively, and the quantum dot probe for an immune sensor, characterized in that it is conjugated by a cyclization reaction of the azide and alkyne.
11. As an immune sensor immobilized on the surface of a detector that specifically binds to a substance to be detected,

    The detector is an immunosensor, characterized in that it is a quantum dot probe for an immunosensor manufactured by performing a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots.
12. The immunodetection method using the immunosensor of claim 11 .
13. 13. The method of claim 12,

    The immunodetection method includes the step of detecting the specific binding of an antigen-antibody by reacting a quantum dot probe for an immune sensor prepared by performing a conjugation reaction between quantum dots and biomolecules and a reaction for forming a cluster of quantum dots with a substance to be detected. Immunodetection method, characterized in that.

Images