KR20160097432A - Immunoassay using disulfide-confined aryldiazonium salt compound as a linker - Google Patents

Abstract

The present invention relates to an immunoassay method using a disulfide-confined aryldiazonium (DASD) salt compound as a linker. More specifically, the present invention relates to a novel immunoassay method using a DASD salt compound as a linker, in which a particular structure DSAD salt compound simultaneously including a disulfide part activated by electrochemical oxidation and an aryldiazonium part capable of being grafted on an electrode (ITO) or a reduced graphene oxide (RGO) is used as a linker molecule for fixing a biomolecule of an immunosensor platform, so spatially-selective surface fixing is possible during a sandwich immunoassay and an electrochemical catalyst activity can be maximized when a sandwich ELISA using an RGO is performed.

Description

IMMUNOASSAY USING DISULFIDE-CONFINED ARYLDIA ZONIUM SALT COMPOUND AS A LINKER [0002]

The present invention relates to an immunoassay using a disulfide-confined aryldiazonium salt (DSAD) salt as a linker, and more particularly to a disulfide- By using a DSAD salt compound having a specific structure simultaneously containing an aryldiazonium moiety that can be grafted onto reduced graphene oxide (RGO) as a linker molecule for immobilizing a biomolecule of an immune sensor platform, a sandwich immunoassay The present invention relates to a novel immunoassay using a DSAD salt compound as a linker that enables spatially-selective surface immobilization and maximizes electrochemical catalytic activity when sandwich ELISA using RGO is performed.

For chemical or biochemical sensors, adjusting the surface to have the desired final chemical functionality is a very important factor. There are many ways of modifying the surface, and well-known methods include forming a self-assembled monolayer (SAM) of an alkane thiol on gold, a technique of forming an indium tin oxide (ITO) Techniques for performing silanization of organosilanes, and techniques for electrochemically reducing diazonium salts on metals, carbon, or semiconductors.

Among these techniques, electrochemical grafting of diazonium salts offers advantages in terms of simplicity, stability and process time, and attracts a great deal of attention in terms of method diversity.

On the other hand, in order to obtain spatially-selective surface immobilization, it is important to design a highly reactive structure capable of forming a covalent bond between the surface and the molecule to be immobilized. For example, an activated form of disulfide, that is, a thiosulfinate group or a thiosulfonate group can form a covalent bond with a thiol (-SH) group of a biomolecule, Can be very advantageously applied. In this regard, the present inventors have recently reported on disulfide terminated silane molecules for multi-bio-functionalization.

However, there have been no studies on the disulfide-terminated aryldiazonium salt (DSAD) for use as a linker molecule. The disulfide-terminated aryldiazonium (DSAD) has excellent properties in relation to the surface grafting technology, and needs specific research and development.

On the other hand, reduced graphene oxide (RGO), a carbon layer of sp ² - hybrid single atom thickness, is attracting attention as a very interesting material with unique electrical, mechanical and optical properties.

Reduced graphene oxide (RGO) is a material with a very high potential range, but proper functionalization is required to use it in applications such as biosensors. Studies to functionalize graphene have been conducted, for example, to graft various diazoniums onto reduced graphene oxide (RGO).

However, a remarkable technique has not yet been developed that efficiently grafts the diazonium onto reduced graphene oxide (RGO) to functionalize the reduced graphene oxide (RGO) to suit the biosensor application.

In short, a novel disulfide-terminated aryldiazonium (DSAD) salt compound is simultaneously discovered that includes both an activatable moiety and a moiety that can be effectively grafted onto reduced graphene oxide (RGO) (Linker) molecule for biomolecule immobilization, it is possible to maximize the (catalytic) activity required for the sensor in the electrochemical immunoassay using RGO together with the spatially-selective surface immobilization. The development of a new technology is required.

Korea Patent No. 10-0448880 Korean Patent Publication No. 10-1994-0011640

Disclosure of Invention Technical Problem [9] Accordingly, the present invention provides an immunoassay using a disulfide-terminated aryldiazonium (DSAD) salt compound having a novel structure as a linker for immobilizing a biomolecule.

Specifically, the present invention provides two embodiments of the immunoassay using the disulfide-terminated aryldiazonium (DSAD) salt compound as a linker: 1) a sandwich immunoassay capable of spatially-selective surface immobilization; and 2) A sandwich ELISA method capable of maximizing electrochemical catalytic activity and detectability in electrochemical immunoassay using RGO is provided.

Disclosure of the Invention In order to accomplish the above object, the present invention provides a disulfide-confined aryldiazonium (DSAD) salt compound represented by the following formula (1), more specifically 5- (1,2 (Dithiolan-3-yl) pentanamide-4-benzenediazonium chloride is used as a linker for immobilizing biomolecules.

[Chemical Formula 1]

(In the formula 1,

R is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,

Ar is an aryl group having 6 to 30 carbon atoms,

X is a halogen atom.)

(2)

In a first embodiment, the present invention provides a process for preparing a disulfide-terminated aryldiazonium (DSAD) salt, comprising: (1) electrodeposition of the disulfide-terminated aryldiazonium (DSAD) salt compound described above onto a microelectrode array; (2) electrochemically oxidizing the disulfide-terminated aryldiazonium (DSAD) salt compound electrodeposited on some microelectrodes to convert the disulfide groups on the surface to thiosulfinate groups or thiosulfonate groups and activate them; (3) exposing a primary antibody having a thiol group to the microelectrode array to space-selectively immobilize the primary antibody only on the activated portion; (4) exposing and immobilizing the target antigen; (5) exposing and immobilizing the secondary antibody; And (6) detecting fluorescence on the surface of the microelectrode array. The present invention also provides a sandwich immunoassay.

Here, the microelectrode array may be an ITO (Indium Tin Oxide) microelectrode array, and the electrodeposition may be performed by cyclic voltammetry (CV). The primary antibody may be Anti-Rabbit antigen, the target antigen may be Rabbit antigen, and the secondary antibody may be Anti-Rabbit antigen. In addition, the secondary antibody may be labeled with TRITC (Tetramethylrhodamine isothiocyanate).

As a second specific example, the present invention provides (1) preparing an ITO electrode surface (ERGO / PEI / ITO) sequentially modified with PEI (Polyethylenimine) and electrochemically reduced graphene oxide (ERGO) ; (2) Electrodeposition of the disulfide-terminated aryldiazonium (DSAD) salt compound on the ERGO surface of the ITO electrode surface to obtain a DSAD functionalized ERGO surface (DSAD / ERGO / PEI / ITO); (3) The surface of the disulfide-terminated aryldiazonium (DSAD) salt compound of the surface of ERGO functionalized with DSAD is electrochemically oxidized to convert the disulfide group on the surface to a thiosulfinate group or thiosulfonate group (ODSAD / ERGO / PEI / ITO) < / RTI > (4) exposing and immobilizing a primary antibody having a thiol group on the activated surface; (5) exposing and immobilizing the target antigen; (6) exposing and immobilizing the secondary antibody; And (7) a step of exposing the electrolyte to a chemical catalytic reaction, and then detecting the amount of the substance produced through electrochemistry using a cyclic voltammetry (CV) method. do.

In the step (1), the ITO electrode washed with Piranha solution is washed again with water to form a negative charge on the electrode surface, and the positive electrode is positively charged, , A graphene oxide (GO) is formed on the obtained surface, and then the GO is electrochemically reduced. The primary antibody may be Anti-Mouse antigen, the target antigen may be mouse antigen, and the secondary antibody may be Anti-Mouse antigen. In addition, the secondary antibody may be one labeled with HRP (Horseradish peroxidase). Further, in the step (7), the hydrogen peroxide and the hydroquinone solution as the electrolyte are exposed to the electrodes of the electrochemical immunosensor to cause a chemical catalytic reaction, and then the amount of the generated benzoquinone is detected by the cyclic voltammetry have.

Immunoassays using the disulfide-terminated aryldiazonium (DSAD) salt compound according to the present invention as a linker can spatially-selectively immobilize biomolecules (proteins, antibodies, etc.) It is possible to maximize the detection ability while maintaining the catalytic activity.

As a result, the target substance can be efficiently and highly sensitively detected by sandwich immunoassay (fluorescence detection) or sandwich ELISA using RGO.

In addition, when the disulfide-terminated aryldiazonium (DSAD) salt compound provided by the present invention is used as a linker for space-selective immobilization of biomolecules, it is possible to manufacture biochips in various fields such as ultra fast detection or restricted release have.

In addition, when the disulfide-terminated aryldiazonium (DSAD) salt compound provided by the present invention is used as a linker for the preparation of a graphene-based immunoassay platform, graphene (eg, RGO) Proteins, nucleic acids, and the like.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a schematic representation of a process for preparing disulfide-terminated aryldiazonium (DSAD) salt compounds for use in the present invention.
FIG. 2 is a schematic view illustrating a method of spatially-selectively modifying the surface of an ITO electrode through a disulfide-terminal aryldiazonium salt (DSAD) salt compound of the present invention and a method for detecting a Rabbit antigen using the same.
FIG. 3 is a schematic view showing a method for functionalizing the ERGO surface using the disulfide-terminal aryldiazonium (DSAD) salt compound of the present invention and a mouse antigen detection method using the sandwich ELISA technique.
4 (A) shows a CV curve at a scanning speed of 50 mV S ^-1 when 1.5 mM DSAD (0.1 M HCl) was electrodeposited on the ITO microelectrode; (B) shows the CV curve at a scanning speed of 50 mV S ^-1 for electrochemical oxidation of DSAD / ITO using 0.1 M TBAP (95% ACN); (C) shows the S 2p XPS results for the ITO surface modified with DSAD before electrochemical oxidation; (D) shows the S 2p XPS results for the ITO surface modified with DSAD after electrochemical oxidation; (E) is a fluorescence microscope image (dotted line = position of ITO electrode, scale bar 150 μm) obtained from space-selective immobilization of purified anti-rabbit antigen.
5 (A) shows a CV curve at a scanning speed of 50 mV S ^-1 for the case of electrochemical reduction of GO using 0.5 M NaCl; (B) shows the CV curve at a scan rate of 50 mV S ^-1 when 1.5 mM DSAD (0.1 M HCl) was electrodeposited on the ERGO / PEI / ITO electrode; (C) shows the CV curves at a scanning speed of 50 mVS ^-1 for electrochemical oxidation of DSAD / ERGO / PEI / ITO electrodes using 0.1 M TBAP (95% ACN); (D) shows the untreated ITO (solid line), ERGO / PEI / ITO (dotted line), DSADC / ERGO / PEI / ITO (solid line) at a scanning rate of 50 mVS ^-1 in 1 mM Fe (CN) ₆ ^3- / (Dashed line) and ODSADC / ERGO / PEI / ITO (double dotted line) blocking test.
Figure 6 (A) shows the CV curves at a scan rate of 50 mVS < ^-1 > for reduction of 1 mM BQ (0.1 M PBS) at different modified electrodes; (B) shows the CV response results at a scan rate of 50 mV S ^-1 for immune sensors for detection of mouse antigen and rabbit antigen (1 μg / mL) in 0.1 M PBS containing ₂ mM HQ and 1.5 mM H ₂ O ₂ ; (C) shows CV response results at a scan rate of 50 mV S ^-1 for an immune sensor to detect mouse antigens at different concentrations in 0.1 M PBS containing ₂ mM HQ and 1.5 mM H ₂ O ₂ ; (D) is the calibration curve for the immune sensor response at different mouse antigen concentrations;

Hereinafter, the present invention will be described in detail.

Immunoassay of the present invention is a disulfide-confined aryldiazonium (DSAD) salt compound represented by the following formula (1), more specifically, 5- (1,2-dithi Olan-3-yl) pentanamide-4-benzenediazonium chloride as a linker for immobilizing biomolecules.

[Chemical Formula 1]

(In the formula 1,

R is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,

Ar is an aryl group having 6 to 30 carbon atoms,

X is a halogen atom.)

(2)

The disulfide-terminated aryldiazonium (DSAD) salt compound used in the present invention is a compound having a disulfide moiety activated by electrochemical oxidation and an aryldiamine capable of being grafted on an electrode (ITO) to a reduced graphene oxide (RGO) By including the zonium moiety at the same time, spatially-selective surface immobilization is enabled, and biomolecules such as antibodies can be effectively immobilized.

Specifically, the disulfide-terminated aryldiazonium salt (DSAD) salt compound of the present invention can be very usefully used as a linker for immobilizing biomolecules on various immune assay platforms and biosensors for detecting target substances When a disulfide-terminated aryldiazonium (DSAD) salt compound is electrochemically oxidized, for example, the disulfide moiety is activated to a thiosulfinate group or a thiosulfonate group, To form a covalent bond with the thiol (-SH) group of the biomolecule to efficiently fix the biomolecule.

The disulfide-terminated aryldiazonium (DSAD) salt compound synthesizes a carbamate-based compound containing a dithiolane moiety using a lipoic acid as a reactant; Removing the Boc moiety; And introducing a halogen anion.

In one embodiment, the disulfide-terminated aryldiazonium (DSAD) salt compound of Formula 2, according to the present invention,

a) reacting lipoic acid and N-Boc-1,4-phenylenediamine with triethylamine (TEA), hydroxybenzotriazole (HOBt) and EDCI (1-ethyl-3- [3- (dimethylamino) propyl] -carbodiimide hydrochloride to synthesize tert-butyl 4- (5- (1,2-dithiolan-3-yl) pentanamide) phenylcarbamate;

b) dissolving the compound of step a) in a mixture of dichloromethane (DCM) and trifluoroacetic acid (TFA) to form N- (4-aminophenyl) Synthesizing pentanamide; And

c) dissolving the compound of step b) in hydrochloric acid (HCl) and adding sodium nitrite (NaNO ₂ ) to give 5- (1,2-dithiolan-3-yl) pentanamide-4-benzenediazonium chloride (See Fig. 1).

In the immunoassay using the disulfide-terminated aryldiazonium (DSAD) salt compound of the present invention as a linker for immobilizing a biomolecule, the biomolecule can bind a DSAD salt compound activated with a thiol group or the like to the activated DSAD salt compound, Any substance that can specifically bind to a substance can be used. For example, nucleic acids, proteins or oligopeptides, lipids, carbohydrates, etc., including antibodies, antigens, DNA, RNA or PNAs can be used. Herein, the protein includes an antigen or an antibody for the diagnosis of an infectious disease diagnosis (AFP) or an antibody for diagnosis of infectious disease diagnosis (AFP) together with Antibody, HIV p24, Combo, RgpIII, HCV core, NS5, NS3, E1 / E2, HBV surface antigen and IgG- , Antigens for ACNcer diagnosis including PSA, CA1-1, glypeACN, and enzymes for measuring antibodies and activity (enzymes used for toxicity analysis, environmental analysis, food bacteria analysis, etc.). In addition, low molecular substances used in the development of new drugs and natural products such as native plants, microorganisms, fungi, and medicinal plants are also included in the immobilization. The target material includes all of the detectable substances using the immunoassay according to the present invention, and examples thereof include, but are not limited to, antigens, nucleic acids, metal ions, and glucose.

Immunoassays using the disulfide-terminated aryldiazonium (DSAD) salt compound of the present invention as a linker for immobilizing a biomolecule can be largely carried out in the following two ways.

First, the above-mentioned disulfide-terminated aryldiazonium (DSAD) salt compound is applied in a spatially-selective manner for electrodeposition, cathodic deposition and activation on a patterned microelectrode array Based sandwich immunoassay through the treatment of the cells. This approach can be applied specifically for the detection of, for example, Rabbit antigens.

Specifically,

(1) electrodepositing the DSAD salt compound onto an indium tin oxide (ITO) microelectrode array through a cyclic voltammetry (CV);

(2) electrochemically oxidizing the DSAD salt compound electrodeposited on some ITO microelectrodes to convert the disulfide group on the surface to a thiosulfinate group (R ¹ -S-SO-R ² ) or a thiosulfonate group (R ¹ -S- SO _{2 -} R ² );

(3) spatially-selectively immobilizing the biomolecule only in the activated portion by exposing the biomolecule having the thiol group (primary antibody) (for example, Anti-Rabbit antigen);

(4) exposing and immobilizing the target material (target antigen) (e.g., Rabbit antigen);

(5) exposing and immobilizing a secondary antibody (for example, Anti-Rabbit antigen) labeled with TRITC (Tetramethylrhodamine isothiocyanate);

(6) observing the surface with a fluorescence microscope;

The target substance can be fluorescence-detected (see FIG. 2).

Second, as an electrochemical immunoassay, the disulfide-terminated aryldiazonium (DSAD) salt compound is reacted with reduced graphene oxide (RGO) present on the electrode, for example, electrochemically reduced (Electrochemically grafting) electrochemically reduced graphene oxide (ERGO) to maintain and enhance the electrochemical catalytic activity by mutual bonding. This approach can be applied specifically for electrochemical detection of, for example, mouse antigens.

Specifically,

(1) An ITO electrode washed with a Piranha solution was washed with water again to form a negative charge on the electrode surface, and the positive electrode was charged with a positive charge of polyethylenimine (PEI) (ERGO / PEI / ITO) by electrochemically reducing GO by forming a graphene oxide (GO) through electrostatic interaction on the obtained surface after pretreatment with an aqueous solution. ;

(2) electrodepositing the DSAD salt compound onto ERGO to obtain a DSAD functionalized ERGO surface (DSAD / ERGO / PEI / ITO);

(3) The DSAD salt compound of DSAD / ERGO / PEI / ITO is electrochemically oxidized to convert the disulfide group on the surface to a thiosulfinate group (R ¹ -S-SO-R ² ) or a thiosulfonate group (R ¹ -S- SO ₂ -R ² ) and activating (ODSAD / ERGO / PEI / ITO surface) to produce an electrochemical immunosensor;

(4) exposing and fixing a primary antibody (e.g., Anti-Mouse antigen) having a thiol group on the ODSAD / ERGO / PEI / ITO surface;

(5) exposing and immobilizing a target antigen (e. G., Mouse antigen);

(6) exposing and immobilizing a secondary antibody labeled with HRP (e.g., Anti-Mouse antigen);

(7) Exposure of an electrolyte (for example, hydrogen peroxide and hydroquinone solution) causes a chemical catalytic reaction, and then the amount of a substance (for example, benzoquinone) generated through electrochemical reaction is detected by using a cyclic voltammetry Expelling step;

The target antigen can be electrochemically detected by a sandwich ELISA method (see FIG. 3).

In this method, the aryl radical of the disulfide-terminated aryldiazonium (DSAD) salt compound during the electrodeposition process forms a covalent bond with the sp ² carbon atom of RGO. In addition, in the presence of hydrogen peroxide (H ₂ O ₂ ), HRP (Horseradish peroxidase) catalyzes the conversion of hydroquinone (HQ) into benzoquinone (BQ) resulting in a signal current proportional to the amount of target antigen . ELISA is a sensitive diagnostic tool for antigen detection and a technique used for quality control in various industries. An electric signal is generated by an enzyme reaction product linked to a detection reagent, and accurate measurement is possible. And depends on the amplification of such electrical signals. The biosensor platform using the disulfide-terminated aryldiazonium salt (DSAD) salt compound according to the present invention showed excellent sensitivity and specificity to the target substance, particularly mouse antigen, when applied to the electrochemical ELISA technique.

The disulfide-terminated aryldiazonium (DSAD) salt compound provided by the present invention can be prepared by allowing the aryldiazonium moiety to react with various solid surfaces (for example, carbon-based materials such as graphene, graphite and diamond; gold; platinum; , It can be used as a highly useful linker molecule for the modification of various functional nanomaterial surfaces through covalent immobilization.

Hereinafter, the present invention will be described more specifically with reference to Production Examples, Examples and Experimental Examples. However, these production examples, examples and experimental examples are intended to assist the understanding of the present invention, and the scope of the present invention is not limited thereto in any sense.

Manufacturing example : Disulfide -end Aryldiazonium ( DSAD ) Synthesis of salt compound

(1) reagents and devices

(LA), N-Boc-1,4-phenylenediamine, triethylamine (TEA), hydroxybenzotriazole (HOBt), EDCl, tetrabutylammonium perchlorate (TBAP) ) phosphine (TCEP), the graphite powder (<20μm), sulfuric acid (H ₂ SO _4), hydrogen peroxide (H ₂ O _2), hydrochloric acid (HCl), sodium nitrite (NaNO _2), sodium chloride (NaCl), Rabbit antigen, Mouse antigen, Anti-Rabbit antigen, Anti-Mouse antigen, Anti-Rabbit antigen TRITC and Anti-Mouse antigen HRP were obtained from Sigma Aldrich.

The phosphate buffered saline (PBS) solution was composed of 0.01 M phosphate and 0.15 M NaCl, and PBST was composed of PBS and 0.05% (w / w) Tween-20.

All chemicals were analytical grade and were used without further purification.

All buffers and aqueous solutions were prepared with 18.2 MΩ pure water.

Fluorescence imaging, cyclic voltammetry (CV) and X-ray photoelectron spectroscopy (XPS) were performed using the same instrument as reported previously in Haque et al., 2012 Respectively.

(2) Rat -Butyl 4- (5- (1,2- Dithian -3 days) Pentanamide ) Phenylcarbamate  Synthesis: (1a)

N-Boc-1,4-phenylenediamine, triethylamine (TEA), hydroxybenzotriazole (HOBt), EDCl and additional triethylamine (TEA) were successively added to the liposic acid solution dissolved in dichloromethane .

The reaction mixture was then stirred at 25 &lt; 0 &gt; C overnight and diluted with water.

The product was extracted with dichloromethane (DCM), dried over magnesium sulfate, filtered and concentrated under reduced pressure.

The resulting reddish brown powder was suspended in diethyl ether, followed by filtration and rinsing with ether to give a pale pink powder (yield: 80.5%).

1 H-NMR (CDCl ₃ , 400 MHz):? Ppm = 1.25 (s, 1H, CH ₂ ); 1.91 (s, 9 H, BOC); 1.61 (s, 1 H, CH ₂ ); _{1.68-1.78 (m, 4H, CH 2} ); 1.91-1.94 (t, 1H, -S- CH 2); _{2.32-2.36 (t, 2H, CH 2} ); 2.43-12.48 (q, 1H, -S- CH 2); _{3.10-3.18 (m, 2H, CH 2} ), 3.56-3.59 (t, 1H, -S-CH-); 6.46 (s, 1H, CONH); 7.18 (s, 1H, CONH-BOC); 7.26-7.31 (d, 2H, arom); 7.41-7.43 (d, 2H, arom.).

(3) N- (4- Aminophenyl ) -5- (1,2- Dithian -3 days) Pentanamide  Synthesis: (2a)

Compound 1a (210.97 mg, 0.532 mM) was dissolved in 6.666 mL dry DCM: TFA (10: 1) mixture, stirred overnight at room temperature, then washed with water and extracted with DCM.

The resulting solution was dried over magnesium sulfate and concentrated under reduced pressure.

Purification was then performed on a silica-gel column (mobile phase 5% methanol in DCM) to give a beige solid (21% yield).

1 H-NMR (CDCl ₃ , 400 MHz):? Ppm = 0.83-0.85 (d, 1H, CH ₂ ); _{1.23-1.26 (d, 1H, CH 2} ); _{1.36-1.40 (t, 2H, CH 2} ); _{1.56-1.59 (t, 2H, CH 2} ); _{1.68-1.70 (m, 1H, CH 2} ); 1.84-1.89 (m, 1 H, CH ₂ ); 2.20-2.23 (t, 1 H, CH ₂ ); 2.39 - 2.45 (m, 1H, CH ₂ ); _{3.09-3.21 (m, 2H, CH 2} ); 3.59-3.66 (m, 1H, -S-CH-); 4.87 (s, 2H, NH ₂ ); 6.46-6.49 (d, 2H, arom); 7.19-7.21 (d, 2H, arom); 9.45 (s, 1H, CONH).

(4) 5- (1,2- Dithian -3 days) Pentanamide Benzene Diazonium  Synthesis of chloride

Compound 2a (0.889 mg) was dissolved in 1.9 mL of 0.1 M HCl and cooled in an ice bath to prepare 2 mL of a 1.5 mM solution.

Then, 100 μL of cold NaNO ₂ (0.23 mg in 0.1 mL H ₂ O) was added dropwise with stirring.

Stirring was continued for a further 30 minutes to finally obtain a solution of the disulfide-terminated aryldiazonium (DSAD) salt compound, which was used in the electrodeposition process described below.

Example  1: Micro electrode Above the array  Antibody immobilization and sandwich immunoassay

The ITO microelectrode array was washed with Piranha solution (H ₂ SO ₄ : H ₂ O ₂ = 3: 1), then washed again with a large amount of water, and N ₂ And dried with a gas.

Subsequently, the DSAD prepared in the above Preparation Example was electrodeposited on four microelectrodes, and then only one microelectrode was electrochemically oxidized to produce a thiosulfinate group or thiosulfonate group.

The array surface was exposed to cleaved anti-rabbit antigen fragments in PBST for 2 hours, then washed with PBST and water and incubated with N ₂ And dried with a gas.

The array surface was then treated with 50 μL of Rabbit antigen solution (5 μg / mL Rabbit antigen in PBST) for 1 hour, then washed with PBST and water and incubated with N ₂ And dried with a gas.

The surface of the array was exposed to 50 μL of anti-rabbit antigen (20 μg / mL Anti-Rabbit antigen in PBST) labeled with TRITC for 1 hour, and then the surface was observed with a fluorescence microscope.

Example  2: Sandwich ELISA

(One) ODSAD / ERGO / PEI / ITO  Surface preparation

As reported previously in Khan et al., 2014, GO was synthesized and characterized.

The ITO electrode was cut to a size of 2.5 占 cm ² , washed with a pyran solution and water, and dried with N ₂ gas.

Then, an ITO electrode was washed in 25 ℃ exposure for 1 hour in 1% PEI solution 100μL Next, the surface with water, and N ₂ And dried with a gas.

The modified surface was treated with 0.2 mg / mL GO for 3 hours, then washed with water and dried with N ₂ gas.

The resulting GO / PEI / ITO surface was then electrochemically reduced using 0.5 M NaCl.

DSAD was electrodeposited on the ERGO modified surface thus obtained.

Then, oxidized DSAD / ERGO / PEI / ITO (= ODSAD / ERGO / PEI / ITO) surface was obtained by electrochemically oxidizing DSAD / ERGO / PEI / ITO with 0.1M TBAP (95% ACN).

(2) manufacture of immunosensor electrodes and ELISA  Perform

ODSAD / ERGO / PEI / ITO surface-Anti-Mouse antigen cleaves over (100μg / mL) were washed and then added dropwise to 100μL immobilized with Anti-Mouse antigen, incubated for 2 hours, and then the bait, PBST and water and N ₂ And dried with a gas.

Then, the processes for the surface with 1% bovine serum albumin (BSA) solution in PBS 1 time block the non-specific binding sites on the surface, and then washed with PBS and the modified surface with water, and N ₂ And dried with a gas.

It was exposed for 1 hour in PBST Mouse antigen 100μL each having a different density on the surface, and then, washed with water, PBST, and N ₂ And dried with a gas.

Next, 100 μL of Anti-Mouse antigen HRP (20 μg / mL in PBST) was exposed on the modified surface and incubated for 1 hour, then the surface was washed with PBST and water, and N ₂ And dried with a gas.

For comparative experiments, we used negative control instead of mouse antigen or using Rabbit antigen as a negative control.

All of the above procedures were performed at 25 &lt; 0 &gt; C.

(3) In cyclic voltammetry  Of antigens

The active area of the working electrode was 0.45 cm ² .

Enzyme detection was attempted by keeping 1.5 mM H ₂ O ₂ and 2.0 mM hydroquinone (HQ) in 0.1 M PBS in the cell for 5 minutes at room temperature without touched.

As a result, an electrochemical reduction current on the reduction of benzoquinone (BQ) was obtained, and three measurements were performed for each mouse antigen concentration.

Experimental Example : Review results

(One) DSAD Space-selective immobilization

As shown in Figure 2, DSAD was grafted onto four different ITO microelectrodes, after which only electrode 1 was electrochemically oxidized.

Then, the purified anti-rabbit antigen was exposed to the four electrodes, and the anti-rabbit antigens labeled with Rabbit antigen and TRITC were sequentially exposed. The final surface was irradiated with a fluorescence microscope to detect the rabbit antigen.

As a result of electrodeposition of DSAD cations on the ITO microelectrode through cyclic voltammetry (CV), a large irreversible reduction current was observed at -0.35 V during the first cycle and a significant reduction in current in the second cycle 4 (A)). That is, it can be seen that electrodeposition on the ITO electrode was very effective.

Electrochemical oxidation of the ITO electrode modified with DSAD resulted in the electrochemical oxidation of DSAD producing two successive irreversible waves at 0.85 V and 1.2 V during the first cycle and disappeared in the following cycles (B)). These two waves are due to the two steps involved in the oxidation of the disulfide (see Gourcy et al., 1981).

In addition, X- ray photoelectron spectroscopy, a surface; After a review by (X-ray photoelectron spectroscopy XPS) , Sulfur 2p XPS is S 2p _3/2 of the sulfide group, which bar, jinyeotneun two peaks at the binding energy and 163.5 respectively 164.5eV and it is corresponding to the S 2p _1/2 (see Fig. 4 (C)). On the other hand, no peak was observed in the untreated electrode.

Since electrochemical oxidation, S 2p XPS spectrum is non-match was it to indicate the oxidized S 2p _{1/2, 3/2} of the strength reduction bar, which increase of the oxidized S. The double peak at 167.5 and 168.5 eV corresponds to the sulfonate (SO ₂ ) in the thiosulfonate group and the additional peak around 166.5 eV corresponds to the sulfinate (SO) in the thiosulfinate group ) Reference). Thiosulfinate can be seen to be generated from a 2e loss, and thiosulfonate can be seen to be generated from a 4e loss (Terashima et al., 2003). Both functional groups are produced on the electrode surface.

(2) Rabbit antigen Of fluorescence-based detection

The thiol reactivity of the oxidized surface was examined by immobilization of the prepared thiol group-containing antibody according to antibody cleavage using TCEP.

After electrochemical oxidation of electrode 1, a solution containing the cleaved anti-rabbit antigen (c-Ab-R) was exposed to the surface of the array and a solution of Rabbit antigen (Ag-R) Followed by exposure of the anti-rabbit antigen solution labeled with TRITC as a secondary antibody.

The surface obtained by washing and drying was examined with a fluorescence microscope. As a result, red color was observed only on the oxidized surface as shown in FIG. 4 (E). That is, this indicates that the detection of the protein on the high-density microelectrode array has been successful.

(3) DSAD Using ERGO Functionalization of

FIG. 3 shows a process of functionalizing ERGO-modified ITO with DSAD and a method of using the obtained surface as a platform for detecting mouse antigen.

An additional PEI layer of positive (+) charge was formed on the clean ITO, and the GO was electrochemically reduced by electrostatic interactions thereon, and then ERGO was obtained.

As a result of the experiment of scanning the potential from 0.0 to -1.0 V, a remarkable reduction current was observed around -0.8 V during the first cycle, and the current was greatly reduced in the remaining cycles (see FIG. 5 (A)). This cyclic voltammetric behavior is similar to the reported reduction of GO sheet on ITO, which means that the GO has been successfully reduced (Haque et al., 2012) and will refer to this surface as ERGO / PEI / ITO .

DSAD was then electrodeposited on ERGO / PEI / ITO to form DSAD / ERGO / PEI / ITO.

The CV curve for electrodeposition of ERGO phase DSAD is shown in FIG. 5 (B). A high reduction current was observed around -0.3 V during the first cycle, and no peak was observed in the second and third cycles. This means that the DSAD has been electrodeposited through the first cycle.

5C shows the CV curves for the electrochemical oxidation (ODSAD / ERGO / PEI / ITO) of the ERGO surface modified with DSAD. The disulfide group on the ERGO was a thiosulfinate group or a thiosulfonate group It can be seen that it is oxidized.

In addition, the CV curves of Fe (CN) ₆ ³ -redox probes for the ITO electrodes modified to be different from each other in FIG. 5 (D) show the shapes of untreated ITO and ERGO / PEI / ITO And DSG modified ERGO showed high blocking properties for Fe (CN) ₆ ^3- ion. The oxidized surface (ODSAD / ERGO / PEI / ITO) also exhibited high blocking properties for the Fe (CN) ₆ ^3- ions, indicating that the diazonium layer was not only very stable, It means sound.

(4) DSAD Functionalized ERGO  Electrochemical catalytic activity of surface

The electrochemical catalytic activity for the BQ reduction of the ITO electrodes, which were modified to be different from each other, was investigated through CV and is shown in Fig. 6 (A).

As a result, ITO modified with ERGO showed much higher electrochemical catalytic activity than ITO untreated for the reduction of BQ. The high electrochemical catalytic activity of these ERGO surfaces is due to ERGO's large surface area, high electrical conductivity, and fast hetero-electron transport capability (Khan et al., 2013).

The ERGO surface functionalized with DSAD blocks the electrochemical reduction of BQ, but electrochemical oxidation of such surface reverts to high electrochemical catalytic activity.

(5) Electrochemical performance of immunoassay

Sandwich ELISA for detection of mouse antigen was performed on the ODSAD / ERGO / PEI / ITO surface according to Example 2 using a secondary antibody labeled with HRP. Here, the immune sensor measures HQ and H ₂ O ₂ Lt; / RTI &gt; The HRP molecule is H ₂ O ₂ , The resulting BQ is electrochemically reduced on the ODSAD / ERGO / PEI / ITO surface to generate a high signal current.

Figure shows the CV response of the immune sensor in PBS buffer solution containing the HQ and H ₂ O ₂ in the 6 (B).

As a result, a high reduction peak current was obtained for mouse antigen (1 μg / mL), which means that the immobilized HRP molecule exerts excellent catalytic activity through the immune response at the surface.

On the other hand, in the case of the antigen-free control experiment, only a very small reduction peak current was observed on the CV, which is the result of non-specific binding of proteins and electrochemical reduction of H ₂ O ₂ . The control experiment using the Rabbit antigen was also similar to the control experiment without the antigen.

These results confirm that the immunosensor according to the present invention is very specific.

FIG. 6 (C) shows the CV response to the electrode surface under different mouse antigen concentrations from 1 ng / mL to 1 μg / mL.

As a result, it can be seen that the reduction peak current of BQ increases as the concentration of mouse antigen increases.

FIG. 6 (D) shows the background separation reduction peak current vs. background reduction peak current. Calibration plot is shown as log value of mouse antigen concentration.

As a result, the background separation reduction peak current vs. A linear relationship was observed between the logarithm of the mouse antigen concentration and the detection limit for mouse antigen was 1 ng / mL.

Claims (12)

Immunoassay using a disulfide-confined aryldiazonium (DSAD) salt compound represented by the following formula 1 as a linker for immobilizing a biomolecule:
[Chemical Formula 1]

In Formula 1,
R is a hydrogen atom or an alkyl group having 1 to 4 carbon atoms,
Ar is an aryl group having 6 to 30 carbon atoms,
X is a halogen atom.
The method according to claim 1,
Wherein the disulfide-terminated aryldiazonium (DSAD) salt compound is represented by the following formula (2): &lt; EMI ID =
(2)

.
(1) Electrodeposition of the disulfide-terminated aryldiazonium (DSAD) salt compound of claim 1 or 2 onto a microelectrode array;
(2) electrochemically oxidizing the disulfide-terminated aryldiazonium (DSAD) salt compound electrodeposited on some microelectrodes to convert the disulfide groups on the surface to thiosulfinate groups or thiosulfonate groups and activate them;
(3) exposing a primary antibody having a thiol group to the microelectrode array to space-selectively immobilize the primary antibody only on the activated portion;
(4) exposing and immobilizing the target antigen;
(5) exposing and immobilizing the secondary antibody; And
(6) detecting fluorescence on the surface of the microelectrode array;
0.0 &gt; (Sandwich &lt; / RTI &gt; immunoassay).
The method of claim 3,
Wherein the microelectrode array is an indium tin oxide (ITO) microelectrode array.
The method of claim 3,
Wherein the electrodeposition is performed by cyclic voltammetry (CV).
The method of claim 3,
Wherein the primary antibody is Anti-Rabbit antigen, the target antigen is Rabbit antigen, and the secondary antibody is Anti-Rabbit antigen.
The method of claim 3,
Wherein said secondary antibody is labeled with TRITC (Tetramethylrhodamine isothiocyanate).
(1) preparing an ITO electrode surface (ERGO / PEI / ITO) sequentially modified with PEI (Polyethylenimine) and electrochemically reduced graphene oxide (ERGO);
(DSAD / ERGO / PEI / ITO) functionalized with DSAD by electrodeposition of the disulfide-terminated aryldiazonium (DSAD) salt compound of the item 1 or 2 onto the ERGO surface of the ITO electrode surface, ;
(3) The surface of the disulfide-terminated aryldiazonium (DSAD) salt compound of the surface of ERGO functionalized with DSAD is electrochemically oxidized to convert the disulfide group on the surface to a thiosulfinate group or thiosulfonate group (ODSAD / ERGO / PEI / ITO) &lt; / RTI &gt;
(4) exposing and immobilizing a primary antibody having a thiol group on the activated surface;
(5) exposing and immobilizing the target antigen;
(6) exposing and immobilizing the secondary antibody; And
(7) a step of exposing the electrolyte to cause a chemical catalytic reaction, and then detecting the amount of the substance produced through electrochemical reaction using cyclic voltammetry (CV);
Lt; RTI ID = 0.0 &gt; ELISA. &Lt; / RTI &gt;
9. The method of claim 8,
The step (1)
The ITO electrode washed with Piranha solution was again washed with water to form a negative charge on the surface of the electrode and the positive electrode was pretreated with a positive charge of PEI, Wherein a graphene oxide (GO) is formed on the obtained surface, and then the GO is electrochemically reduced.
9. The method of claim 8,
Wherein the primary antibody is Anti-Mouse antigen, the target antigen is mouse antigen, and the secondary antibody is Anti-Mouse antigen.
9. The method of claim 8,
Wherein said secondary antibody is labeled with HRP (Horseradish peroxidase).
9. The method of claim 8,
The step (7)
The method comprising: exposing a solution of hydrogen peroxide and hydroquinone as an electrolyte to an electrode of an electrochemical immunosensor to cause a chemical catalytic reaction, and then detecting the amount of benzoquinone produced by cyclic voltammetry.

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