Novel electrochemiluminescent co-reactants and electrochemiluminescent systems containing the same

Abstract

The present invention provides novel electrochemiluminescent coreactants and electrochemiluminescent systems containing them. [Solution] When measuring electrochemiluminescence using a specific 4-nitrogen-substituted pyridine derivative with a specific luminescent chemical species (such as a polycyclic aromatic hydrocarbon compound, a metal complex compound, a quantum dot, or nanoparticles) as an electrochemiluminescence label, it exhibits an excellent detection signal, allows for improvement of voltage application conditions, and can be widely applied to immunoassay and related diagnostic equipment, as well as chemical analysis and related general diagnostic equipment.

Description

[Technical Field]

[0001] The present invention relates to a novel electrochemiluminescence co-reactant and an electrochemiluminescence system containing the same. When an electrochemiluminescence (ECL) co-reactant containing the compound represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof, is measured using a specific luminescent chemical species (such as a polycyclic aromatic hydrocarbon compound, a metal complex compound, a quantum dot, or a nanoparticle) as an electrochemiluminescence label, it exhibits an excellent detection signal, allows for improvement of voltage application conditions, and can be widely applied to immunoassay and related diagnostic equipment, as well as chemical analysis and related general diagnostic equipment.

[0002] [Chemical formula 1] TIFF2026136148000002.tif7141

[0003] In the above chemical formula 1, the R 1 or R 2 Each of these elements may be the same or different, and represents one selected from the group consisting of a hydrogen atom; a halogen atom; a C1-C6 linear, branched, or cyclic alkyl group; a C1-C6 alkoxy group; and a C1-C6 haloalkyl group. [Background technology]

[0004] Research continues to advance for rapid, highly specific, sensitive, and accurate methods for detecting and quantifying chemical, biochemical, and biological substances. Improving analytical performance, such as sensitivity, is crucial because typical biological samples contain very small amounts of specific analytes.

[0005] One approach to improving analytical sensitivity is to utilize methods that can be used for highly sensitive photodetection (e.g., photomultiplier tubes). In this regard, the use of luminescent indicator molecules is considered a very important element. For example, the presence of an analyte (or its binding partner) can be quantitatively detected using a luminescent label associated with the analyte (or its binding factor).

[0006] The amount of analyte can be quantitatively determined when the analyte participates in a reaction that induces the modulation of luminescence, as described below. Specifically, i) the analyte can react with other species to modulate the luminescence properties of the second species, ii) the analyte can undergo chemical modifications that modulate its own luminescence properties, iii) the analyte may be a catalyst (e.g., an enzyme) that induces the reaction of other species, and iv) the analyte can participate in a reaction that produces a species and then participate in a subsequent reaction that induces the modulation of luminescence.

[0007] Methods for detecting luminescent indicator molecules include photoluminescence, chemiluminescence, and electrochemiluminescence (ECL).

[0008] Among various photodetection systems, electrochemiluminescence (ECL) systems do not require large, expensive light sources, making it possible to construct low-cost, compact diagnostic systems, and they can minimize signal interference with background signals from other interfering elements.

[0009] More specifically, electrochemiluminescence (ECL) is an electrically-induced photogenerating phenomenon discovered around 1960. This phenomenon involves i) inducing an oxidation reaction of a specific luminescent substance through the application of voltage, ii) then transforming the resulting intermediate reactant into an excited final product through a secondary chemical reaction, and iii) the excited state being converted back to the ground state to generate light. Such ECL is used as a detection method in high-end medical immunodiagnostic equipment.

[0010] In terms of applications, the only immunodiagnostic devices based on electrochemiluminescence (ECL) are the COBAS® diagnostic devices and the Mesoscale Discovery ECL Systems series from Roche, which utilize the Elecsys method and monopolize the global high-end immunodiagnostic device market. These methods utilize the ruthenium compound [Ru(bpy)3] as the luminescent material. 2+ ECL is generated using tripropylamine (TPA), a co-reactant with [a specific component]. However, the problem is that tripropylamine is hydrophobic, resulting in poor luminescence efficiency under platinum and gold electrode conditions. Furthermore, in the 60-year history of electrochemiluminescence, no co-reactant has yet been found that exhibits superior luminescence efficiency to tripropylamine.

[0011] Specifically, tripropylamine (TPrA) is commonly used as a co-reactant because it allows efficient electrochemiluminescence (ECL) not only in organic media but also in aqueous media and at physiological pH 7.4. However, TPrA has volatile toxicity and has the disadvantage that it must be used at a high concentration (generally up to 100 mM) to obtain a high electrochemiluminescence (ECL) signal. In addition, since TPrA has a slow electrochemical oxidation rate, it limits the electrochemiluminescence (ECL) efficiency, and since it is basic, there is the complication that a high-concentration buffer solution is required to produce the solution. Also, there is a relatively large variation between the same detection signals and it has the disadvantage of chemically reacting with carbon dioxide in the air.

[0012] In addition, the electrochemiluminescence (ECL) efficiency of ruthenium pyridine [Ru(bpy)3 2+ and tripropylamine (TPrA) depends on the electrode materials. In the potential region where electrochemiluminescence occurs, platinum (Pt) electrodes and gold (Au) electrodes are covered with a positive oxide layer, which has been shown to suppress the direct oxidation of tripropylamine, and the electrochemiluminescence intensity is calculated to be even lower. On the other hand, polished glassy carbon (GC) electrodes have a relatively fast electrochemical oxidation rate of tripropylamine, which increases the amount of tripropylamine oxidized on the electrode surface and results in a particularly high emission intensity.

[0013] Under such circumstances, the inventors attempted to develop a new electrochemiluminescent co-reactant that can improve the emission intensity and detection reproducibility of an electrochemiluminescence (ECL) system.

Summary of the Invention

Problems to be Solved by the Invention

[0014] The inventors of the present invention have been developing a novel co-reactant that has a fast reaction rate, is less affected by electrode materials, and has excellent luminescence efficiency, in relation to electrochemiluminescence (ECL) that can be widely applied to blood glucose, cholesterol sensors, molecular diagnosis, or antibody detection via immunoassay, etc. Instead of conventional tripropylamine that has been mainly used as a co-reactant. In the case of the pyridine derivative represented by Chemical Formula 1, they found that it has excellent luminescence intensity in an electrochemiluminescence system using ruthenium pyridine as a label, and thus completed the present invention.

[0015] Therefore, an object of the present invention is to provide an electrochemiluminescence (ECL) co-reactant containing a compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0016] Another object of the present invention is to provide an electrochemiluminescence system that excites the co-reactant and an electrochemiluminescence label.

[0017] Another object of the present invention is to provide a detection method using the electrochemiluminescence system.

[0018] Yet another object of the present invention is to provide a kit for electrochemiluminescence immunoassay or molecular diagnosis containing the co-reactant.

Means for Solving the Problems

[0019] To achieve the above object, the present invention provides an electrochemiluminescence (ECL) co-reactant containing a compound represented by the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[0020] [Chemical Formula 1] TIFF2026136148000003.tif7141

[0021] In the above Chemical Formula 1, the R 1 or R 2Each of these elements may be the same or different, and represents one selected from the group consisting of a hydrogen atom; a halogen atom; a C1-C6 linear, branched, or cyclic alkyl group; a C1-C6 alkoxy group; and a C1-C6 haloalkyl group.

[0022] Furthermore, the present invention provides an electrochemiluminescence system comprising an electrochemical cell filled with an electrolyte solution containing an electrochemiluminescence (ECL) coreactant containing the compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof, and an electrochemiluminescence label; and a photodetector connected to the electrochemical cell.

[0023] Furthermore, the present invention provides a method for detecting an electrochemiluminescence system, comprising the steps of (a) placing a sample into an electrochemiluminescence (ECL) system including an electrochemiluminescence coreactant containing the compound represented by chemical formula 1 or a pharmaceutically acceptable salt thereof, and an electrochemiluminescence label, and allowing the system to react; and (b) measuring the electrochemiluminescence intensity (ECL intensity) of the reaction sample from step (a) using an electrochemiluminescence-based detector based on an input potential (or voltage), and detecting an optical signal.

[0024] Furthermore, the present invention provides a kit for electrochemiluminescence immunoassay or molecular diagnostics comprising the aforementioned co-reactants; and electrochemiluminescence labeling; [Effects of the Invention]

[0025] The pyridine derivative according to the present invention can be rapidly and accurately detected by electrochemiluminescence signals, and can replace the conventional co-reactant tripropylamine (TPrA).

[0026] In detail, it offers excellent handling characteristics as a solid compound, allows for improvement of potential (or voltage) conditions for luminescence, improves luminescence efficiency even at low concentrations, and provides broad potential applications in various bioanalyses such as immunoassay. [Brief explanation of the drawing]

[0027] [Figure 1] Figure 1 is a schematic diagram of an electrochemiluminescence system including a potentiostat and a photomultiplier tube, as an embodiment of the present invention. [Figure 2] Figure 2 shows the results of measuring the cyclic voltammogram (CV) in an electrochemiluminescence system containing a 5 mM co-reactant and 1 μM [Ru(bpy)3]2 + ruthenium pyridine in 1XPBS (pH 7.4) (Scan rate: 0.1 V / s, WE: GC, CE: Pt, RE: Ag / AgCl). [Figure 3] Figure 3 shows the results of measuring ECL intensity in an electrochemiluminescence system containing a 5 mM co-reactant and 1 μM [Ru(bpy)3]2 + ruthenium pyridine in 1XPBS (pH 7.4) (Scan rate: 0.1 V / s, WE: GC, CE: Pt, RE: Ag / AgCl). [Figure 4] Figure 4 shows the results of measuring the cyclic voltammogram (CV) in an electrochemiluminescence system containing 5 mM co-reactant and 1 μM [Ru(bpy)3]2+ in 1XPBS (pH 7.4) (Scan rate: 0.1 V / s, WE: Pt, CE: Pt, RE: Ag / AgCl). [Figure 5] Figure 5 shows the results of measuring ECL intensity in an electrochemiluminescence system containing 5 mM co-reactant and 1 μM [Ru(bpy)3]2+ in 1XPBS (pH 7.4) (Scan rate: 0.1 V / s, WE: Pt, CE: Pt, RE: Ag / AgCl). [Figure 6] Figure 6 is a plot showing the dependence of the ECL intensity of 1 μM [Ru(bpy)3]2+ ruthenium pyridine on the concentrations of 4-DMAP and TPrA (1–100 mM) in 1XPBS (pH 7.4). The potential was increased stepwise from 0 V to 1.6 V (WE: Pt, CE: Pt, RE: Ag / AgCl). [Figure 7]Figure 7 is a plot showing the dependence of the ECL intensity of 10 μM [Ru(bpy)3]2+ ruthenium pyridine on the concentrations (1–100 mM) of 4-DMAP and TPrA in 1XPBS (pH 7.4). The potential was increased stepwise from 0 V to 1.6 V. (WE: Pt, CE: Pt, RE: Ag / AgCl). [Figure 8] Figure 8 shows the results of measuring ECL intensity under various pH conditions using [Ru(bpy)3]2+ ruthenium pyridine (1 μM and 10 μM) (WE: Pt, CE: Pt, RE: Ag / AgCl). [Figure 9] Figure 9 is a graph comparing the electrochemiluminescence intensity when 4-DMAP and TPrA are used as co-reactants in an acetonitrile (ACN) solution, and glassy carbon (A), platinum (B), and gold (C) are used as working electrodes (CE: Pt, RE: Ag / AgCl). [Figure 10] Figure 10 shows the linear sweep potential curve and electrochemiluminescence intensity when 4-DMAP is used as a co-reactant in an acetonitrile (ACN) solution, with glassy carbon used as the working electrode (CE: Pt, RE: Ag / Ag+ (3M AgNO3), scan rate: 0.1 V / s). [Figure 11] Figure 11 shows the difference in electrochemiluminescence signals generated by changes in the concentration of Ru(bpy)3²⁺ luminescent material, obtained in the form of a calibration curve, when 7 mM 4-DMAP and 7 mM tripropylamine were used as co-reactants, respectively. [Figure 12] Figure 12 shows the results of electrochemiluminescence immunodiagnosis for "anti-SARS-CoV-2" antibodies present in saliva samples from 10 vaccinated individuals, using either 4-DMAP or tripropylamine as co-reactants, respectively. [Modes for carrying out the invention]

[0028] Electrochemiluminescence is a light-emitting process in which compounds generated at electrodes undergo high-energy electron transfer reactions to produce light in an excited state. Luminescent labeling reagents used in electrochemiluminescence include transition metal complex compounds, luminescent organic semiconductors, quantum dot materials, perovskite nanoparticles, metal nanoparticles, or carbon nanoparticles. Such organic and inorganic luminescent labeling has been widely applied in bioanalysis to date.

[0029] The oxidation reaction, the fundamental principle of electrochemiluminescence, involves the loss of electrons at the electrode surface during the reaction process by the luminescent substrate and composition. The electron donor becomes a strong reducing agent, reducing the luminescent substrate to an excited state by releasing hydrogen ions (H₂O₂). + The luminescent substrate loses its emission and subsequently emits photons to return to its ground state. This process is repeated on the electrode surface, and photons are generally emitted continuously to maintain a constant substrate concentration.

[0030] One example is the electrochemiluminescence system using ruthenium pyridine and tripropylamine. Electrochemiluminescence is a specific chemiluminescent reaction induced by electrochemistry at the surface of an electrode. An antigen-antibody complex and a ruthenium pyridine conjugate are electrochemically excited in the presence of tripropylamine, triggering a redox reaction that emits photons, which can be detected by a photomultiplier tube. This process is repeated to produce many photons, which amplifies the optical signal. Generally, labels used in electrochemiluminescence analysis can bind to antibody or antigen molecules with different chemical structures to produce labeled antibodies or antigens.

[0031] Currently available electrochemiluminescence methods primarily use tripropylamine (TPrA) as a co-reactant. The disadvantages of TPrA are that it is liquid, difficult to handle, has a slow reaction rate, requires high concentrations, is highly susceptible to electrode materials, has limited luminescence efficiency, and is toxic and unstable.

[0032] The inventors have confirmed that when the solid compound 4-dimethylaminopyridine (4-DMAP) is used as a co-reactant in electrochemiluminescence, it participates in the luminescence reaction very rapidly and exhibits excellent luminescence efficiency.

[0033] Accordingly, the present invention provides an electrochemiluminescent (ECL) co-reactant comprising a compound represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof.

[0034] [Chemical formula 1] TIFF2026136148000004.tif7141

[0035] In Chemical Formula 1 above, the R 1 or R 2 may each be the same or different and each represents any one selected from the group consisting of a hydrogen atom; a halogen atom; a C1-C6 linear or branched or cyclic alkyl group; a C1-C6 alkoxy group; and a C1-C6 haloalkyl group.

[0036] In Chemical Formula 1 above, the R 1 or R 2 may each be the same or different and each represents any one selected from the group consisting of a hydrogen atom; a halogen atom; a C1-C4 linear or branched or cyclic alkyl group; a C1-C4 alkoxy group; and a C1-C4 haloalkyl group.

[0037] Preferably, the R 1 or R 2 may each be the same or different and may be a C1-C4 linear or branched alkyl group; or a C1-C4 haloalkyl group.

[0038] More preferably, Chemical Formula 1 may be 4-dimethylaminopyridine.

[0039] The present invention also provides an electrochemical cell filled with an electrolyte solution containing the coreactant and the electrochemiluminescent label; and an electrochemiluminescent system including a photodetector connected to the electrochemical cell.

[0040] The coreactant is a compound having the structure of Chemical Formula 1 described above.

[0041] The electrochemiluminescent label may be one or more selected from the group consisting of transition metal complex compounds, luminescent organic semiconductors, quantum dot materials, perovskite nanoparticles, metal nanoparticles, and carbon nanoparticles, but is not necessarily limited to these.

[0042] More specifically, the transition metal compound can be one or more selected from the group consisting of ruthenium (Ru), iridium (Ir), rhenium (Re), platinum (Pt), osmium (Os), copper (Cu), and iron (Fe).

[0043] In detail, the ionic transition metal complex compounds are tris(2,2'-bipyridine)ruthenium(II)bis(hexafluorophosphate), Ru(bpy)3(PF6)2, tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II)bis(hexafluorophosphate), Ru(bpy)3(PF6)2, and tris(4,7-diphenyl-1,10-phenanthroline). [nanthroline)ruthenium(II)bis(hexafluorophosphate,Ru(dp-phen)3(PF6)2], bis(2-phenylpyridine)(2,2'-dipyridine)iridium(III)(hexafluorophosphate)[bis(2-phenylpyridine)(2,2'-dipyridine)iridium(III)(hexafluorophosphate,Ir(ppy)2(bpy)PF6], bis(2-phen Lupyridine)(4,4'-di-tert-butyl-2,2'-dipyridyl)iridium(III)(hexafluorophosphate)[bis(2-phenylpyridine)(4,4'-di-tert-butyl-2,2'-dipyridyl)iridium(III)(hexafluorophosphate),Ir(dtbbpy)(ppy)2PF6], 4'-di-tert-butyl-2,2'-dipyridyl-bis[2-(2',4'-difluorophenyl)pyridyl Iridium(III)(hexafluorophosphate){4'-di-tert-butyl-2,2'-dipyridyl-bis[2-(2',4'-difluorophenyl)pyridine]iridium(III)(hexafluorophosphate), Ir(ppy-F2)2(dtbbpy)PF6}, Iridium bis(5-(trifluoromethyl)-2-(4-trifluoromethyl)phenyl)pyridine)picolinateIt may include, but is not limited to, one or more species selected from the group consisting of Ir(ppy-(CF3)2)2(pico)}, iridium(III){Tris[2-(p-tolyl)pyridine]iridium(III), Ir(mppy)3}, 1,10-[phenanthroline]rhenium(I)(hexafluorophosphate), Re(phen)PF6], platinum(II) coproporphyrin, PtCP, and tris(2,2'-bipyridine)osmium(II)(hexafluorophosphate), Os(bpy)3(PF6)2.

[0044] In particular, the luminescent organic semiconductor may include luminescent conjugated organic semiconductors such as luminescent monomolecules or polymers. In particular, luminol and rubrene and their derivatives, anthracene and its derivatives, pyrene and its derivatives, dicyclooxyphenyl-substituted poly(1,4-phenylene vinylene), super It may contain, but is not limited to, one or more selected from the group consisting of [yellow], poly(2-methoxy-5-(2-ethylhexyloxy)-1,4-phenylenevinylene), MEH-PPV, poly(2-methoxy-5-(3',7'-dimethyloctyloxy)-1,4-phenylenevinylene), MEMO-PPV, and poly(9,9-dioctylfluorene-alt-benzothiadiazole), F8BT.

[0045] The quantum dot material may contain inorganic compounds of group 13-15 or group 12-15 elements. More specifically, the quantum dot material containing the inorganic compound may contain, but is not limited to, one or more elements selected from the group consisting of cadmium selenide (CdSe), cadmium sulfide (CdS), zinc selenide (ZnSe), indium phosphide (InP), lead sulfide (PbS), and lead selenide (PbSe).

[0046] The perovskite nanoparticles may include a halide perovskite. More specifically, the halide perovskite may be represented by the chemical formula ABX3, A2BX6, or A3B2X9. In this case, A may be an organic or inorganic cation, B may be a metal cation, and X may be a halide anion.

[0047] The metal nanoparticles may include metal atom clusters having dimensions of 1 nm or less that exhibit discontinuous energy levels. More specifically, they may include gold (Au) nanoparticles, silver (Ag), copper (Cu), or silver (Ag)-gold (Au) binary metal nanoparticles.

[0048] The carbon particles may include, but are not limited to, graphene quantum dots (GQDs) or carbon quantum dots (CQDs).

[0049] Furthermore, the electrolyte solution may include, but is not limited to, a liquid electrolyte containing salt, water, and an organic solvent; a solid electrolyte in which salt is dissolved in a polymer; a gel-like electrolyte containing a polymer, salt, water, and an organic solvent; or an ion gel electrolyte containing a block copolymer and an ionic liquid. The salt is an or inorganic ionic compound salt and may include, but is not limited to, one or more selected from the group of phosphates, nitrates, hydrochlorides, sulfates, lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, ammonium salts, etc.

[0050] More specifically, the electrolyte solution used in the electrochemiluminescence system of the present invention may contain, but is not limited to, a solution comprising one or more water or organic solvents selected from the group consisting of phosphate buffer saline (PBS), Tris buffer solution, acetonitrile (ACN), dichloromethane, ethanol, methanol (methanol), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethylene carbonate (EC), and propylene carbonate (PC).

[0051] The pH of the electrolyte solution may be 5 to 12, and preferably 7.4 to 10.

[0052] Figure 1 shows an example of an electrochemiluminescence system for measuring electrochemiluminescence according to the present invention, which includes an electrochemical cell containing an electrolyte solution, a potentiostat, and a photomultiplier tube (PMT). In this system, the photomultiplier tube (PMT) is connected to the potentiostat and driven simultaneously. Therefore, to measure electrochemiluminescence, an electrochemiluminescence reaction can be induced by using the potentiostat to induce a reaction in the electrochemical cell. To measure electrochemiluminescence, the potentiostat and electrochemiluminescence measurement software can be used to measure the emission intensity of the electrochemiluminescence.

[0053] The electrodes constituting the electrochemical cell may include, but are not limited to, a working electrode, a reference electrode, and a counter electrode.

[0054] The working electrode may be one or more electrodes selected from the group consisting of carbon, platinum (Pt), gold (Au), silver (Ag), nickel (Ni), stainless steel, palladium, tin, indium, and silicon, but is not necessarily limited to these.

[0055] The counter electrode may be one or more electrodes selected from the group consisting of elements carbon, platinum (Pt), gold (Au), silver (Ag), nickel (Ni), stainless steel, palladium, tin, indium, and silicon, but is not necessarily limited to these.

[0056] The reference electrode may be one or more selected from the group consisting of a silver (Ag)-based silver pseudo-reference electrode (Ag pseudo-reference), an Ag / AgCl electrode, an Ag / AgNO3 electrode, a mercury (Hg) calomel electrode, an Hg / HgO electrode, and an Hg2SO4 electrode, but is not necessarily limited to these.

[0057] Furthermore, the present invention provides a detection method for an electrochemiluminescence system, comprising the steps of (a) placing an electrolyte solution containing a sample into an electrochemical cell within the electrochemiluminescence system and allowing it to react; and (b) measuring the electrochemiluminescence intensity (ECL intensity) of the reaction sample from step (a) using an electrochemiluminescence-based detector based on an input voltage and detecting an optical signal.

[0058] Step (a) is a step in which the sample is placed in an electrolyte solution containing a co-reactant and ruthenium pyridine, which is an electrochemiluminescent label, and the sample is reacted, and the sample may be, but is not limited to, serum, urine, or tissue fluid.

[0059] Step (b) is a step in which the electrochemiluminescence intensity (ECL intensity) of the reaction sample from step (a) is measured using an electrochemiluminescence-based photodetector based on an input potential (or voltage). There are various types of photodetectors, which are equipment for measuring such luminescence. For example, the photodetector may be one or more selected from the group consisting of silicon, germanium, germanium-phosphide, indium-gallium-arsenide, and lead-sulfide based photodiodes; photomultiplier tubes (PMTs); charge-coupled devices (CCDs); electron-multiplying charge-coupled devices (EMCCDs); and scientifically complementary metal-oxide-semiconductors (sCMOS), but is not necessarily limited to these.

[0060] Furthermore, the present invention can provide an electrochemiluminescence immunoassay detection method using an electrochemical method. Accordingly, the present invention provides a kit for electrochemiluminescence immunoassay or molecular diagnostics comprising the aforementioned co-reactants and an electrochemiluminescence label.

[0061] More specifically, the electrochemiluminescence immunoassay or molecular diagnostic kit of the present invention may include an electrolyte solution, which may contain, but is not limited to, a solution containing one or more water or organic solvents selected from the group consisting of phosphate buffer saline (PBS), Tris buffer solution, acetonitrile (ACN), dichloromethane, ethanol, methanol (methanol), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethylene carbonate (EC), and propylene carbonate (PC).

[0062] The pH of the electrolyte solution may be 5 to 12, and preferably 7.4 to 10.

[0063] Furthermore, the kit may contain 4-dimethylaminopyridine at concentrations of greater than 0 mM and less than or equal to 20 mM, greater than 0 mM and less than or equal to 15 mM, greater than 0 mM and less than or equal to 10 mM, or greater than 0 mM and less than or equal to 7 mM.

[0064] As an embodiment of the present invention, electrochemiluminescence labels containing ruthenium compounds are used to label antigens or antibodies, and electrochemiluminescence immunoassays can be performed by immunoassays and ECL reactions. For a specific chemiluminescence reaction electrochemically induced at the electrode surface, the antibody (Ab) is labeled with ruthenium pyridine, which is an electrochemiluminescent reagent, and the carrier is coated with the antigen or antibody that has the corresponding antigen or antibody in the sample and forms a complex in a specific manner for immunoassays. The labeled complex is separated from the free labels by separation techniques. The antigen (Ag) or antibody (Ab) can be measured quantitatively or qualitatively by the emission intensity of ruthenium pyridine from the electrode. The pyridine derivative, which is a co-reactant, flows into an electrochemical cell, and a voltage may be applied to initiate the ECL reaction.

[0065] The electrochemiluminescence immunoassay or molecular diagnostic kit according to the present invention can significantly reduce the amount of antigen and antibody when applied to diagnostic equipment. The ECL emission intensity from the electrochemical reaction between the pyridine derivative and ruthenium pyridine according to the present invention is more than 20 times better than when using tripropylamine, and detection is possible even with very small amounts of antigen and antibody, thus efficiently reducing the amount of antibody. Furthermore, the price of the kit or analytical equipment is relatively lower.

[0066] Furthermore, the electrochemiluminescence immunoassay or molecular diagnostic kit according to the present invention can achieve a better detection signal and superior detection sensitivity for the analyte compared to using tripropylamine.

[0067] As described above, the pyridine derivative represented by chemical formula 1 according to the present invention can be widely applied to in vitro diagnostic devices such as immunoassays by co-reacting with ruthenium pyridine, an electrochemiluminescent label, and significantly increasing the luminescence intensity.

[0068] The following are preferred embodiments to aid in understanding the present invention. However, the following embodiments are provided solely to facilitate understanding of the present invention and do not limit the scope of the present invention.

[0069] <Experimental Example> Common Experiment and Measurement Method Before starting the electrochemiluminescence measurement with the ECL detector of the example, the cell was cleaned with a cleaning solution (ethanol and water and dried with N2 gas). The surface of the working electrode was also polished with alumina (0.05 μm) slurry, ultrasonically treated with a mixture of deionized water (DI) and ethanol (1:1 v / v) for 5 minutes, cleaned, and dried with N2 gas.

[0070] After cleaning, the electrochemical cell was filled with an electrolyte solution and attached to a photomultiplier tube (PMT). Next, power was supplied between the working electrode and the reference electrode, applying a voltage. Through this, a predetermined current was charged to the liquid sample in the cell by a control signal, and the reaction at the working electrode began. The ECL light generated at the working electrode passed through the photomultiplier tube (PMT), and the ECL light from the ECL reaction at the working electrode was detected by optical detectors such as photomultiplier tubes (PMTs) located on and adjacent to the electrochemical cell. Furthermore, the main body was completely surrounded in a darkroom environment (not shown in the Maxi-form diagram), through which the photomultiplier tubes (PMTs) could receive the ECL light generated in the cell without external interference.

[0071] Figure 1 shows an example of an electrochemiluminescence system for measuring electrochemiluminescence according to the present invention. A potentiostat is used to induce a reaction in an electrochemical cell for measuring electrochemiluminescence, and the potentiostat and electrochemiluminescence measurement software are run to measure the emission intensity of the electrochemiluminescence.

[0072] In detail, the inventors investigated the performance and influential mediating variables of 4-DMAP, a newly discovered co-reactant, through experiments, and compared its performance with that of conventional co-reactants, TPrA and DBAE. ECL emission was measured by supplying 7 mL of electrolyte to a cell and applying a potential to the electrode, and the intensity of the ECL light was recorded using a photomultiplier tube (PMT).

[0073] Furthermore, the ECL detection method via potential control was performed by sweeping from 0V to 1.6V at a speed of 0.1V / sec. The above voltage values ​​were determined between the working electrode (glassy carbon electrode, platinum electrode, or gold electrode) and the reference electrode (Ag / AgCl or Ag / AgCl). + It was assigned between ) and ). Below, all important parameters are listed. [Examples]

[0074] Confirmation of the electrochemical behavior of various electrochemiluminescent co-reactants Ru(bpy)3 is an electrochemiluminescent label. 2+ Tripropylamine (TPrA), dibutylethanolamine (DBAE), and 4-dimethylaminopyridine (4-DMAP), which are electrochemiluminescent coreactants, were prepared along with phosphate buffer solution (PBS) and acetonitrile (ACN) as solvents. These were mixed to prepare each sample, and the cyclic voltammogram (CV) and ECL were measured and recorded for these samples. A potentiostat was used for the experiment. Voltage scanning started from 0.0V at a speed of 0.1V / s. Glassy carbon electrodes, platinum electrodes, and silver electrodes were used as working electrodes, and Ag / AgCl or Ag / AgNO3 was used as the reference electrode. The upper voltage limit was 1.6V, the lower voltage limit was 0V, and the final voltage was 0V. The electrochemical behavior of various electrochemiluminescent coreactants was measured and is shown in Figures 2 to 5.

[0075] Figures 2 and 3 show Ru(bpy)3 using a glassy carbon material as the working electrode during periodic potential scanning between 0V and 1.6V in PBS. 2+ / TPrA and Ru(bpy)3 2+ The cyclic voltammogram (CV) and ECL intensity were measured for the 4-DMAP electrochemiluminescence system, and the results are shown in Figures 2 and 3. 2+ The ECL intensity of / 4-DMAP is Ru(bpy)3 2+ We were able to confirm that it was higher than / TPrA.

[0076] Figures 4 and 5 show Ru(bpy)3 using a platinum material as the working electrode during periodic potential scanning between 0V and 1.6V in PBS. 2+ / TPrA and Ru(bpy)3 2+ The cyclic voltammogram (CV) and ECL intensity were measured for a 4-DMAP electrochemiluminescence system, and the results are shown below.

[0077] The results in Figures 4 and 5 show Ru(bpy)3 2+ The anode current of the / 4-DMAP is Ru(bpy)3 2+ We were able to confirm that it was higher than the anode current of / TPrA. Also, Ru(bpy)3 2+ The ECL intensity of / 4-DMAP is Ru(bpy)3 2+ The ECL intensity was significantly higher than that of / TPrA.

[0078] These results confirmed that platinum electrodes are the most favorable working electrodes for 4-DMAP oxidation in phosphate-buffered saline (PBS), followed by glassy carbon electrodes. [Examples]

[0079] ECL characteristics depending on the concentration of electrochemiluminescent co-reactants PBS samples are processed using Ru(bpy)3. 2+ Sample solutions were prepared by maintaining a constant concentration of the active ingredient while varying the concentration of 4-DMAP, an electrochemiluminescent coreactant. Voltage scanning was performed using the same procedure as described in Example 1, and the electrochemiluminescence intensity was measured by taking three readings for each concentration of the electrochemiluminescent coreactant. The measurement results are shown in Figure 6.

[0080] Figure 6 shows the results of measuring ECL intensity using a platinum electrode as the working electrode, depending on the 4-DMAP concentration. It was confirmed that the ECL intensity significantly increased when 4-DMAP was used in the concentration range of greater than 0 to 5 mM. However, when the 4-DMAP concentration was higher than 5 mM, the ECL intensity decreased, confirming that the optimal concentration is 5 mM or less. In the case of TPrA, it was confirmed that the ECL intensity continued to increase as the TPrA concentration increased.

[0081] Through these results, we were able to confirm that when using 4-DMAP as an electrochemical co-reactant, using it in a concentration range of over 0 mM and up to 5 mM yields the best emission intensity. [Examples]

[0082] Various Ru(bpy)3 2+ ECL properties of electrochemiluminescent coreactants depending on concentration Ru(bpy)3 in various concentrations 2+ A PBS sample containing Ru(bpy)3 was prepared. Voltage scanning was performed using the same procedure as described in Example 1. 2+ The electrochemiluminescence intensity was measured at each concentration, and the results are shown in Figure 7. As shown in Figure 7, a platinum (Pt) electrode was used as the working electrode, and 10 μM Ru(bpy)3 was used. 2+ Furthermore, when measuring ECL using a 7 mM 4-DMAP concentration, the highest intensity values ​​were observed, and it was confirmed that the intensity decreased when the 4-DMAP concentration exceeded 7 mM.

[0083] Overall, 10 μM Ru(bpy)3 2+ In a PBS solution containing [the specified compound], the most appropriate concentration of the co-reactant 4-DMAP was found to be around 7 mM. [Examples]

[0084] ECL properties of electrochemiluminescent coreactants depending on pH We attempted to confirm the ECL properties of the electrochemiluminescent coreactant at various pH values. Except for the change in pH from 5 to 12, we used a constant concentration of Ru(bpy)3. 2+ Electrochemiluminescence from electrochemiluminescent co-reactants was measured for PBS samples containing [component name]. Voltage scanning was performed using the same procedure as described in Example 1. Electrochemiluminescence intensity was measured three times for different pH values, and the results are shown in Figure 8.

[0085] Figure 8 shows the results of investigating 4-DMAP performance at different pH values, with 1 μM Ru(bpy)3 2+ We were able to confirm that the pH range of 7.4 to 10 is the most optimal pH range for 4-DMAP performance when using 5 mM 4-DMAP. Similarly, with 10 μM Ru(bpy)3 2+ Furthermore, when using 7 mM 4-DMAP, we observed that ECL intensity was inferior when the pH value was below 7.4 or above 10. Therefore, we were able to confirm that the optimal pH is between 7.4 and 10. [Examples]

[0086] ECL properties of electrochemiluminescent co-reactants under various solvents Sample preparation was performed using acetonitrile (ACN) solution instead of PBS solution.

[0087] Figure 9 shows the results of measuring electrochemiluminescence intensity using acetonitrile (ACN) solution instead of PBS. When a glassy carbon electrode (Figure 9A), a platinum electrode (Figure 9B), and a gold electrode (Figure 9C) were used as working electrodes, the results were obtained at relatively low concentrations of Ru(bpy)3 at 3 mM, 5 mM, and 7 mM, respectively. 2+The ECL intensity of / 4-DMAP is Ru(bpy)3 2+ We were able to confirm that it was higher than / TPrA. [Examples]

[0088] ECL properties of electrochemiluminescent co-reactants involving various luminescent materials iridium-based transition metal complex compounds used as electrochemiluminescence labels include iridium bis[5-(trifluoromethyl)-2-(4-trifluoromethyl)phenyl)pyridine]picolinate, (Ir(ppy-(CF3)2)2(pico)), and bis(2-phenylpyridine)(4,4'-di-tert-butyl-2,2'-dipyridyl)iridium(III)(hexafluorophosphate)[bis(2-phenylpyridine)(4,4'-di-tert-butyl-2,2'-dipyridyl)iridi Samples were prepared by mixing um(III)(hexafluorophosphate), Ir(dtbbpy)(ppy)2PF6, Tris[2-(p-tolyl)pyridine]iridium(III), Ir(mppy)3, 4-dimethylaminopyridine (4-DMAP), and the supporting electrolyte tetrabutylammonium hexafluorophosphate (TBAPF6) with acetonitrile. Since the luminescence properties of these samples are highly sensitive to oxygen and moisture, measurements were carried out under a glove box in a nitrogen environment.

[0089] Linear sweep voltammograms (LSVs) and electrochemical charges (ECLs) were measured and recorded for these samples. The sweep rate was 0.1 V / s, starting from 0.0 V and sweeping within a voltage range of 2.0 V. A glassy carbon electrode was used as the working electrode, with Ag / Ag as the reference electrode. + (3M AgNO3) was used, with Pt as an auxiliary electrode, and the results are shown in Figure 10.

[0090] Figure 10 shows the linear sweep potential curve and electrochemiluminescence intensity when 4-DMAP is used as a co-reactant and an iridium-based transition metal complex compound in an acetonitrile (ACN) solution, with glassy carbon used as the working electrode. The results in Figure 10 confirm that even when an iridium-based transition metal complex compound is used as an electrochemiluminescence label, high ECL intensity and high anode current are observed.

[0091] These results demonstrate that electrochemiluminescence labeling can utilize a variety of transition metal complex compounds. [Examples]

[0092] Ru(bpy)3 using electrochemiluminescent coreactants 2+ Comparison of ECL detection curves based on concentration Prepare separate PBS solutions containing the co-reactant at a concentration of 7 mM, and then use Ru(bpy)3 2+ The electrochemiluminescence signals generated when the concentration of was varied were observed. Voltage scanning was performed using the same procedure as described in Example 1. The electrochemiluminescence intensity was measured by taking three readings for each condition.

[0093] Figure 11 shows Ru(bpy)3 in the respective solutions using 4-DMAP and tripropylamine as co-reactants. 2+ This is the electrochemiluminescence intensity generated depending on the concentration of Ru(bpy)3. In the case of tripropylamine coreactants (right side, black graph), Ru(bpy)3 in the concentration range of 0-10 nM 2+ An electrochemiluminescence detection signal can be obtained for Ru(bpy)3 2+The detection limit (LOD) for 4-DMAP was 0.63 nM (630 pM). On the other hand, for 4-DMAP, the detection limit for Ru(bpy)3 at concentrations of 0-0.1 nM was 0.63 nM. 2+ It exhibits a high electrochemiluminescence signal, with a detection limit of 0.0415 nM (41.5 pM), demonstrating detection sensitivity more than 15 times better than that of tripropylamine.

[0094] These results demonstrate that electrochemiluminescence signals can be provided at even lower concentrations of the luminescent material compared to tripropylamine, thereby offering even better detection sensitivity when used in immunodiagnosis or molecular diagnostics. [Examples]

[0095] Ru(bpy)3 using electrochemiluminescent coreactants 2+ Comparison of ECL detection curves based on concentration Electrochemiluminescence immunodiagnosis was performed on "anti-SARS-CoV-2" antibodies present in human saliva using 4-DMAP and tripropylamine as co-reactants. Saliva samples were collected from 10 vaccinated individuals, and a three-stage electrochemical immunoanalysis was performed. Stage 1) 30 μL of supernatant from centrifuged human saliva was mixed with a pre-prepared SARS-CoV-2 antigen-coated magnetic bead reagent (capture reagent) (2 μm in diameter). At this stage, the neutralizing antibodies present in human saliva form a sandwich immunoconjugate with the antigen pre-immobilized on the magnetic bead. ii) After incubation (37°C) of this mixture, only the magnetic beads were collected and washed twice with PBS. Subsequently, these were subjected to Ru(bpy)3 2+Mix with 30 μL of labeled 5 μg / mL human IgG antibody reagent (ECL signal generating reagent). iii) Next, collect only the magnetic beads from the mixture in ii) above and place them on a gold-printed electrode. Add 7 mM DMAP or 7 mM tripropylamine buffer solution (PBS) to the magnetic beads on the electrode surface, apply a voltage to each, and measure the electrochemiluminescence signal.

[0096] Figure 12 shows the intensity of the electrochemiluminescence signal measured when 4-DMAP was used as a co-reactant and when tripropylamine was used as a co-reactant in electrochemical immunoassay for anti-SARS-CoV-2 neutralizing antibodies using saliva samples from 10 vaccinated individuals. The results showed that using 4-DMAP as a co-reactant resulted in a detection signal that was more than 15 times better than that using tripropylamine for the same 10 saliva samples.

[0097] This demonstrates that using 4-DMAP as a co-reactant in electrochemiluminescence immunoassay or molecular diagnostics can yield even better detection sensitivity.

[0098] Therefore, it was confirmed that the luminescence intensity between the pyridine derivative represented by chemical formula 1 and ruthenium pyridine according to the present invention is at least 20 times higher than that obtained using tripropylamine, demonstrating superior luminescence sensitivity. Consequently, it can replace the conventional co-reactant tripropylamine, allows for voltage adjustment for luminescence, and improves luminescence efficiency, offering broad potential applications in various bioanalyses such as immunoassay.

[0099] Having described in detail certain aspects of the present invention above, it will be clear to those with ordinary skill in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. Electrochemiluminescent co-reactants containing the compound represented by the following chemical formula 1, or a pharmaceutically acceptable salt thereof: [Chemical formula 1] In the above chemical formula 1, the R １ or R ２ These may be the same or different, and include hydrogen atoms; halogen atoms; and C. １ ~C ６ Linear, branched, or cyclic alkyl groups; C １ ~C ６ Alkoxy groups; and C １ ~C ６ This indicates one selected from the group consisting of haloalkyl groups.

2. The aforementioned R １ or R ２ may be the same or different, respectively, and is a C １ -C ４ linear or branched alkyl group; or a C １ -C ４ The coreactant for electrochemiluminescence according to claim 1, characterized in that it is a haloalkyl group.

3. The electrochemiluminescent coreactant according to claim 1, characterized in that the chemical formula 1 is 4-dimethylaminopyridine (4-DMAP).

4. An electrochemiluminescence system comprising: an electrochemical cell filled with an electrolyte solution containing a co-reactant according to any one of claims 1 to 3 and an electrochemiluminescent label; and a photodetector connected to the electrochemical cell.

5. The electrochemical luminescence system according to claim 4, characterized in that the electrochemical cell includes one or more working electrodes selected from the group consisting of elements carbon, platinum (Pt), gold (Au), silver (Ag), nickel (Ni), stainless steel, palladium, tin, indium, and silicon.

6. The electrochemiluminescence system according to claim 4, characterized in that the electrolyte solution is one or more selected from the group consisting of phosphate buffer solution (PBS), acetonitrile (ACN), dichloromethane, ethanol (ethanol), methanol (ethanol), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethylene carbonate (EC), and propylene carbonate (PC).

7. The electrochemiluminescence system according to claim 4, characterized in that the pH of the electrolyte solution is 5 to 12.

8. The electrochemiluminescence system according to claim 4, characterized in that the electrochemiluminescence label is one or more selected from the group consisting of transition metal complex compounds, luminescent organic semiconductors, quantum dot materials, perovskite nanoparticles, metal nanoparticles, and carbon nanoparticles.

9. (a) The step of placing an electrolyte solution containing the sample into an electrochemical cell in the electrochemiluminescence system according to claim 4 and allowing it to react; and (b) A step in which the reaction sample from step (a) is measured using an electrochemiluminescence-based detector to determine the electrochemiluminescence intensity (ECL intensity) based on the input voltage, and an optical signal is detected; A method for detecting an electrochemiluminescence system that includes [specific components / systems].

10. A kit for electrochemiluminescence immunoassay or molecular diagnostics comprising a co-reactant according to any one of claims 1 to 3; and an electrochemiluminescence label.

11. The kit according to claim 10, characterized in that it contains one or more electrolyte solutions selected from the group consisting of phosphate buffer solution (PBS), acetonitrile (ACN), dichloromethane, ethanol (ethanol); methanol (ethanol), tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethylene carbonate (EC), and propylene carbonate (PC).

12. The kit according to claim 11, characterized in that the electrolyte solution has a pH of 5 to 12.

13. The kit according to claim 10, characterized in that it contains 4-dimethylaminopyridine at a concentration of more than 0 mM and less than or equal to 20 mM.

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