Electrochemiluminescence co-reactants and electrochemiluminescence systems comprising the same

Abstract

The present application relates to an electrochemiluminescence co-reactant and an electrochemiluminescence system comprising the same, and the electrochemiluminescence (ECL) co-reactant and specific luminescent chemical species (polycyclic aromatic hydrocarbon compounds, metal complexes, quantum dots or nanoparticles, etc.) comprising a compound represented by the following Chemical Formula I or a pharmaceutically acceptable salt thereof are used as electrochemiluminescence labels, which can make the detection signal very excellent and improve the voltage application condition in chemiluminescence determination, thereby can be widely applied in immunoassay and diagnostic instruments based thereon and chemical analysis and diagnostic instruments based thereon. In the above Chemical Formula I, the above R 1 or R 2 Each can be the same or different, and represents any one selected from the group consisting of a hydrogen atom, a halogen atom, a C1-C6 straight chain or branched chain or cyclic alkyl group, a C1-C6 alkoxy group, and a C1-C6 haloalkyl group.

Description

Technical Field

[0001] This invention relates to novel electrochemiluminescence co-reactants and electrochemiluminescence systems containing the same. The co-reactant comprises a compound represented by the following chemical formula I or a pharmaceutically acceptable salt thereof, and a specific luminescent chemical (polycyclic aromatic hydrocarbon compound, metal complex, quantum dot or nanoparticle, etc.) as an electrochemiluminescence label. This allows for excellent detection signals and improved voltage application conditions during chemiluminescence assays, thus enabling its widespread application in immunoassays and related diagnostic instruments, as well as in chemical analysis and related diagnostic instruments.

[0002] Chemical Formula I

[0003]

[0004] In the above chemical formula I, the above R 1 or R 2 Each can be the same or different, and represents any one of the following groups: hydrogen atom, halogen atom, C1-C6 straight-chain or branched or cyclic alkyl group, C1-C6 alkoxy group, and C1-C6 haloalkyl group. Background Technology

[0005] The industry is constantly conducting research to find rapid, highly specific, sensitive, and accurate methods for detecting and quantifying chemical, biochemical, and biological substances. This is because the amount of a specific analyte in a biological sample is quite small; therefore, improvements in analytical performance, such as sensitivity, are particularly important.

[0006] One approach to improving analytical sensitivity is to utilize methods applicable to high-sensitivity optical detection (e.g., photomultiplier tubes). Relatedly, the use of luminescent indicator molecules is particularly important. For example, the presence of an analyte (or its binding factor) can be quantitatively detected by using a luminescent label associated with the analyte of interest (or its binding factor).

[0007] As described below, when an analyte participates in a reaction that modulates induced luminescence, the amount of the analyte can be determined quantitatively. Specifically, i) the analyte can modulate a second type of luminescence property through reactions with other types of substances, ii) it can be through chemical modifications that modulate the luminescence properties of the analyte itself, iii) it can be a catalyst (e.g., an enzyme) for inducing other types of reactions, and iv) the analyte can participate in a reaction of the generating type and in subsequent reactions that modulate luminescence after induction.

[0008] Methods for detecting luminescent indicator molecules include photoluminescence, chemiluminescence, and electrochemiluminescence.

[0009] Among many optical detection systems, electrochemiluminescence systems do not require large and expensive light sources, thus enabling the construction of inexpensive and compact diagnostic systems that minimize signal interference caused by background signals from other interferometers.

[0010] More specifically, electrochemiluminescence is an electroluminescent phenomenon discovered around 1960. This phenomenon involves: i) inducing an oxidation reaction of a specific luminescent substance by applying a voltage; ii) then, the resulting intermediate reactants undergo a secondary chemical reaction to transform into an excited-state final product; and iii) this excited state transitions to the ground state, emitting light. Such electrochemiluminescence is used as a detection method in high-end medical immunodiagnostic instruments.

[0011] As an application, electrochemiluminescence-based immunodiagnostic instruments include those from Roche utilizing the Elecsys method. The diagnostic instruments and the Mesoscale Discovery ECL Systems series are unique and monopolize the world's high-end immunodiagnostic instrument market. These methods utilize ruthenium compounds (Ru(bpy)3) as luminescent agents. 2+ Electrochemiluminescence is achieved by reacting tripropylamine (TPA) with a base as a co-reactant. However, the problem is that tripropylamine, as a co-reactant, is hydrophobic, resulting in poor luminescence efficiency under platinum and gold electrode conditions. Furthermore, in the past sixty years of electrochemiluminescence of bases, no co-reactant has shown superior luminescence efficiency compared to tripropylamine.

[0012] In detail, tripropylamine is not only an organic medium but also an aqueous medium, and it allows for effective electrochemiluminescence even at physiological pH 7.4, thus it is often used as a co-reactant. However, tripropylamine (TPrA) is volatile and toxic, and has the disadvantage of requiring high concentrations (typically up to 100 mM) to obtain high electrochemiluminescence signals. Furthermore, the slow electrochemical oxidation rate of tripropylamine limits its electrochemiluminescence efficiency, and its alkaline nature necessitates the complexity of preparing high-concentration buffer solutions. Additionally, it exhibits significant deviations between identical detection signals and reacts chemically with atmospheric carbon dioxide.

[0013] Furthermore, ruthenium pyridine (Ru(bpy)3) 2+ The electrochemiluminescence efficiency of tripropylamine depends on the electrode materials. In the electrochemiluminescence region, platinum (Pt) and gold (Au) electrodes are covered by a positive oxide coating, which inhibits the direct oxidation of tripropylamine and results in lower electrochemiluminescence intensity. Conversely, polished glassy carbon (GC) electrodes allow for relatively faster electrochemiluminescence oxidation of tripropylamine, resulting in a greater amount of tripropylamine oxidized on the electrode surface and thus significantly higher luminescence intensity.

[0014] Against this backdrop, the inventors aim to develop a novel electrochemiluminescent co-reactant that can improve the luminescence intensity and detection reproducibility of electrochemiluminescence systems. Summary of the Invention

[0015] Technical issues

[0016] In the field of electrochemiluminescence, which has wide applications in blood glucose and cholesterol sensors and antibody detection through molecular diagnostics or immunoassay, the inventors, in their efforts to develop a novel co-reactant that can replace tripropylamine, which has been mainly used as a co-reactant, has a rapid reaction, is less affected by electrode materials, and has excellent luminescence efficiency, discovered that in the case of pyridine derivatives represented by chemical formula I, the luminescence intensity of the electrochemiluminescence system labeled with ruthenium pyridine is excellent, thus completing the present invention.

[0017] Therefore, the object of the present invention is to provide an electrochemiluminescent co-reactant comprising a compound represented by chemical formula I or a pharmaceutically acceptable salt thereof.

[0018] Furthermore, another object of the present invention is to provide an electrochemiluminescence system for excitation of the aforementioned co-reactants and electrochemiluminescence labels.

[0019] Furthermore, another object of the present invention is to provide a detection method utilizing the above-described electrochemiluminescence system.

[0020] Furthermore, another object of the present invention is to provide a kit comprising the above-mentioned co-reactants for electrochemiluminescence immunoassay or molecular diagnostics.

[0021] Technical solution

[0022] To achieve the above objectives, the present invention provides an electrochemiluminescent co-reactant comprising a compound represented by the following chemical formula I or a pharmaceutically acceptable salt thereof.

[0023] Chemical Formula I

[0024]

[0025] In the above chemical formula I, the above R 1 or R 2 Each can be the same or different, and represents any one of the following groups: hydrogen atom, halogen atom, C1-C6 straight-chain or branched or cyclic alkyl group, C1-C6 alkoxy group, and C1-C6 haloalkyl group.

[0026] Furthermore, the present invention provides an electrochemiluminescence system comprising: an electrochemical cell filled with an electrolyte solution containing an electrochemiluminescence co-reactant containing a compound represented by the above chemical formula I or a pharmaceutically acceptable salt thereof and an electrochemiluminescence label; and a photodetector connected to the electrochemical cell.

[0027] Furthermore, the present invention provides a detection method for an electrochemiluminescence system, comprising: step (a), placing a sample into an electrochemiluminescence system containing an electrochemiluminescence co-reactant and an electrochemiluminescence label containing a compound represented by the above chemical formula I or a pharmaceutically acceptable salt thereof to react; and step (b), using an electrochemiluminescence-based detector to measure the electrochemiluminescence intensity (ECL intensity) of the reaction sample in step (a) with respect to an input potential (or voltage) to detect an optical signal.

[0028] Furthermore, the present invention provides a kit for electrochemiluminescence immunoassay or molecular diagnostics comprising the above-mentioned co-reactants and electrochemiluminescence labels.

[0029] The effects of the invention

[0030] The pyridine derivative of the present invention can be detected rapidly and accurately as an electrochemiluminescence signal, thereby replacing the existing co-reactant tripropylamine.

[0031] In detail, as a solid compound, it exhibits excellent processability and can improve the potential (or voltage) conditions for luminescence, enabling improved luminescence efficiency even at low concentrations, thus providing potential for wide application in various biological analyses such as immunoassays. Attached Figure Description

[0032] Figure 1 A schematic diagram of an electrochemiluminescence system including a potentiostat and a photomultiplier tube is shown as an example of the present invention.

[0033] Figure 2 To contain 5 mM of the co-reactant and 1 μM of [Ru(bpy)3] in 1X phosphate buffer solution (pH 7.4) 2+ Results of cyclic voltammograms (CV) measured in the ruthenium pyridine electrochemiluminescence system (scan rate: 0.1 V / s, WE: GC, CE: Pt, RE: Ag / AgCl).

[0034] Figure 3 To contain 5 mM of the co-reactant and 1 μM of [Ru(bpy)3] in 1X phosphate buffer solution (pH 7.4) 2+ Results of electrochemiluminescence intensity determination in ruthenium pyridine electrochemiluminescence system (scan rate: 0.1 V / s, WE: GC, CE: Pt, RE: Ag / AgCl).

[0035] Figure 4 To prepare a solution containing 5 mM of co-reactant and 1 μM of [Ru(bpy)3] in 1X phosphate buffer (pH 7.4) 2+ Results of cyclic voltammograms (CV) measured in an electrochemiluminescence system of ruthenium pyridine (scan rate: 0.1 V / s, WE: Pt, CE: Pt, RE: Ag / AgCl).

[0036] Figure 5 To prepare a solution containing 5 mM of co-reactant and 1 μM of [Ru(bpy)3] in 1X phosphate buffer (pH 7.4) 2+ Results of electrochemiluminescence intensity determination in ruthenium pyridine electrochemiluminescence system (scan rate: 0.1 V / s, WE: Pt, CE: Pt, RE: Ag / AgCl).

[0037] Figure 6 To illustrate the [Ru(bpy)3] at 1 μm in 1X phosphate buffer solution. 2+Plot of the electrochemiluminescence intensity of ruthenium pyridine as a function of the concentrations of 4-dimethylaminopyridine (4-DMAP) and tripropylamine (1 mM–100 mM) (pH 7.4). Potential increases in stages from 0 V to 1.6 V (WE: Pt, CE: Pt, RE: Ag / AgCl).

[0038] Figure 7 To illustrate the [Ru(bpy)3] at 10 μm in 1X phosphate buffer solution. 2+ Plot of the electrochemiluminescence intensity of ruthenium pyridine as a function of the concentrations of 4-dimethylaminopyridine (4-DMAP) and tripropylamine (1 mM–100 mM) (pH 7.4). Potential increases in stages from 0 V to 1.6 V (WE: Pt, CE: Pt, RE: Ag / AgCl).

[0039] Figure 8 To use [Ru(bpy)3] 2+ Results of electrochemiluminescence intensity determination of ruthenium pyridine (1 μm and 10 μm) under various pH conditions (WE: Pt, CE: Pt, RE: Ag / AgCl).

[0040] Figure 9 shows the electrochemiluminescence intensity curves comparing the results of using 4-dimethylaminopyridine and tripropylamine as co-reactants in acetonitrile (ACN) solution and using glassy carbon (part A), platinum (part B), and gold (part C) as working electrodes, respectively (CE: Pt, RE: Ag / AgCl).

[0041] Figure 10 To illustrate the use of 4-dimethylaminopyridine as a co-reactant in acetonitrile (ACN) solution, linear scan potential curves and electrochemiluminescence intensity graphs (CE: Pt, RE: Ag / Ag) are shown when glassy carbon is used as the working electrode. + (3M silver nitrate (AgNO3)), scan rate: 0.1V / s).

[0042] Figure 11 The results show the differences in electrochemiluminescence signals resulting from changes in the concentration of the pyridine ruthenium luminescent agent, obtained by measuring the calibration curve morphology when 7 mM 4-dimethylaminopyridine and 7 mM tripropylamine were used as co-reactants, respectively.

[0043] Figure 12The results are presented by electrochemiluminescence immunoassay of the presence of "SARS-CoV-2 neutralizing antibody (anti-SARS-CoV-2)" in saliva samples from 10 vaccinated individuals, using 4-dimethylaminopyridine or tripropylamine as co-reactants, respectively. Detailed Implementation

[0044] Electrochemiluminescence (ECL) is a luminescent process characterized by the high-energy electron-moving reaction of compounds generated at electrodes to produce light in an excited state. The luminescent labeling reagents used in ECL include transition metal complexes, luminescent organic semiconductors, quantum dots, perovskite nanoparticles, metal nanoparticles, or carbon nanoparticles. To date, such organic and inorganic luminescent labels have been widely used in bioanalysis.

[0045] The oxidation reaction, the fundamental principle of electrochemiluminescence, involves the loss of electrons by the luminescent matrix and its composition at the electrode surface during the reaction. To enable the electron donor to act as a strong reducing agent, reducing the luminescent matrix to an excited state and causing it to lose hydrogen ions (H+), further oxidation is necessary. + After that, photons are released to return the luminescent matrix to its ground state. This process is repeated on the surface of the electrode, and photons are typically released continuously to maintain a specified matrix concentration.

[0046] As an example, there is an electrochemiluminescence system using ruthenium pyridine and tripropylamine. Electrochemiluminescence is a specific chemiluminescent reaction induced by electrochemistry on the surface of an electrode. The antigen-antibody complex and the ruthenium pyridine conjugate are electrochemically excited in the presence of tripropylamine, undergoing a redox reaction to release photons, which can be sensed by a photomultiplier tube. This process is repeated to generate a large number of photons, amplifying the optical signal. Typically, the labels used in electrochemiluminescence analysis can bind to antibody or antigen molecules with different chemical structures used to generate the labeled antibodies or antigens.

[0047] Currently available electrochemiluminescence methods primarily use tripropylamine as a co-reactant. However, tripropylamine has several drawbacks, including difficulty in handling it in a liquid state, slow reaction time, requirement for high concentrations, significant dependence on electrode materials, limited luminescence efficiency, and limitations in toxicity and stability.

[0048] The inventors have confirmed that when the solid compound 4-dimethylaminopyridine (4-DMAP) is used as a co-reactant in electrochemiluminescence, it quickly participates in the luminescence reaction and exhibits excellent luminescence efficiency.

[0049] Therefore, the present invention provides an electrochemiluminescent co-reactant comprising a compound represented by the following chemical formula I or a pharmaceutically acceptable salt thereof.

[0050] Chemical Formula I

[0051]

[0052] In the above chemical formula I, the above R 1 or R 2 Each can be the same or different, and represents any one of the following groups: hydrogen atom, halogen atom, C1-C6 straight-chain or branched or cyclic alkyl group, C1-C6 alkoxy group, and C1-C6 haloalkyl group.

[0053] In the above chemical formula I, the above R 1 or R 2 Each can be the same or different, and represents any one of the following groups: hydrogen atom, halogen atom, C1-C4 straight-chain or branched or cyclic alkyl group, C1-C4 alkoxy group and C1-C4 haloalkyl group.

[0054] Preferably, the above-mentioned R 1 or R 2 Each can be the same or different, and can be a C1-C4 straight-chain or branched alkyl group, or a C1-C4 haloalkyl group.

[0055] More preferably, the above chemical formula I can be 4-dimethylaminopyridine.

[0056] Furthermore, the present invention provides an electrochemiluminescence system comprising: an electrochemical cell filled with an electrolyte solution containing the aforementioned co-reactant and the electrochemiluminescence label; and a photodetector connected to the aforementioned electrochemical cell.

[0057] The aforementioned co-reactants are compounds with the structure of chemical formula I.

[0058] The aforementioned electrochemiluminescent label can be one or more selected from the group consisting of transition metal complexes, luminescent organic semiconductors, quantum dot materials, perovskite nanoparticles, metal nanoparticles and carbon nanoparticles, but is not limited thereto.

[0059] In detail, the aforementioned transition metal compound may include one or more elements selected from the group consisting of ruthenium (Ru), iridium (Ir), rhenium (Re), platinum (Pt), osmium (Os), copper (Cu), and iron (Fe).

[0060] In detail, the aforementioned ionic transition metal complexes may include 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(dp-phen)3(PF6)2, and bis(2-phenylpyridine)(2,2'-dipyridine)iridium(III)(hexafluorophosphate), Ir(ppy)2(bpy)PF6. Bis(2-phenylpyridine)(4,4′-di-tert-butyl-2,2′-bipyridine)iridium(III) (hexafluorophosphate), Ir(dtbbpy)(ppy)2PF6, 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]pyridinecarboxylate bis[5-(trifluoromethyl)-2-(4-(trifluoromethyl)phenyl)pyridine]picolinate, Ir(ppy-(CF3)2)2(pico)), iridium(III)(Tris[2-(p-tolyl)pyridine]iridium(III), Ir(mppy)3), 1,10-[phenanthroline]rhenium(I) (hexafluorophosphate) salt (1,The substance is one or more of the group consisting of 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, but is not limited thereto.

[0061] Specifically, the aforementioned light-emitting organic semiconductors may comprise light-emitting conjugated organic semiconductors such as light-emitting monomolecules or polymers. Specifically, they may include luminol and rubrene and their derivatives, anthracene and its derivatives, pyrene and its derivatives, decycloxyphenyl-substituted poly(1,4-phenylene vinylene), super... One or more of the following groups, but not limited thereto: 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).

[0062] The aforementioned quantum dot materials may contain inorganic compounds of elements from groups 13-15 or 12-15. Specifically, quantum dot materials containing the aforementioned inorganic compounds may contain 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), but are not limited thereto.

[0063] The aforementioned perovskite nanoparticles may comprise halide-based perovskites. Specifically, the halide-based perovskites may be represented by the chemical formulas ABX3, A2BX6, or A3B2X9. In this case, A can be an organic or inorganic cation, B can be a metal cation, and X can be a silver halide ion.

[0064] The aforementioned metal nanoparticles may comprise metal atom clusters with a size of less than 1 nm exhibiting discrete energy levels. Specifically, they may comprise gold (Au) nanoparticles, silver (Ag), copper (Cu), or silver (Ag)-gold (Au) binary metal nanoparticles.

[0065] The aforementioned carbon particles may include graphene quantum dots (GQDs) or carbon quantum dots (CQDs), but are not limited to these.

[0066] Furthermore, the aforementioned electrolyte solution may include: a liquid electrolyte containing a salt, water, and an organic solvent; a solid electrolyte in which a salt is dissolved in a polymer; a gel electrolyte containing a polymer, a salt, water, and an organic solvent; or an ion gel electrolyte containing a block copolymer and an ionic liquid. However, it is not limited to these categories. The aforementioned salt is an organic or inorganic ionic compound, and may be one or more of the following groups: phosphates, nitrates, hydrochlorides, sulfates, lithium salts, sodium salts, potassium salts, calcium salts, magnesium salts, and ammonium salts, but is not limited to these.

[0067] In detail, the electrolyte solution used in the electrochemiluminescence system of the present invention may be a solution containing one or more water or organic solutes selected from the group consisting of phosphate buffer saline (PBS), Tris buffer solution, acetonitrile (ACN), dichloromethane, ethanol, methanol, tetrahydrofuran (THF), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ethylene carbonate (EC), and propylene carbonate (PC), but is not limited thereto.

[0068] The pH of the electrolyte solution can be from 5 to 12, preferably from 7.4 to 10.

[0069] Figure 1An example of an electrochemiluminescence system for performing the electrochemiluminescence measurement of the present invention is shown, comprising an electrochemical cell containing an electrolyte solution, a potentiostat, and a photomultiplier tube (PMT). In this case, the photomultiplier tube is connected to the potentiostat for simultaneous driving. Therefore, for electrochemiluminescence measurement, the electrochemiluminescence reaction can be induced by the potentiostat inducing a reaction in the electrochemical cell. For electrochemiluminescence measurement, the luminescence intensity, etc., can be measured using the potentiostat and electrochemiluminescence measurement software.

[0070] The electrodes constituting the above-mentioned electrochemical cell may include a working electrode, a reference electrode, a working electrode, and a counter electrode, but are not limited to these.

[0071] The aforementioned 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 limited thereto.

[0072] The aforementioned counter electrode is selected from one or more electrodes composed of carbon, platinum, gold, silver, nickel, stainless steel, palladium, tin, indium, and silicon, but is not limited thereto.

[0073] The aforementioned reference electrode may be one or more electrodes selected from the group consisting of silver-based silver sample reference electrode (Ag pseudo-reference), Ag / AgCl electrode, Ag / AgNO3 electrode, mercury (Hg) electrode, Hg / HgO electrode and Hg2SO4, but is not limited thereto.

[0074] Furthermore, the present invention provides a detection method for an electrochemiluminescence system, comprising: step (a), placing an electrolyte solution containing a sample into an electrochemical cell within the electrochemiluminescence system to react; and step (b), using an electrochemiluminescence-based detector to measure the electrochemiluminescence intensity of the reaction sample in step (a) with respect to the input voltage to detect an optical signal.

[0075] Step (a) above is a step of placing a sample into an electrolyte solution containing co-reactants and ruthenium pyridine as an electrochemiluminescent label to react. The sample can be serum, urine or tissue fluid, but is not limited to these.

[0076] Step (b) above involves detecting an optical signal by measuring the intensity of electrochemiluminescence of the reaction sample from step (a) with respect to an input potential (or voltage) using an electrochemiluminescence-based detector. Several types of photodetectors can be used as equipment for measuring luminescence. For example, the photodetector may be one or more of the following: a photodiode based on silicon, germanium, germanium-phosphide, indium-potassium-arsenide, or lead-sulfide; a photomultiplier tube; a charge-coupled device (CCD); an electron-multiplying charge-coupled device (EMCCD); and a scientific complementary metal-oxide-semiconductor (sCMOS), but is not limited thereto.

[0077] Furthermore, the present invention provides an electrochemiluminescence immunoassay detection method using electrochemical methods. Therefore, the present invention provides a kit for electrochemiluminescence immunoassay or molecular diagnostics comprising the above-mentioned co-reactants and electrochemiluminescence labels.

[0078] In detail, the kit for electrochemiluminescence immunoassay or molecular diagnostics of the present invention may include an electrolyte solution, which may be a solution of water or an organic solute selected from the group consisting of phosphate buffer, Tris buffer, acetonitrile, dichloromethane, ethanol, methanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, ethylene carbonate and propylene carbonate, but is not limited thereto.

[0079] The pH of the electrolyte solution can be from 5 to 12, preferably from 7.4 to 10.

[0080] Furthermore, the above kit may contain 4-dimethylaminopyridine at concentrations 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 or greater than 0 mM and less than or equal to 7 mM.

[0081] As an example of the present invention, for labeling antigens or antibodies, electrochemiluminescent labels containing ruthenium compounds can be used, and electrochemiluminescent immunoassay can be performed via an immunoreaction and an electrochemiluminescent reaction. To perform a specific chemiluminescent reaction on the surface of the electrode using electrochemical micro-inducers, antibodies (Abs) are labeled with ruthenium pyridine as an electrochemiluminescent reagent. A carrier in the sample contains the corresponding antigen or antibody and is coated with the antigen or antibody complex formed in a specific manner via an immunoreaction. The conjugated complex is separated from the free labels using a separation technique. The antigen (Ag) or antibody (Ab) can be quantitatively or qualitatively detected in the electrode based on the luminescence intensity of the ruthenium pyridine. A pyridine derivative, as a co-reactant, flows into the electrochemical cell, and a voltage can be applied to initiate electrochemiluminescence.

[0082] When the kit of the present invention for electrochemiluminescence immunoassay or molecular diagnostics is applied to diagnostic instruments, the amount of antigen and antibody can be greatly reduced. Compared with the use of tripropylamine, the electrochemiluminescence intensity of the electrochemical reaction between the pyridine derivative of the present invention and ruthenium pyridine is almost 20 times better, allowing detection even with relatively small amounts of antigen and antibody, thereby effectively reducing the amount of antibody. Furthermore, the price of the kit or analytical instrument can also be relatively low.

[0083] Furthermore, compared to the use of tripropylamine, the kit of the present invention for electrochemiluminescence immunoassay or molecular diagnostics can obtain a better signal for the analyte and a better detection sensitivity.

[0084] As described above, the pyridine derivative represented by chemical formula I of the present invention reacts with ruthenium pyridine as an electrochemiluminescence marker to significantly improve the luminescence intensity, thereby enabling its widespread application in in vitro diagnostic instruments using immunoassay.

[0085] The following embodiments are provided to aid in understanding the present invention. However, the present invention is not limited to the embodiments described below, as they are intended to facilitate a better understanding of the invention.

[0086] Experimental Example. Common Experiments and Measurement Methods

[0087] Before starting the electrochemiluminescence measurement in the electrochemiluminescence detection device of the example, the electrode was first washed with a washing solution (using ethanol and water, and drying with nitrogen (N2)). The working electrode surface 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 five minutes, rinsed, and then dried with nitrogen.

[0088] After washing, the electrochemical cell is filled with an electrolyte solution and then attached to a photomultiplier tube. A voltage is then applied between the working electrode and the reference electrode, and a pre-set current is charged to the liquid sample in the cell via a control signal, thus initiating a reaction in the working electrode. The electrochemiluminescent light generated in the working electrode passes through the photomultiplier tube, and the electrochemiluminescent light from the electrochemiluminescent reaction at the working electrode is sensed by optical detection instruments such as photomultiplier tubes arranged adjacent to the electrochemical cell. Furthermore, the entire assembly is completely enveloped in a dark chamber environment not shown in a Markush format, allowing the photomultiplier tube to receive the electrochemiluminescent light without external interference.

[0089] Figure 1 To illustrate an example of an electrochemiluminescence system for performing the electrochemiluminescence measurement of the present invention, for the purpose of electrochemiluminescence measurement, a reaction can be induced in an electrochemical cell using a potentiostat, and the luminescence intensity of electrochemiluminescence can be measured by using a potentiostat and electrochemiluminescence measurement software.

[0090] In detail, the inventors investigated the performance and influential parameters of the novel co-reactant 4-dimethylaminopyridine, discovered through experiments, and compared its performance with that of tripropylamine and dibutylethanolamine (DBAE), which are existing co-reactants. Electrochemiluminescence was measured by supplying 7 mL of electrolyte to the cell and applying a potential to the electrodes, and the intensity of the electrochemiluminescence was recorded using a photomultiplier tube.

[0091] Meanwhile, the potential-controlled electrochemiluminescence detection method is performed by scanning the potential from 0V to 1.6V at a rate of 0.1V / sec. These voltage values ​​are distributed between the working electrode (glassy carbon electrode, platinum electrode, or gold electrode) and the reference electrode (Ag / AgCl or Ag / Ag). + Between ), and then all the important parameters are listed below.

[0092] Example 1. Confirmation of the electrochemical activity of various electrochemiluminescence co-reactants

[0093] Ruthenium pyridine was prepared as the electrochemiluminescence label; tripropylamine, dimethyl ethanolamine (DBAE), and 4-dimethylaminopyridine were prepared as co-reactants; and phosphate buffered solution (PBS) and acetonitrile (ACN) were prepared as solvents. These were mixed to prepare individual samples. Cyclic voltammetry (CV) and electrochemiluminescence were measured and recorded for these samples. Experiments were conducted using a potentiostat. Voltage scans were performed at 0.1 V / s, starting from 0.0 V. Glassy carbon, platinum, and silver electrodes were used as working electrodes, and Ag / AgCl or Ag / AgNO3 was used as the reference electrode. The upper limit voltage was 1.6 V, the lower voltage limit was 0 V, and the final voltage was 0 V. The electrochemical activity of various electrochemiluminescence co-reactants was measured. Figures 2 to 5 As shown.

[0094] Figure 2 and Figure 3 To measure the potential of Ru(bpy)3 using glassy carbon as the working electrode during periodic potential scans from 0V to 1.6V in phosphate buffer solution. 2+ / TPrA and Ru(bpy)3 2+ The cyclic voltammetry curves and electrochemiluminescence intensities of / 4-DMAP were determined using an electrochemiluminescence system, and the results are shown below. Figure 2 and Figure 3 The result confirms that Ru(bpy)3 2+ / 4-DMAP has a higher electrochemiluminescence intensity than Ru(bpy)3 2+ / TPrA high.

[0095] on the other hand, Figure 4 and Figure 5 To measure the potential of Ru(bpy)3 using platinum as the working electrode during periodic potential scans from 0V to 1.6V in phosphate buffer solution. 2+ / TPrA and Ru(bpy)3 2+ The cyclic voltammetry curves and electrochemiluminescence intensity of the / 4-DMAP electrochemiluminescence system are measured and the results are shown.

[0096] pass Figure 4 and Figure 5 The results confirm that Ru(bpy)3 2+ / 4-DMAP's positive current ratio Ru(bpy)3 2+ / TPrA has a high positive current. Furthermore, Ru(bpy)3 2+ / 4-DMAP has a higher electrochemiluminescence intensity than Ru(bpy)3 2+ / TPrA exhibits significantly higher electrochemiluminescence intensity.

[0097] These results confirm that the platinum electrode is the most favorable working electrode for the oxidation of 4-dimethylaminopyridine in phosphate buffer solution, followed closely by the glassy carbon electrode.

[0098] Example 2. Electrochemiluminescence characteristics as a function of the concentration of the electrochemiluminescence co-reactant

[0099] The Ru(bpy)3 of the sample in phosphate buffer solution 2+ While maintaining the specified concentration, sample solutions were prepared with different concentrations of 4-dimethylaminopyridine as the electrochemiluminescence co-reactant. Voltage scanning was performed using the same procedure described in Example 1, and the electrochemical intensity was measured three times for each concentration of the electrochemiluminescence co-reactant. The measurement results are as follows: Figure 6 As shown.

[0100] Figure 6 To determine the electrochemiluminescence intensity as a function of 4-dimethylaminopyridine concentration when using a platinum electrode as the working electrode, we found that the electrochemiluminescence intensity limit increases when using 4-dimethylaminopyridine in a concentration range greater than 0 mM to less than or equal to 5 mM. However, if the concentration of 4-dimethylaminopyridine exceeds 5 mM, the electrochemiluminescence intensity decreases, thus confirming that the optimal concentration is below 5 mM. On the other hand, in the case of tripropylamine, we found that the electrochemiluminescence intensity continues to increase with increasing tripropylamine concentration.

[0101] These results confirm that when using 4-dimethylaminopyridine as the electrochemical co-reactant, the best luminescence intensity is achieved when used at a concentration range greater than 0 mM and less than or equal to 5 mM.

[0102] Example 3. Electrochemiluminescence characteristics of electrochemiluminescence co-reactants with varying concentrations of pyridine and ruthenium.

[0103] Prepare Ru(bpy)3 containing multiple concentrations 2+ The phosphate buffer solution was used. Voltage scanning was performed using the same procedure as described in Example 1. The determination of Ru(bpy)3... 2+ The electrochemiluminescence intensities at different concentrations are shown in the following figures. Figure 7 As shown. (Through) Figure 7 The results confirm that using a platinum electrode as the working electrode and employing 10 μm Ru(bpy)3... 2+When the electrochemiluminescence was measured with 7 mM 4-dimethylaminopyridine, the intensity was the highest. If the concentration of 4-dimethylaminopyridine exceeded 7 mM, the intensity decreased.

[0104] In summary, it can be seen that Ru(bpy)3 containing 10μm... 2+ In phosphate buffer solution, the optimal concentration of 4-dimethylaminopyridine as a co-reactant is around 7 mM.

[0105] Example 4. Electrochemiluminescence characteristics of electrochemiluminescence co-reactants with pH

[0106] The electrochemiluminescence properties of the electrochemiluminescent co-reactants were confirmed at various pH values. Except for pH variations from 5 to 12, the electrochemiluminescence properties of Ru(bpy)3 at specified concentrations were also confirmed. 2+ The electrochemiluminescence (ECL) of a phosphate buffer solution sample was measured in conjunction with the co-reactant. The process scan voltage was used with the same technique as in Example 1. The ECL intensity was measured three times for different pH values, and the results are as follows: Figure 8 As shown.

[0107] Figure 8 To investigate the properties of 4-dimethylaminopyridine at different pH values, it can be confirmed that using 1 μM Ru(bpy)3 2+ When using 5 mM 4-dimethylaminopyridine, the pH range of 7.4 to 10 is the optimal pH range for the performance of 4-dimethylaminopyridine. Similarly, when using 10 μM Ru(bpy)3... 2+ When 7 mM of 4-dimethylaminopyridine was used, weak electrochemiluminescence intensity was observed at pH values ​​below 7.4 and above 10. Therefore, the optimal pH range can be identified as 7.4 to 10.

[0108] Example 5. Electrochemiluminescence properties of electrochemiluminescence co-reactants in various solvents. Acetonitrile solution was used instead of phosphate buffer solution to prepare the sample.

[0109] Figure 9 shows the results of measuring electrochemiluminescence intensity using acetonitrile solution instead of phosphate buffer solution. When using a glassy carbon electrode (part A of Figure 9), a platinum electrode (part B of Figure 9), and a gold electrode (part C of Figure 9) as working electrodes, it can be confirmed that at relatively low concentrations of 3 mM, 5 mM, and 7 mM, Ru(bpy)3... 2+ / 4-DMAP has a higher electrochemiluminescence intensity than Ru(bpy)3 2+ / TPrA high.

[0110] Example 6. Electrochemiluminescence co-reactants and their electrochemiluminescence properties with various luminescent substances

[0111] Samples were prepared by mixing iridium-based transition metal complexes bis[5-(trifluoromethyl)-2-(4-(trifluoromethyl)phenyl)pyridine]pyridinecarboxylate, bis(2-phenylpyridine)(4,4′-di-tert-butyl-2,2′-bipyridine)iridium(III) (hexafluorophosphate), tris[2-(p-tolyl)pyridine]iridium(III) (Tris[2-(p-tolyl)pyridine]iridium(III), Ir(mppy)3), and 4-dimethylaminopyridine (4-DMAP) as electrochemiluminescence markers, and tetrabutylammonium hexafluorophosphate (TBAPF6) as the supporting electrolyte with acetonitrile. Because the luminescence properties of these samples are highly sensitive to oxygen and moisture, the preparation was carried out in a glove box under nitrogen atmosphere.

[0112] For these samples, linear scan potential curves and electrochemiluminescence were measured and recorded. Scans were performed at a rate of 0.1 V / s, starting at 0.0 V and continuing within a voltage range up to 2.0 V. In this case, a glassy carbon electrode was used as the working electrode, employing Ag / Ag... + Using 3M AgNO3 as the reference electrode and platinum as the auxiliary electrode, the results are as follows: Figure 10 As shown.

[0113] Figure 10 Linear scan potential curves and electrochemiluminescence intensity results are presented for the use of 4-dimethylaminopyridine as a co-reactant and a transition metal complex in acetonitrile solution, with glassy carbon as the working electrode. Figure 10 The results confirmed that when using iridium-based transition metal complexes as electrochemiluminescence markers, high electrochemiluminescence intensity and high positive current were also observed.

[0114] These results show that electrochemiluminescence labels can utilize a variety of transition metal complexes.

[0115] Example 7. Comparison of electrochemiluminescence detection curves with varying concentrations of ruthenium pyridine when using electrochemiluminescence co-reactants.

[0116] Prepare phosphate buffer solutions containing 7 mM of the co-reactant and observe the changes in Ru(bpy)3. 2+ The electrochemiluminescence signal occurred at a concentration of [specific value]. Voltage scanning was performed using the same procedure as described in Example 1. The electrochemiluminescence intensity was measured three times under each condition.

[0117] Figure 11To determine the reaction of Ru(bpy)3 in solutions using 4-dimethylaminopyridine and tripropylamine as co-reactants 2+ The electrochemiluminescence intensity occurs at a certain concentration. In the case of tripropylamine co-reactant (right, black curve), Ru(bpy)3 can be observed in the concentration range of 0 nM-10 nM. 2+ To obtain the electrochemiluminescence detection signal, Ru(bpy)3 2+ The limit of detection (LOD) was shown to be 0.63 nM (630 pM). Conversely, in the case of 4-dimethylaminopyridine, the limit of detection was 0 nM–0.1 nM for Ru(bpy)3. 2+ It exhibits a high electrochemiluminescence signal and a detection limit of 0.0415 nM (41.5 pM), demonstrating a detection sensitivity that is more than 15 times better than that of tripropylamine.

[0118] The results above show that an electrochemiluminescence signal can be provided in a lower concentration of luminescent material than tripropylamine, which can provide better detection sensitivity when used in immunodiagnostics or molecular diagnostics.

[0119] Example 8. Comparison of the effects of electrochemiluminescence co-reactants on Ru(bpy)3 2+ Electrochemiluminescence detection curve of concentration

[0120] Electrochemiluminescence immunoassay was performed on SARS-CoV-2 neutralizing antibodies (anti-SARS-CoV-2) present in human saliva using 4-dimethylaminopyridine and tripropylamine as co-reactants. Saliva samples were collected from ten vaccinated individuals, and a three-step electrochemiluminescence immunoassay was performed. Step i): 30 μL of supernatant from centrifuged human saliva was mixed with pre-prepared magnetic beads (magnetiCbeads, 2 μm in diameter) attached with SARS-CoV-2 antigen (capture reagent). In this step, the neutralizing antibodies present in the human saliva formed a sandwich immunoconjugate with the antigen pre-immobilized on the magnetic beads. Step ii): After incubating the mixture at 37°C for 30 minutes, only the magnetic beads were collected and washed twice with phosphate buffer. Then, they were mixed with 30 μL of Ru(bpy)3... 2+ A 5 μg / mL human IgG antibody reagent (electrochemiluminescence signal generating reagent) containing the labeled substance is mixed. In step iii), finally, only the magnetic beads from the mixture in step ii) are captured onto a gold-printed electrode. 7 mM dimethylaminopyridine (DMAP) or 7 mM tripropylamine buffer (phosphate buffer) is dropped onto the magnetic beads on the electrode surface, and a voltage is applied to measure the electrochemiluminescence signal.

[0121] Figure 12 The image shows the intensity of the electrochemiluminescence signal measured when using 4-dimethylaminopyridine as a co-reactant and when using tripropylamine as a co-reactant in electrochemiluminescence diagnostics of anti-SARS-CoV-2 neutralizing antibodies in saliva samples from 10 vaccinated individuals. The results indicate that using 4-dimethylaminopyridine as a co-reactant on the same 10 saliva samples showed a detection signal that was more than 15 times better than that of tripropylamine.

[0122] This demonstrates that using 4-dimethylaminopyridine as a co-reactant in electrochemiluminescence immunoassay or molecular diagnostics can achieve superior detection sensitivity.

[0123] Therefore, it can be confirmed that the luminescence intensity of the pyridine derivative represented by Formula I of the present invention and ruthenium pyridine is at least 20 times better than the luminescence sensitivity obtained when using tripropylamine. Therefore, it can replace the existing co-reactant tripropylamine, and the luminescence efficiency can be improved by adjusting the voltage used for luminescence, thus providing potential for wide application in various biological analyses such as immunoassays.

[0124] The foregoing has described specific aspects of the present invention in detail. It should be understood by those skilled in the art that these specific descriptions are merely preferred embodiments of the invention, and the scope of the invention is not limited to these embodiments. Therefore, the range of indications of the present invention should be defined by the appended claims and their equivalents.

Claims

Use of 1,4-dimethylaminopyridine or a pharmaceutically acceptable salt thereof as an electrochemiluminescent co-reactant for electrochemiluminescent labeling. The electrochemiluminescent label is a ruthenium-based or iridium-based transition metal complex.

2. An electrochemiluminescence system, characterized in that, include: An electrochemical cell filled with an electrolyte solution containing an electrochemiluminescent co-reactant and an electrochemiluminescent label; and a photodetector connected to the electrochemical cell. The electrochemiluminescence co-reactant is 4-dimethylaminopyridine or a pharmaceutically acceptable salt thereof. The electrochemiluminescent label is a ruthenium-based or iridium-based transition metal complex.

3. The electrochemiluminescence system according to claim 2, characterized in that, The aforementioned electrochemical cell includes one or more working electrodes selected from the group consisting of carbon, platinum, gold, silver, nickel, stainless steel, palladium, tin, indium, and silicon.

4. The electrochemiluminescence system according to claim 2, characterized in that, The electrolyte solution mentioned above is selected from one or more of the group consisting of phosphate buffer solution, acetonitrile, dichloromethane, ethanol, methanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, ethylene carbonate and propylene carbonate.

5. The electrochemiluminescence system according to claim 2, characterized in that, The pH of the above electrolyte solution is between 5 and 12.

6. A detection method for an electrochemiluminescence system, characterized in that, include: Step (a): An electrolyte solution containing the sample is placed into the electrochemical cell within the electrochemiluminescence system of claim 2 to react; as well as Step (b) uses an electrochemiluminescence-based detector to measure the electrochemiluminescence intensity of the reaction sample from step (a) as a function of the input voltage to detect the optical signal.

7. A kit for electrochemiluminescence immunoassay or molecular diagnostics, characterized in that, Include: Electrochemiluminescence co-reactants; and electrochemiluminescence labels, The electrochemiluminescence co-reactant is 4-dimethylaminopyridine or a pharmaceutically acceptable salt thereof. The electrochemiluminescent label is a ruthenium-based or iridium-based transition metal complex.

8. The kit for electrochemiluminescence immunoassay or molecular diagnostics according to claim 7, characterized in that, The above kit contains one or more electrolyte solutions selected from the group consisting of phosphate buffer, acetonitrile, dichloromethane, ethanol, methanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, ethylene carbonate, and propylene carbonate.

9. The kit for electrochemiluminescence immunoassay or molecular diagnostics according to claim 8, characterized in that, The pH of the above electrolyte solution is between 5 and 12.

10. The kit for electrochemiluminescence immunoassay or molecular diagnostics according to claim 7, characterized in that, The above kit contains 4-dimethylaminopyridine at a concentration greater than 0 mM and less than or equal to 20 mM.

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