Method for enhancing the performance of electrochemiluminescence immunoassay by using electrically neutral metal complex

Abstract

The present invention relates to a method for bioanalysis, in particular, to a method for generating electrochemiluminescence signal at low voltage to achieve high signal-to-noise ratio and improved concentration-signal response relationship, thereby improving electrochemiluminescence (ECL) immunoassay performance.

Description

TECHNICAL FIELD

[0001] The present invention relates to a method for improving the performance of electrochemiluminescence immunoassay by adjusting electrochemical reaction conditions. BACKGROUND

[0002] Electrochemiluminescence (ECL) immunoassay is a widely used immunoassay method in clinical testing. In ECL immunoassay, the succinimidyl ester (NHS ester, see Figure 1 A) of tris (2, 2'-bipyridyl) ruthenium (II) (commonly denoted as Ru (bpy) 3 2+ ) or other luminescent metal complexes is used as a label to tag the antibody or antigen of the analyte. After the antibody reacts with the analyte in the sample under certain conditions to form an antibody / antigen complex, the luminescent metal complex undergoes electrochemical reaction and a series of subsequent chemical reactions in an electrochemical flow cell, ultimately leading to the formation of the luminescent excited state of the luminescent metal complex, resulting in a detectable luminescent signal.

[0003] ECL immunoassay involves many technical details such as how the antibody (in sandwich method) or analyte (in competition method) is labeled, how the analyte is captured, how the ECL reaction is triggered, and how the working electrode is regenerated. Taking ECL sandwich method immunoassay as an example, in a typical commercial ECL assay, the labeling molecule shown in Figure 1 A is used to label the antibody (signal antibody) at the lysine residue's -amino site of one antibody of the analyte, while another antibody (capture antibody) is biotinylated. After a clinical sample is mixed with the two types of antibodies and streptavidin-coated magnetic beads for a predetermined period of time at a certain temperature, a sandwich structure immunocomplex is formed on the surface of the magnetic beads. Then, the magnetic beads are brought into an electrochemiluminescence measurement cell (flow cell) and captured on the surface of the electrochemical working electrode by a movable magnet located below the measurement cell. A buffer solution containing tri- n -propylamine (TPA or N(C3H7)3) washes away unwanted substances and provides a chemical environment for the ECL reaction of the Ru (bpy) 3 2+ luminescent group described in the following reaction path one.

[0004] At a certain voltage (such as 1.4 V vs. Ag / AgCl), tripropylamine in the buffer solution is oxidized to the cationic radical N(C3H7)3 •+ (reaction 1) and further loses a proton to become a neutral radical H6C3 •N(C3H7)2 (Reaction 2). This neutral radical has strong reducing power and can reduce Ru(bpy)3 2+ to Ru(bpy)3 1+ (Reaction 3). The cationic radical N(C3H7)3 •+ has oxidizing power and can oxidize Ru(bpy)3 1+ to the excited state (Reaction 3). and then back to the original Ru(bpy)3 2+ ground state (Reaction 5).

[0005] Reaction Pathway One

[0006]

[0007] Thus, in the ECL process, Ru(bpy)3 2+ is not consumed but undergoes a cyclic change of oxidation state, i.e., Ru(bpy)3 2+ → Ru(bpy)3 1+ → → Ru(bpy)3 2+ (see W. Miao, J.-P. Choi, A. J. Bard, J. Am. Chem. Soc . 2002, 124 , 14478-14485). This cycle repeats continuously during the measurement, resulting in a long-lasting ECL signal that is detected. The integral of the total ECL light emission over a certain period of time (e.g., 0.5-5 seconds) can be used as a measure of the ECL intensity and correlated to the amount of the analyte. After the measurement, the magnetic beads and the attached immunocomplexes are washed away by an aqueous stream, the measurement cell is cleaned, and the electrode surface is regenerated electrochemically to prepare it for the next test. Details of these time-sequenced experiments are disclosed in U.S. Patents 5,147,806, 5,538,687 and 6,599,473.

[0008] In fact, Reaction Path One is just one of the possible reaction paths that can generate ECL. Other possible reaction paths (e.g., Reaction Path Two below) Reaction Pathways Two to Four have been proposed to explain the formation of the excited state and ECL generation under different conditions (see J. K. Leland and M. J. Powell, J. Electrochem. Soc. 1990, 137 , 3127-3131,and W. Miao, J.-P. Choi, A. J. Bard, J. Am. Chem. Soc2002, 124 , 14478-14485).

[0009] Reaction Pathway Two

[0010]

[0011] Reaction Pathway Three

[0012]

[0013] Reaction Pathway Four

[0014]

[0015] The above reaction pathways two, three, and four occur when the electrode voltage is high enough to react Ru(bpy)3. 2+ Oxidation to Ru(bpy)3 3+ Under the conditions of (i.e., reaction 6), Ru(bpy)3 is not present in the reactions involved in reaction pathway 1. 2+ It is oxidized to Ru(bpy)3 3+ Reaction 6. However, under high voltage conditions that can lead to reaction pathways two, three, and four, the reactions in reaction pathway one also occur simultaneously. Although researchers in this field tend to believe that the vast majority of ECL light emission originates from reaction pathway one, the actual operating voltage used in ECL immunoassay systems is 1.4 V (relative to the Ag / AgCl reference electrode), at which reaction pathways one, two, three, and four can all occur.

[0016] The above Ru(bpy)3 2+ The ECL reaction pathway also applies to its derivatives and analogues. However, due to differences in the redox properties and chemical reactivity of different compounds, the contribution of different reaction pathways to the total ECL may vary. Regardless of the pathway from which the luminescence originates, the rate-controlling step of the ECL process is the electrochemical oxidation of TPA (i.e., reaction 1). The higher the operating voltage, the faster the reaction proceeds, resulting in a stronger luminescence signal. However, at high operating voltages, singlet oxygen (…) is generated due to side reactions. 1 Δ g O2 also generates high background noise (SS Kumar and AJ Bard, Anal. Chem. 2013, 85, 292-295). In the early development of ECL technology, the working voltage applied was much higher than 1.4 V, as disclosed in US Patent 5,147,806, the applied voltage was greater than 1.8 V in TPA solution on platinum electrode, and the maximum luminescence value was obtained at 2.2 V. US Patent 5,538,687 disclosed that the working voltage could be reduced to 1.4 V in an improved measuring cell. Since then, the working voltage applied in commercialized ECL immunoassay systems (Elecsys and cobas) is 1.4 V (see E. Faatz, A. Finke, H.-P. Josel, G. Prencipe, S. Qunit, M. Windfuhr, Automated immunoassays for the detection of biomarkers in body fluids, in Analytical Electrogenerated Chemiluminescence: From Fundamental to Bioassays, N. Sojic, Ed., Royal Socity of Chemistry, 2019; pp 443 - 470).

[0017] US Patent US 10203333 and Chinese Patent ZL 201480045420 disclosed a class of coordination compounds of electrically neutral metal ruthenium (Neutral Ruthenium Complexes, abbreviated as NRC). These electrically neutral ECL labels can reduce the non-specific signal in immunoassays, and some electrically neutral ECL label molecules (such as Figure 1 C NRC) also produce stronger luminescence. US Patent Application US 2021 / 0130876 A1 and WO 2021 / 084472 A1 further disclosed label molecules containing two or more ECL luminophores (e.g. Figure 2 ). These better performing ECL luminophores and the label molecules they constitute enrich the electrochemiluminescence immunoassay methodology - just as chemiluminescence has multiple platform technologies based on different chemiluminophores, electrochemiluminescence also has multiple platforms based on different ECL luminophores (e.g. Figure 3 as shown).

[0018] Due to Figure 1C, Ru(2,2'-bipyridine)(ruthenium pyrromethene disulfonate) [4-(2,2'-bipyridine-4-yl)butyric acid], which has both high luminescence efficiency and the ability to reduce non-specific adsorption in immunoassays, in the process of optimizing its ECL electrochemical reaction conditions, the present inventors unexpectedly found that, unlike the property of the luminescence signal of Ru(bpy)3 2+ increasing with the working voltage, the ECL signal produced by NRC at higher working voltage is not sensitive to voltage. Table 1 shows the ECL (Relative Light Unit) of 10 nM of Ru(bpy)3 2+ and NRC in TPA-containing phosphate buffer solution with the change of working voltage. As shown, the ECL signal values are not much different between working voltages of 1.3-1.5 V, and even at higher than 1.4 V, the luminescence of NRC does not increase, but rather decreases slightly. This feature can also be seen from Figure 4 Figure 4 The ECL produced by several different concentrations of Ru(bpy)3 2+ and NRC solutions at different voltages is shown with the change of time. It is clear that the background noise of the TPA phosphate buffer solution without luminescent substance changes the most with the voltage, while the ECL of NRC changes the least with the voltage.

[0019] Since there is background emission in the TPA phosphate buffer solution (S. S. Kumar and A. J. Bard, Anal. Chem. , 2013, 85 , 292-295), the higher working voltage required for stronger ECL will produce higher background (Table 1 also gives the background emission values at different voltages). In terms of signal-to-noise ratio, the optimal working voltage is 1.1 V. At this voltage, the signal-to-noise ratios of both Ru(bpy)3 2+ and NRC compounds (2732 and 7076, respectively) reach maximum values.

[0020] Table 1

[0021]

[0022] Further surprising to the present inventors is that at 1.1 V, the signal value of NRC (RLU = 120292) is still higher than that of Ru(bpy)3 2+ ​maximum signal value (RLU = 112037, 1.5 V). This finding suggests that in immunoassay using NRC as the signal molecule, one does not have to pursue a stronger emission, but rather maintain the ECL signal at a lower voltage to the level of Ru(bpy)3 2+ in the existing commercial system to obtain a higher signal-to-noise ratio.

[0023] The experimental results in Table 1 and Figure 4 were obtained using a homogeneous solution of Ru(bpy)3Cl2and NRC, in which all four ECL reaction pathways, 1, 2, 3 and 4, can occur at high voltage. However, in immunoassay, the ECL emitter is immobilized on the surface of magnetic beads through antibody-antigen immunoassay, and only a small fraction of NRC, which is about 2 nm from the electrode surface, can be oxidized to cation on the surface of magnetic beads with a diameter of microns (e.g., 1-5 microns) (corresponding to reaction 6 of Ru(bpy)3 2+ , so reaction pathway 1 is the main pathway for signal generation (W. Miao, J.-P. Choi, A. J. Bard, J. Am. Chem. Soc . 2002, 124 , 14478-14485). On the other hand, in immunoassay, the noise generated by the sample without the analyte (the blank value of the zero-value sample) mainly comes from three parts: one is the background emission of the TPA phosphate buffer solution, which is the same as the homogeneous solution (due to the occupation of part of the electrode surface by magnetic beads, this part of the emission should be lower than in the homogeneous solution), two is the ECL from NRC due to non-specific adsorption, and three is the oxidizable redox groups present in the biomolecules on the surface of the magnetic beads (streptavidin, antibodies and other biomolecules adsorbed non-specifically) that can participate in the emission. Therefore, the results in Table 1 obtained in the homogeneous solution cannot be directly extrapolated to the heterogeneous immunoassay on the electrode / magnetic bead surface.

[0024] The present applicant realized the value of improving the signal-to-noise ratio by generating ECL at low voltage, and demonstrated through the immunoassay of procalcitonin (PCT) that, similar to (but not exactly the same as) the performance of NRC in the homogeneous solution, in the ECL immunoassay process, when the working voltage is between 1.2 and 1.4 V, the signal-to-noise ratio changes little (84-90 for a 2 ng / mL sample and 1071-1154 for a 20 ng / mL sample), and when the working voltage is lowered to 1.1 V, the signal-to-noise ratio is significantly improved (118 for a 2 ng / mL sample and 1465 for a 20 ng / mL sample); and when the voltage is further lowered to 1.05 V, the signal-to-noise ratio is further doubled (Table 2).

[0025] Table 2

[0026]

[0027] The experimental results in Table 2 suggest that in heterogeneous immunoassays, although noise (blank value excluding the analyte) has multiple possible sources, an optimal balance between signal value and signal-to-noise ratio (SNR) can still be found by adjusting the voltage. Table 2 shows that the voltage range that causes approximately a three-fold change in SNR is 1.05–1.2 V, while within this range, the ECL signal value (RLU) changes approximately two-fold. In other words, if SNR is more important in evaluating immunoassay performance, sacrificing half the signal value can yield a three-fold improvement in SNR. Table 2 shows the change in the original ECL signal over time (…). Figure 5 The sampling and integration times vary slightly depending on the voltage, and based on these characteristics, the sampling and integration times are also parameters that can be optimized.

[0028] Ru(bpy)3 2+ The redox potentials of NRC and many of its derivatives and analogues (including NRC used in this invention) are around 1.25 V (vs. Ag / AgCl) (see M. Zhou and J. Roovers, Macromolecules, 2001, 34 ,244-252; M. Zhou, et al, Anal. Chem., 2003, 75 , 6708-6717; L. Yu, Y. Liu, M. Zhou, Anal. Bioanal. Chem. 2016, 408 (7095-7103). The generation of ECL at low voltages below its redox potential has been reported before (Y. Zu and AJ Bard, ). Anal. Chem 2000, 72 (3223-3232), reaction pathway one is based on the absence of Ru(bpy)3 2+ It is oxidized to Ru(bpy)3 3+ This was proposed based on the reaction. However, existing knowledge indicates that the oxidation of TPA at low voltage (reaction 1, a key step affecting ECL) is too slow, resulting in a very low ECL signal. Furthermore, no researchers have discovered that ECL generated at low voltage has a high signal-to-noise ratio and recognized its value. Therefore, commercial ECL immunoassay systems all use a working voltage of 1.4 V (higher than Ru(bpy)3). 2+ The redox potential is 1.25 V. Currently, no existing technology has been found to develop low-voltage (below Ru(bpy)3) redox potential. 2+The application of ECLs with a redox potential of 1.25 V is not discussed, and low-voltage ECLs are not used for the purpose of obtaining a high signal-to-noise ratio.

[0029] In view of the above findings, the applicant of this invention proposes that, in order to improve the detection sensitivity of certain immunoassays, a portion of the signal value can be sacrificed, thereby achieving a higher signal-to-noise ratio. The NRC and other high-efficiency ECL luminescent materials used in the specific embodiments of this invention (such as the compounds disclosed in US Patent 10203333, Chinese Patent ZL 201480045420, and WO 2021 / 084472A1) have higher signal-to-noise ratios than Ru(bpy)3. 2+ When it's necessary to find an optimal balance between signal strength and signal-to-noise ratio, sacrificing some signal strength, these high-efficiency ECL emitters outperform traditional Ru(bpy)3. 2+ It can better meet the requirements.

[0030] When NRC was used as an ECL marker to perform ECL immunoassay at a lower voltage and concentration-signal response curves were plotted, the applicant of this invention was even more surprised to find that, in addition to obtaining a higher signal-to-noise ratio, the low solution background generated by the lower voltage significantly improved the concentration-signal response at the low concentration end. Figure 6 This helps improve the detection capabilities of immunoassays based on low-voltage ECL signals. Data supporting this unexpected finding are detailed in specific embodiments. Summary of the Invention

[0031] This invention provides a method for achieving a higher signal-to-noise ratio by sacrificing a portion of the ECL signal value. The method involves using an electrically neutral metal coordination compound as a marker for the ECL and generating the ECL under conditions below the redox potential of the electrically neutral metal coordination compound. When using the low-voltage generated ECL for quantitative analysis of analytes, the concentration-signal response relationship and detection capability are significantly improved. Attached Figure Description

[0032] The invention will be described in conjunction with the following drawings, in which the same reference numerals denote the same elements, and wherein:

[0033] Figure 1 Chemical structural formulas of several ECL markers (ECL marker molecules disclosed in U.S. Patent 5,744,367(A), U.S. Patent 6,808,939(B), WO 2014203067A1(C) and U.S. Patent Application US2016 / 0145281A1(D)).

[0034] Figure 2Multi-label ECL labeling molecules disclosed in Chinese patent application 202010983872.X, U.S. patent application 2021 / 0130876 Al and WO 2021 / 084472 Al

[0035] Figure 3 Conventional immunoassay methods based on different detection signals

[0036] Figure 4 Electrochemiluminescence of Ru(bpy)3 in homogeneous solution at different voltages 2+ and buffer solution background noise

[0037] Figure 5 Time decay curve of electrochemiluminescence of NRC at different voltages in immunoassay

[0038] Figure 6 Concentration-signal curve of NRC at low voltage in immunoassay is superior to Ru(bpy)3 2+

[0039] Figure 7 An electrochemiluminescence signal generation procedure that includes pre-treatment and test steps (working voltage reduced from 1.4 V of prior art to 1.1 V)

[0040] Figure 8 Concentration-signal curve of PCT immunoassay at different voltages. DETAILED DESCRIPTION

[0041] Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. These terms and meanings are well-explained in technical literature, such as Bioconjugate Techniques (G.T. Hermanson, Elsevier, Amsterdam, 2008) and The Immunoassay Handbook (D. Wild et al., 4th Edition, Elsevier, Amsterdam, 2013).

[0042] Within the scope of this invention, substances referred to as "labels," "labeled molecules," "ruthenium (II) labels," and "ECL labels" can be covalently bound to other substances, such as bioactive analytes or their analogues, bioaffinity-based analyte recognition partners or their analogues (e.g., analyte-specific reagents), other binding partners of the aforementioned recognition partners, or a reactive chemical substance capable of forming a covalent bond with the analyte, or its analogues or binding partners as described above. These substances can also be bound to combinations of one or more binding partners and / or one or more reactive components. Furthermore, these substances can also be bound to analytes or their analogues that are linked to a binding partner, a reactive component, or a combination of one or more binding partners and / or one or more reactive components. The direct binding of multiple of the above substances to an analyte or its analogue, or binding to it via other molecules as described above, is also within the scope of this invention.

[0043] As used herein, the term "label" refers to any chemical or biochemical substance that, either alone or through physical / chemical interactions with other reagents, generates a detectable signal (whether a visible signal or a signal detectable by using a suitable instrument) that can be correlated with the amount of a target analyte. Labels include, but are not limited to, molecules containing radioactive atoms (radioactive), luminescent compounds (emitting light through photoexcitation or through chemical reactions), electroactive compounds (generating electrical signals through redox reactions), magnetic particles (magnetic signals), enzymes (generating detectable substances or optical signals through reactions with substrates), and enzymes or enzymatic substrates (catalyzing chemical / biochemical reactions). A label may consist of one or more signal-generating units and one or more reactive groups.

[0044] The term "luminescence" refers to the energy released in the form of electromagnetic radiation (light emission) when an electron transitions from a low-energy state to an "excited" high-energy state and then falls back to a lower-energy state. This light emission typically occurs in the visible or near-visible range of the electromagnetic spectrum. The term "luminescence" generally includes, but is not limited to, luminescent phenomena such as phosphorescence, fluorescence, bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence, and thermoluminescence; however, in this invention, unless otherwise specified, luminescence refers to electrochemiluminescence.

[0045] Within the scope of this invention, the terms "luminescent group" and "luminescent body" refer to the functional group in a compound responsible for producing luminescence. In compounds with complex structures, such as those having multiple functional groups (e.g., reactive groups, hydrophilic / hydrophobic / amphiphilic groups, electron-withdrawing / electron-donating groups, electrobalancing groups, spacer groups, linking groups, branching groups, etc.), the luminescent group is the smallest structural component required to produce luminescence (e.g., see [link to other documentation]). Figure 1 (The circled part in the middle).

[0046] The term "luminescent label" refers to a label composed of one or more luminescent groups and one or more reactive groups, which readily forms covalent bonds with the chemical or biochemical molecule to be labeled. Luminescent labels can be, for example, fluorescent molecules, phosphorescent molecules, radioluminescent molecules, electrochemiluminescent molecules (i.e., ECL labels) of the present invention, or quantum dots with reactive groups on their surface. However, in this invention, unless otherwise specified, "luminescent label" refers to an electrochemiluminescent label (ECL label). Examples of electrochemiluminescent (ECL) labels having one luminescent group and one reactive group are most frequently disclosed in the prior art (see, for example, Figure 1 Examples of luminescent markers include those in WO2003002974A2, WO2014203067A1 (other ruthenium complex markers), and WO2014019711A1 (iridium complex markers). US2005 / 0059834 A1 discloses examples of luminescent markers having three luminescent groups (three ruthenium complex units) and one reactive group (-COOH or NHS ester). US Patent 6140138 discloses examples of luminescent markers having one luminescent group (one ruthenium complex) and two reactive groups (-COOH or NHS ester). Chinese Patent Application 202010983872.X, US Patent Application 2021 / 0130876 A1, and WO 2021 / 084472 A1 disclose ECL marker molecules containing two or more luminescent groups.

[0047] Measurable "analytes" include, but are not limited to, whole cells, cell surface antigens, protein complexes, cell signaling factors and / or components, second messengers, second messenger signaling factors and / or components, subcellular particles (e.g., organelles or membrane fragments), viruses, prions, house dust mites or fragments thereof, viroids, immune factors, antibodies, antibody fragments, antigens, haptens, fatty acids, nucleic acids (and synthetic analogs), ribosomes, proteins (and synthetic analogs), lipoproteins, polysaccharides, inhibitors, cofactors, haptens, cell receptors, receptor ligands, lipopolysaccharides, glycoproteins, peptides, polypeptides, enzymes, etc. Enzyme substrates, enzyme products, nucleic acid processing enzymes (e.g., polymerases, nucleases, integrases, ligases, helicases, telomerases, etc.), protein processing enzymes (e.g., proteases, kinases, protein phosphatases, ubiquitin-protein ligases, etc.), cell metabolites, endocrine factors, paracrine factors, autocrine factors, cytokines, hormones, pharmacological drugs, drugs, therapeutic drugs, synthetic organic molecules, organometallic molecules, sedatives, barbiturates, alkaloids, steroids, vitamins, amino acids, sugars, lectins, recombinant or derived proteins, biotin, avidin, streptavidin, or inorganic molecules present in the sample.

[0048] According to the method and reagents of the present application, an "analyte specific reagent" (ASR) is a molecule or biological molecule with the ability to specifically bind to an analyte, such as antibodies, polyclonal and monoclonal antibodies, specific receptor proteins, ligands, nucleic acid sequences and the like. They are intended for use in biological analysis applications for the identification and quantification of individual chemical or biochemical substances or ligands in a biological sample by specific binding or specific chemical reaction with substances in a sample.

[0049] According to the present application, a "detection reagent" includes an analyte specific reagent (ASR) labeled with at least one ECL luminescent group, or an analyte analog / congener labeled with one ECL luminescent group. It is known to the skilled person that in an assay, the detection reagent is finally immobilized on a solid phase. A "solid phase", also referred to as "solid support", refers to a non-fluid substance, such as magnetic beads and particles (including microparticles and beads), made of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass, and ceramics; gel substances, such as silica, alumina, and polymeric gels; capillaries, which can be made of polymers, metals, glass and / or ceramics; zeolites and other porous substances; electrodes; microtiter plates; solid strips; and cuvettes, test tubes, wafers or other sample containers of a spectrometer. The solid phase component in an assay process differs from an inert solid surface that the assay process can come into contact with in that the "solid phase" contains at least one moiety on its surface that is intended to interact with a capture antibody or capture molecule. The solid phase can be a fixed component, such as a test tube, strip, cuvette, wafer, or microtiter plate, or a non-fixed component, such as a magnetic bead, bead, and microparticle.

[0050] In one embodiment, the method can be performed in a sandwich assay format. In one embodiment, the method can be performed in a competitive assay format. In one embodiment, the method can also be performed in a double antigen bridging assay format (DAGS). Known immunoassay formats are described in detail in the following books: D. Wild et al., The Immunoassay Handbook, 4thEdition, Elsevier, Amsterdam (2013) and E. P. Diamondis and T. K. Christopoulos, Immunoassays, San Diego, Academic Press (1996).

[0051] “Electrochemiluminescence immunoassay” or “ECL immunoassay” is an analytical method that generates a luminescent signal from an ECL luminescent group by electrochemical excitation. A voltage between a working electrode and a reference electrode electrochemically initiates luminescence from an ECL luminescent group bound to an ASR or an analyte analog / congener. The light emitted from the ECL luminescent group is measured by a photodetector and indicates the presence or amount of the target analyte. U.S. Pat. Nos. 5,543,112, 5,935,779, and 6,316,607 describe the ECL method in detail.

[0052] The term “working voltage” in the present invention is a key concept and experimental parameter of the present invention. In a three-electrode electrochemical measurement system consisting of a working electrode, a counter electrode, and a reference electrode, the “working voltage” is the voltage between the working electrode and the reference electrode. Unless specified, the working voltage or voltage mentioned in the present invention is relative to a silver / silver chloride (Ag / AgCl, saturated potassium chloride) reference electrode, which has an electrode potential of +0.197 V relative to the standard hydrogen electrode (SHE). When generating electrochemiluminescence or conducting electrochemiluminescence immunoassay, different instrument systems can be equipped with different reference electrodes, and the voltage between the reference electrode and the working electrode is different from the working voltage in the present invention. The difference can be converted by the electrode potential of the reference electrode. Those skilled in the art know that the electrode potential of different reference electrodes relative to the standard hydrogen electrode (SHE) and its conversion method can be found in literature and books related to electrochemistry. The core spirit of the present invention is to change the background noise and control the ECL reaction speed by adjusting the “working voltage”, so as to obtain better signal-to-noise ratio and concentration-response curve.

[0053] In the ECL analysis procedure, magnetic beads can be suspended in the sample and detection reagent to effectively bind the analyte. The magnetic beads can have a diameter of 0.05 μm to 200 μm, 0.1 μm to 100 μm, or 0.5 μm to 10 μm, and have a surface component capable of binding a biomolecule. In the ECL analysis system (Anseros YnY 2020, YnY 2050, and YnY 3030 system) used by the present applicant, the diameter of the magnetic beads is 2.8 μm. The magnetic beads can be formed of an organic polymer, polystyrene, a polystyrene copolymer such as a polystyrene / butadiene copolymer, a polystyrene / butadiene / acrylonitrile copolymer, a vinyl acrylate copolymer, a vinyl chloride / acrylate copolymer, an inert inorganic material, chromium dioxide, an iron oxide, silica, a silica mixture, a proteinaceous material, or a mixture thereof,

[0054] According to the present application, a "reagent component" comprises reagents that support ECL signal production, such as co-reactants (e.g., tripropylamine, TPA), buffers for pH control, surfactants, preservatives or antimicrobial agents, and optionally other components. The skilled artisan is aware of the components present in the reagent composition required for ECL signal production in electrochemiluminescence detection methods.

[0055] As used herein, "aqueous solution" is a homogeneous solution of a particulate, substance, or liquid compound dissolved in water, or a heterogeneous suspension having microparticles (from 0.05 pm to 200 pm in diameter) suspended in an aqueous solution. The aqueous solution can also comprise an organic solvent. Organic solvents are known to those skilled in the art, such as amines, methanol, ethanol, dimethylformamide, or dimethylsulfoxide. As used herein, it is also understood that the aqueous solution can comprise up to 50% of an organic solvent.

[0056] In the present context, a substance that participates in the ECL process in conjunction with an ECL label is referred to as an ECL "co-reactant". Commonly used co-reactants for ECL include tertiary amines (e.g., tri-n-propylamine, TPA) and analogs / congeners thereof (e.g., 2-(dibutylamino)ethanol, etc.), oxalates, and persulfates. The skilled artisan is aware of co-reactants that can be used in ECL detection methods.

[0057] As used herein, "transition metal complex" relates to an ECL luminophore comprising a transition metal ion bound to a suitable complexing or chelating agent. In one embodiment, the transition metal is selected from the group consisting of ruthenium, iridium, rhenium, osmium, europium, terbium, dysprosium; in another embodiment, the transition metal is ruthenium, iridium, rhenium, or osmium; in a further embodiment, the transition metal is ruthenium or iridium.

[0058] In one embodiment, the ECL luminophore is a class of electrically neutral metal ruthenium coordination compounds disclosed in U.S. Patent US 10203333 and Chinese Patent ZL201480045420.

[0059] In another embodiment, the ECL light-emitting group is an iridium complex and is selected from the following ECL labels. Ir(6-phenylphenanthridine)2-pyridine-2-carboxylic acid or its derivatives, including, for example, Ir(6-phenylphenanthridine)2-3-hydroxypyridine-2-carboxylic acid, Ir(6-phenylphenanthridine)2-4-(hydroxymethyl)pyridine-2-carboxylic acid, Ir(6-phenylphenanthridine)2-2-(carboxyethyl-phenyl)pyridine-2-carboxylic acid, Ir(6-phenylphenanthridine)2-5-(methoxyl)pyridine-2-carboxylic acid, or Ir(6-phenylphenanthridine)2-2-(carboxyethyl-phenyl)pyridine-2-carboxylic acid ester, or its derivatives, for example, iridium complexes in which the ligand is substituted with one or more sulfonic acids, or as described in WO2012107419(A1), WO2012107420(A1), WO2014019707(A2), WO2014019708(A1), WO2014019709(A2), WO2014019710(A1), WO2014019711(A1). It is well known to those skilled in the art that iridium (III) complexes have poor solubility in aqueous solutions, and hydrophilic derivatives of the above ECL compounds can be used. Thus, in another embodiment, the above iridium (III) ECL light-emitting groups can be modified with hydrophilic substituents. In another embodiment, the ECL light-emitting group is an iridium complex with two phenylphenanthridine ligands having two sulfonylpropoxy substituents, two sulfomethyl groups, including 2,9-phenanthridine dimethylsulfonic acid, 6-phenyl-sodium salt (CAS Registry Number 1554465-50-7), or two polyethylene glycol substituents, or three of the above groups in each phenylphenanthridine ligand, or a combination of the above groups in each phenylphenanthridine ligand.

[0060] In another embodiment, the ECL label is a multi-label ECL labeling molecule disclosed in Chinese Patent Application 202010983872.X, U.S. Patent Application 2021 / 0130876 Al and WO 2021 / 084472 Al (e.g., as exemplified in Figure 2

[0061] Since all of the above classes of ECL labels exhibit stronger luminescence than Ru(bpy)3 2+ at the expense of some signal for higher signal-to-noise ratio.

[0062] ​Those skilled in the art know that in an ECL immunoassay, the light signal is triggered by a constant working voltage to excite the ECL reaction, which eventually leads to the light signal generated by the ECL luminophore immobilized on the surface of the magnetic bead. The time of constant voltage excitation is usually between 0.2 and 10 seconds, preferably between 1.0 and 3.0 seconds. During this voltage excitation, the ECL light signal decays gradually (as shown in Figure 5 The area under the ECL light decay curve within a certain time is the relative light unit (RLU). In a particular immunoassay, different RLU corresponds to different concentrations of the analyte. The relationship between the signal response expressed in RLU and the concentration is usually described by a fitted function (linear or nonlinear). Figure 6 The two fitted curves in FIG. 1 fit the four-parameter logistics equation.

[0063] Those skilled in the art also know that in an automated ECL testing system, there is usually a pre-treatment procedure for the electrode surface, which is applied before the sample test to ensure that the electrode surface maintains the same surface chemical state before the test. After a test, the electrochemical measuring cell and the electrode surface need to be cleaned and regenerated. These steps are disclosed in U.S. Patent 5,147,806, and further improved in U.S. Patent 6,599,473. In describing these steps, the terms used in the prior patents are preoperative, conditioning, cleaning. Figure 7 An electrochemiluminescence signal generation procedure is described, which includes the pre-treatment and test steps, but does not include the cleaning procedure after the test step. The pre-treatment and cleaning procedures can be different in different ECL systems, for example, a voltage pulse is added in the electrochemical procedure disclosed in U.S. Patent 6,599,473, which is believed to improve the deposition state of the magnetic bead on the electrode. The present invention does not involve any improvement of the pre-treatment and cleaning procedures, the gist of the present invention is to lower the working voltage in the test step of the electrochemical procedure to below the redox potential of the ECL luminophore, as shown in Figure 7 from 1.4 V to 1.1 V.

[0064] The present invention is further illustrated by the following description of specific embodiments, which are not intended to limit the present invention, and those skilled in the art can make different modifications to each embodiment according to the basic spirit of the present invention, as long as the modifications do not deviate or depart from the basic spirit of the present invention (i.e., applying a working voltage below the redox potential of the ECL luminophore to generate the ECL signal), the embodiment is within the scope of the present invention.

[0065] In one embodiment, the present invention relates to a method for detecting an analyte in a sample, the method comprising the steps of:

[0066] a) incubating the sample with a detection reagent to provide detection reagent bound to the analyte,

[0067] wherein the sample contains the analyte,

[0068] wherein the detection reagent is labeled with one or more ECL labels, comprising one or more luminescent groups;

[0069] wherein the ECL luminescent groups are transition metal complexes, and

[0070] wherein the detection reagent bound to the analyte is immobilized on a solid phase;

[0071] b) separating the detection reagent bound to the analyte from the detection reagent not bound to the analyte and other non-immobilized substances to provide separated detection reagent bound to the analyte, wherein the separated detection reagent bound to the analyte is immobilized on the solid phase;

[0072] c) contacting the separated detection reagent bound to the analyte with an aqueous buffer solution, wherein the aqueous buffer solution comprises at least one tertiary amine;

[0073] d) applying a working voltage between a working electrode and a reference electrode, which is lower than 1.25 V (vs. Ag / AgCl reference electrode in a potassium chloride solution) to trigger the electrochemical oxidation of the tertiary amine and the subsequent ECL reaction, thereby releasing an ECL signal; and

[0074] e) detecting the electrochemiluminescence signal and correlating the signal intensity with the analyte concentration by an appropriate algorithm.

[0075] In one embodiment, the method further comprises the initial step of providing the detection reagent by labeling an analyte-specific reagent with one or more ECL labels.

[0076] In another embodiment, the method further comprises the initial step of providing the detection reagent by labeling the analyte or an analyte analog / congener / derivative with an ECL label.

[0077] In another embodiment, the ECL groups comprise ruthenium or iridium complexes, and the working electrode is platinum, gold, and carbon.

[0078] In another embodiment, the plurality of luminescent groups are the same.

[0079] In another embodiment, the plurality of luminescent groups can be different.

[0080] In another embodiment, the tertiary amine is an alkyl tertiary amine.

[0081] In another embodiment, the tertiary amine is a branched amine.

[0082] In another embodiment, the tertiary amine is tri-n-propylamine (TPA), tributylamine, triethylamine, or N,N-dibutylethanolamine.

[0083] In another embodiment, the analyte-specific reagent is a monoclonal antibody.

[0084] In another embodiment, the analyte-specific reagent is a protein or nucleic acid recognition buddy of the analyte or its analogues.

[0085] In another embodiment, the analyte-specific reagent is an analogue / homologous / derivative of the analyte.

[0086] In another embodiment, the tertiary amine is at least 10 times more concentrated than the ECL marker. For example, the concentration of the tertiary amine is from 10 μM to 1 M, preferably from 10 mM to 500 mM.

[0087] In another implementation, the operating voltage below 1.25 V is maintained for a period of time, typically between 0.2 seconds and 10 seconds, preferably between 1.0 seconds and 3.0 seconds.

[0088] However, in all implementations, the operating voltage used to trigger the ECL reaction does not exceed 1.25 V.

[0089] The following examples illustrate the spirit of the invention. That is, by using an operating voltage lower than the redox potential of the ECL luminescent material to generate the ECL signal, an improved concentration-signal relationship curve is obtained while achieving a high signal-to-noise ratio.

[0090] Example

[0091] Example 1 - Signal Antibody Labeling

[0092] use Figure 1 The NRC shown in C, namely Ru(2,2'-bipyridine)(red phenanthrene-line disulfonate) [4-(2,2'-bipyridine-4-yl)butyric acid], is labeled with a PCT antibody to form an NRC-labeled signal antibody.

[0093] 2.5 mg (2.5 μmol) of NRC was administered at 5.0 mmol / L. -1 The concentration was dissolved in 500 μL of MES buffer (0.1 mol / L). 1in 0.1 mol L 1 , pH = 4.7). To this solution, 1.0 mg (5.2 μmol) of EDC and 3.0 mg (13.8 μmol) of sulfo-NHS were added to obtain a concentration of about 10 mmol L 1 of EDC and 27 mmol L 1 of sulfo-NHS. This solution was shaken at room temperature for 10 minutes. 0.7 μL (10 μmol) of 2-mercaptoethanol was added to the above reaction solution (final concentration of 20 mmol L 1 ). After 5 minutes at room temperature, 8.0 μL (containing 40 nmol of NRC) of this incubation solution was added to 500 μL of AFP antibody (1.2 mg / mL, about 4 nmol of pure PCT antibody, molar reaction ratio of 10) in PBS (0.1 mol L

[0094] The above obtained solution (about 0.5 ml) was loaded onto a PD-10 column previously equilibrated with PBS. Two yellow bands were formed during the separation process. The first elution band corresponding to the labeled antibody was collected (about 0.75 ml). The binding ratio of the label NRC to the antibody was determined to be 6.1:1.

[0095] Example 2 - Biotinylation of Capture Antibody

[0096] To 2 mL of CBS buffer (pH 9.5) containing 2.1 mg of desalted PCT capture antibody, an aliquot (15 μL) of 20 mM NHS-LC-biotin (MW 454.54) in DMF was added, and the mixture was incubated for 1 hour, followed by a total of 20 hours of dialysis in PBS buffer. The final concentration of biotinylated PCT capture antibody was determined to be 1.72 mg mL by BCA assay. The degree of biotinylation was determined using a competition method (see Y. Xu, Y. Pan, L. Li and M. Zhou, ACS Omega , 2020, 5 , 32591-32596).

[0097] Example 3 - Immunoassay

[0098] The NRC-labeled PCT signal antibody and the biotinylated PCT capture antibody were diluted to 1 μg mL -1 and 4 μg mL -1Dynabeads® M-280 coated with streptavidin as magnetic medium for capturing the biotinylated antibody / antigen / ruthenium labeled antibody immune complex.

[0099] On a fully automated immunoassay instrument with variable working voltage (ProScientia 2020, Shenzhen Ansay Diagnostic Technology Co. Ltd.), 50 μΐ of different concentrations of PCT (analyte) in PBS solution were mixed with 85 μΐ of biotinylated PCT capture antibody in PBS solution at a concentration of 4 μg mL -1 and 85 μΐ of NRC labeled signal antibody solution at a concentration of 1 μg mL -1 . Each mixture was incubated at 37.2 degrees Celsius for 10 minutes. 20 μΐ of a suspension of streptavidin coated Dynabeads® M-280 (0.75 mg mL -1 ) was added to the above reaction mixture solution, and after 10 minutes of continued reaction at 37.2 degrees Celsius, 150 μΐ of the above reaction suspension was injected into the three electrode measuring cell of the fully automated immunoassay instrument, with a photomultiplier tube above the working electrode and a movable magnet below the working electrode. ECL was generated by a redox process in phosphate buffer (pH 6.8, 0.18 mol L -1 trispropylamine solution). After each measurement, the measuring cell was cleaned and the electrodes were electrochemically regenerated using the methods described in U.S. Patents 5,538,687 and 6,599,473 Bl.

[0100] Table 3 is the result of ECL measurements of PCT solutions of different concentrations with adjusted working voltage.

[0101] Table 3

[0102]

[0103] The data in Table 3 were plotted and fitted with a four parameter logistics equation to obtain the concentration-signal curve Figure 8 . It is evident that the curve at the low concentration end becomes steeper as the working voltage is lowered. This is especially true at 1.05 V and 1.1 V. This indicates that a lower detection limit can be obtained. For PCT, the detection limit obtained at 1.4 V is 0.02 ng mL -1 , while at a working voltage of 1.05 V, the lowest detection limit reaches 0.005 ng mL -1 .

[0104] From the foregoing description, it will be apparent to those skilled in the art that the improved signal-to-noise ratio and the improved concentration-signal response relationship of the low voltage ECL disclosed herein are readily apparent. Those skilled in the art will therefore recognize that changes or modifications can be made to the above-described embodiments without departing from the broad inventive concepts thereof, and that such changes or modifications are intended to be included within the scope and spirit of the application. It is to be understood that the application is not limited to the specific embodiments described herein, but encompasses all changes and modifications that fall within the scope and spirit of the application.

Claims

1. An immunoassay method employing low operating voltage to excite electrochemiluminescence, characterized in that, The electrochemiluminescence is derived from the metal complex, and the low operating voltage is lower than the redox potential of the metal complex; a) The low operating voltage mentioned above is the voltage between the working electrode and the reference electrode. When the reference electrode of the test system is an Ag / AgCl electrode containing potassium chloride solution, it is between 1.0 V and 1.25 V. When the reference electrode of the test system is not the Ag / AgCl electrode containing potassium chloride solution, according to the potential conversion relationship between different reference electrodes, the low operating voltage after conversion to an Ag / AgCl electrode containing potassium chloride solution is between 1.0 V and 1.25 V. b) The metal complex is Ru(2,2'-bipyridine)(bathophenanthroline disulfonate)[4-(2,2'-bipyridin-4-yl)butanoic acid].

2. The method according to claim 1, characterized in that, During the time interval in which electrochemiluminescence is generated, the low operating voltage is a constant voltage between 1.0 V and 1.25 V.

3. The method according to claim 1, characterized in that, During the time interval in which electrochemiluminescence is generated, the low operating voltage is a voltage that varies between 1.0 V and 1.25 V.

Images