Ultrasensitive chemiluminescent substrates for peroxidase

Abstract

A method for increasing the light emission produced by the chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, a peroxidase or a conjugate thereof, an enhancer, a co-enhancer and a peroxide oxidant, wherein the enhancer is an anionic N-alkylphenothiazine and the co-enhancer is selected from 4-dialkylaminopyridine or N-azole, and wherein the method comprises the steps of: i. achieving a chemiluminescent substrate by mixing together 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, the enhancer, the co-enhancer and the peroxide oxidant, and ii. adding the peroxidase or a conjugate thereof to the chemiluminescent substrate.

Description

Technical Field

[0001] The present invention relates to a chemiluminescent substrate for use in determining peroxidase in an assay for determining an analyte in a sample, wherein the chemiluminescent substrate allows quantitative peroxidase detection with very high sensitivity. Background Art

[0002] The chemiluminescent oxidation of luminol catalyzed by horseradish peroxidase (HRP) is widely used in analytical tests for antigens, antibodies and nucleic acids, and especially in blot tests such as dot blots, Western blots (proteins), Southern blots and Northern blots (nucleic acids).

[0003] It is known that the chemiluminescent oxidation of luminol catalyzed by HRP can be made faster and more efficient by adding an electron mediator or enhancer, as described, for example, by Kricka LJ (1991), Clinical Chemistry, 37: 1472-1481; or by Kricka LJ, Voyta JC and Bronstein I in "Chemiluminescent Methods for Detecting and Quantitating Enzyme Activity" (2000), Methods Enzymol; 305: 370-390.

[0004] The mechanism of the enhanced chemiluminescence reaction (ECL) in which luminol and enhancer are oxidized simultaneously is described as follows [Lind J, Merenyi G and Eriksen TE (1983), J Am Chem Soc, 105: 7655-7661]. In the first step of ECL, the substrate-enhancer (E), which is more active than luminol for HRP, is oxidized by hydrogen peroxide in the presence of HRP according to a "ping-pong" mechanism:

[0005] HRP+H2O2→HRP-I (1)

[0006] HRP-I+E→HRP-II+E · (2)

[0007] HRP-II+E→HRP+E · (3)

[0008] Where E is the enhancer, E ·is the free radical product of the one-electron oxidation of the enhancer. HRP is horseradish peroxidase in its Fe(III) resting state. HRP-I and HRP-II are oxidative intermediates of peroxidase, which are two and one oxidation equivalents higher than the calcium resting state, respectively. Then, the free radical product of the enhancer (E · ) and the luminol molecule (LH - ) reacts reversibly [Easton PM, Simmonds AC, Rakishev A, Egorov AM and Candeias LP (1996), JAm Chem Soc; 118: 6619-6624]:

[0009]

[0010] Thermodynamically, the position of the redox equilibrium (4) is determined by the difference in reduction potential between the enhancer and the luminol radical under the experimental conditions. ·- ) is disproportionated to luminol anion (LH - ) and diazaquinone intermediate (L):

[0011] 2L ·- →LH - +L (5)

[0012] The diazaquinone intermediate (L) reacts with hydrogen peroxide to form luminol peroxide (LO2 2- ), which collapses with the expulsion of molecular nitrogen into the excited state of 3-aminophthalate ([AP 2- ]*). [AP 2- ]*Then return to the ground state (AP 2- ), and emits a photon (hν) at 425nm:

[0013] L+H2O2→LO2 2- →[AP 2- ]*+N2→AP 2- +hν (6)

[0014] The intensity of the emitted light is related to the luminol free radical (L ·- ) is proportional to the square of the rate of generation of the luminol radical. The quadratic relationship is a consequence of the excited species generation mechanism, which involves the disproportionation of two luminol radicals, Equation 5. In turn, the rate of generation of the luminol radical is given by the enzyme conversion rate, which results in the luminol radical (L ·-). The enzyme conversion rate is controlled by the rate-determining step, namely the reduction of HRP-II to ferroenzyme (HRP), equation (3). In summary, the enhancement of chemiluminescence can be well approximated by considering (a) the promotion of enzyme conversion by the reaction of the enhancer with HRP-II, and (b) the reversible electron transfer reaction between the enhancer radical and luminol.

[0015] Many compounds have been successfully used to enhance HRP-induced chemiluminescence, including: luciferin, 6-hydroxybenzotriazole, p-iodophenol, p-coumaric acid and other phenolic enhancers (Thorpe GHG and Kricka LJ (1986), Methods Enzymol; 133:331); aromatic amines (U.S. Pat. No. 4,279,950); acetanilide (European Patent Application No. 603953); N-substituted phenothiazines (U.S. Pat. No. 5,171,688 and U.S. Pat. No. 6,432,662); boric acid (U.S. Pat. No. 5,629,168).

[0016] A fundamental limitation in the chemiluminescent output of luminol-based substrates relates to their relatively low luminescence quantum yield, Equation (6). For this reason, a number of luminol analogs have been developed, the most notable being 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, also known as L-012 (European Patent Application No. 491477), which has the following chemical structure:

[0017]

[0018] Thus, in a p-iodophenol- and borate-enhanced chemiluminescence assay for horseradish peroxidase, this compound showed up to a 10-fold increase in light emission compared to luminol [Ji X, Kondo K, Aramaki Y and Kricka LJ (1996) J Biolumin Chemilumin; 11: 1-7].

[0019] Recently, several compounds belonging to the lophanine class were tested as enhancers in the 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione substrate. In particular, using 2-(4-hydroxyphenyl)-4,5-di(2-pyridyl)imidazole as an enhancer, the chemiluminescent system was found to be 20 times more sensitive than the conventional enhanced luminol system using 4-iodophenol [Ichibangase T, Ohba Y, Kishikawa N, Nakashima K, and Kuroda N (2014) Luminescence 29:118-121].

[0020] However, recent developments in luminol-based technology have resulted in chemiluminescent HRP substrates that are at least as sensitive as those based on L-012 [U.S. Patent No. 7803573 (2010), U.S. Patent No. 9040252 (2015)].

[0021] Therefore, given that the chemiluminescent efficiency of the chemiluminescent compound L-012 is higher than that of luminol, there is still sufficient potential to further increase the sensitivity of peroxidase detection.

[0022] OBJECTIVE AND SUMMARY OF THE INVENTION

[0023] An object of the present invention is to provide an ultrasensitive chemiluminescent substrate for the quantitative determination of peroxidase in an assay for detecting an analyte in a sample.

[0024] According to the invention, the above objects are achieved thanks to the kit specified in the appended claims, which are to be understood as forming an integral part of the present description.

[0025] According to one embodiment, the present disclosure relates to a method for increasing the light emission produced by the chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, peroxidase, enhancer, co-enhancer and peroxide oxidant, wherein the enhancer is an anionic N-alkylphenothiazine and the co-enhancer is selected from 4-dialkylaminopyridine or N-azole.

[0026] The present invention also relates to a kit for performing a method for increasing the light emission produced by the chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione as disclosed herein, wherein the kit comprises 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, a peroxide oxidant, an enhancer and a co-enhancer, wherein the enhancer is an anionic N-alkylphenothiazine and the co-enhancer is selected from 4-dialkylaminopyridine or N-azole. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0028] - Figure 1 Shown are the structures of anionic N-alkylphenothiazine enhancers of the present invention.

[0029] - Figure 2 A graph showing the pH dependence of the chemiluminescent signal of the L-012 / peroxide / SPTZ system is shown.

[0030] - Figure 3 Shown is a bar graph of the initial chemiluminescent signal of the L-012 / perborate substrate enhanced by SPTZ and CPTZ in the presence of the co-enhancers imidazole (IMDZ); 1-methylimidazole (MIMDZ), 1,2,3-triazole (TRIAZ), 4-morpholinopyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinylpyridine (PPY).

[0031] - Figure 4 The dependence of the chemiluminescent signal on the concentration of L-012 in the L-012 / perborate / SPTZ / MORP substrate is shown.

[0032] - Figure 5 The dependence of the chemiluminescent signal on the SPTZ concentration in the L-012 / perborate / SPTZ / MORP substrate is shown.

[0033] - Figure 6 The dependence of the chemiluminescent signal on the MORP concentration in the L-012 / perborate / SPTZ / MORP substrate is shown.

[0034] - Figure 7 The dependence of the chemiluminescent signal on the perborate concentration in the L-012 / perborate / SPTZ / MORP substrate is shown.

[0035] - Figure 8 The pH dependence of the chemiluminescent signal in the L-012 / perborate / SPTZ / MORP substrate is shown.

[0036] - Fig. 9 The pH dependence of the chemiluminescent signal in the L-012 / perborate / SPTZ / imidazole substrate is shown.

[0037] - Fig.10 The dependence of the chemiluminescent signal on the imidazole concentration in the L-012 / perborate / SPTZ / MORP substrate is shown.

[0038] - Fig.11 Shown is a comparison between the initial chemiluminescent signals in the luminol / perborate / SPTZ and L-012 / perborate / SPTZ substrates in the absence and presence of MORP and imidazole co-enhancer.

[0039] - Fig.12 A graph showing the chemiluminescent signal of the L012 / B3 and luminol / A3 substrates as a function of the amount of horseradish peroxidase (HRP) is shown.

[0040] - Fig.13Shown is a Western blot of human IKBα obtained with L012 / B3 substrate and the luminol-based substrate WestarSupernova.

[0041] - Fig.14 ELISA dose response curves for human HDAC-1 against L012 / B3 substrate and the luminol-based substrate SuperSignal Femto ELISA are shown.

[0042] Detailed Description of the Invention

[0043] In the following description, many specific details are given to provide a thorough understanding of the embodiments. The embodiments described may be practiced without one or more of the specific details, or with other methods, components, materials, etc. In other cases, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the embodiments.

[0044] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics may be combined in any suitable manner.

[0045] The headings provided herein are for convenience only and do not interpret the scope or meaning of the embodiments.

[0046] The present disclosure discloses a method for performing a chemiluminescent assay for detecting an analyte in a sample, comprising:

[0047] a. reacting the analyte with an analyte detection reagent, wherein the detection reagent is directly or indirectly conjugated with a peroxidase to form a composite analyte-detection reagent;

[0048] b. reacting the complexed analyte-detection reagent with a chemiluminescent substrate comprising an enhancer, a co-enhancer, a peroxide oxidant, and 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, which co-react in a chemiluminescent reaction with light emission, wherein the enhancer is an anionic N-alkylphenothiazine and the co-enhancer is selected from 4-dialkylaminopyridines and N-oxazoles; and

[0049] c. Detecting the analyte by measuring light emission.

[0050] In one embodiment, the present invention relates to a method for increasing the light emission produced by the chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, a peroxidase or a conjugate thereof, an enhancer, a co-enhancer and a peroxide oxidant, wherein the enhancer is an anionic N-alkylphenothiazine and the co-enhancer is selected from 4-dialkylaminopyridine or N-azole, wherein the method comprises the following steps:

[0051] (i) a chemiluminescent substrate is produced by mixing 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, an enhancer, a co-enhancer and a peroxide oxidant, and

[0052] (ii) adding peroxidase or its conjugate to the chemiluminescent substrate.

[0053] In one embodiment, the anionic N-alkyl phenothiazines are selected from 3-(10H-phenothiazin-10-yl)propane-1-sulfonic acid (SPTZ), 4-(10H-phenothiazin-10-yl)butane-1-sulfonic acid (SBTZ), 3-(10H-phenothiazin-10-yl)propionic acid (CPTZ) and 4-(10H-phenothiazin-10-yl)butanoic acid (CBTZ) and salts thereof. The chemical structures of these N-alkyl phenothiazines are shown in Figure 1 Shown in.

[0054] A co-enhancer or second enhancer is defined as a compound that does not enhance the chemiluminescent reaction of luminol or its analogs by itself, but increases the light output when used in combination with an enhancer, see Marzocchi E, Grilli S, Della Ciana L, Prodi L, Mirasoli M and Roda A (2008) Anal biochem, 377: 189-194; Vdovenko MM, Della Ciana L and Sakharov IY (2009) Anal Biochem, 392: 54-58. The increase in light output is attributed to a corresponding increase in peroxidase enzyme conversion, see Sakharov IY and Vdovenko MM (2013) Anal Biochem, 434: 12-14.

[0055] The co-enhancers of the present invention belong to two different classes of compounds: 4-dialkylaminopyridines (as described in US 7,803,573) and N-azoles (as described in US 9,040,252). Among the 4-dialkylaminopyridines, preferred compounds are 4-morpholinopyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinylpyridine (PPY). Among the N-azoles, preferred compounds are imidazole and 1-methylimidazole.

[0056] The peroxide oxidant can be hydrogen peroxide or any complex of hydrogen peroxide that releases hydrogen peroxide after dissolving in an aqueous solution, such as perborate, percarbonate, or a urea / hydrogen peroxide complex with a molar ratio equal to 1: 1. In a preferred embodiment, the peroxide oxidant is selected from hydrogen peroxide, urea / hydrogen peroxide complex, perborate, and percarbonate.

[0057] Peroxidase is any peroxidase suitable for chemiluminescent assays. According to one embodiment, the peroxidase is selected from horseradish peroxidase (e.g., Sigma VIA or IX type), or anionic peroxidases such as soybean peroxidase and sweet potato peroxidase. The peroxidase may be conjugated or not conjugated with a detection reagent for an analyte to be detected.

[0058] According to one embodiment, the concentration of the chemiluminescent compound 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione in the chemiluminescent substrate is between 0.01 mM and 1 mM, preferably between 0.05 and 0.5 mM.

[0059] According to one embodiment, the concentration of the anionic N-alkylphenothiazine enhancer in the chemiluminescent substrate is comprised between 0.1 and 10 mM, preferably between 0.5 and 5 mM.

[0060] According to one embodiment, the concentration of the co-enhancer in the chemiluminescent substrate is comprised between 0.1 and 100 mM, preferably between 0.5 and 50 mM.

[0061] According to one embodiment, the concentration of peroxide in the chemiluminescent substrate is comprised between 0.1 and 10 mM, preferably between 0.5 and 8 mM.

[0062] According to one embodiment, the pH of the chemiluminescent substrate is comprised between 5.0 and 9.0, preferably between 6.0 and 8.5.

[0063] According to a preferred embodiment, the chemiluminescent substrate having a pH comprised between 5.0 and 9.0, preferably between 6.0 and 8.5, contains:

[0064] i) 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione at a concentration between 0.01 mM and 1 mM, preferably between 0.05 and 0.5 mM;

[0065] ii) anionic N-alkylphenothiazine enhancers at a concentration between 0.1 and 10 mM, preferably between 0.5 and 5 mM;

[0066] iii) a co-enhancer at a concentration between 0.1 and 100 mM, preferably between 0.5 and 50 mM;

[0067] iv) a peroxide oxidizing agent at a concentration between 0.1 and 10 mM, preferably between 0.5 and 8 mM.

[0068] According to one embodiment, the method further comprises the step of detecting the analyte in the sample by measuring the light emission produced by the chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, peroxidase, peroxide oxidant, enhancer and co-enhancer.

[0069] The present invention also relates to a kit for performing the method for increasing the light emission produced by the chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione as disclosed herein, wherein the kit comprises 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, a peroxide oxidant, an enhancer and a co-enhancer, wherein the enhancer is an anionic N-alkylphenothiazine and the co-enhancer is selected from 4-dialkylaminopyridine or N-azole.

[0070] In one embodiment, the anionic N-alkylphenothiazine is selected from 3-(10H-phenothiazin-10-yl)propane-1-sulfonic acid, 4-(10H-phenothiazin-10-yl)butane-1-sulfonic acid, 3-(10H-phenothiazin-10-yl)propionic acid and 4-(10H-phenothiazin-10-yl)butanoic acid, and salts thereof.

[0071] In one embodiment, the co-enhancer is selected from imidazole, 1-methylimidazole, 4-morpholinopyridine (MORP), 4-dimethylaminopyridine (DMAP), and 4-pyrrolidinylpyridine (PPY).

[0072] In one embodiment, the peroxide oxidant is selected from hydrogen peroxide, a complex of urea and hydrogen peroxide in a molar ratio of 1:1, perborate, and percarbonate.

[0073] In another embodiment, the kit further comprises a peroxidase. Preferably, the peroxidase is selected from horseradish peroxidase, soybean peroxidase and sweet potato peroxidase. In various embodiments, the peroxidase (or its conjugate) can be added by the end user.

[0074] In one embodiment, the kit contains 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione in a first vial and a peroxide oxidant in a second vial, and wherein the enhancer and co-enhancer are present in the first vial or the second vial or both vials.

[0075] Kit reagents are present in vials in liquid or solid form. If the kit reagents are solid, they may be in powder or tablet form and must be reconstituted in liquid phase by adding water or a buffer solution when used.

[0076] It is best to prepare the 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione and the peroxide oxidant in separate vials to extend their shelf life. The enhancer and co-enhancer, as well as other additives such as chelators and stabilizers, can be added to either the 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione or peroxide oxidant vial, or to both vials. The two vials also contain a buffer substance that, when mixed, produces a chemiluminescent substrate or "working solution" at a pH appropriate for the assay.

[0077] If peroxidase or its conjugate is present in the kit, it is not stored with its substrate component (ie, the peroxide oxidizing agent), but rather is stored in a third vial.

[0078] The term "assay" refers to semi-quantitative and quantitative detection of an analyte. Typically, the implementation of an assay requires correlating the light output with the amount of peroxidase used. Thus, light emission is detected or measured so that the presence or amount of the analyte is related to the generation of light.

[0079] According to the present disclosure, the implementation of the assay requires correlating the light output generated by the chemiluminescent reaction of the chemiluminescent compound reacted with the peroxidase, enhancer, co-enhancer and peroxide oxidant to the amount of any reaction partner (i.e., any one of (8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, peroxidase, peroxide oxidant, enhancer and co-enhancer). According to a preferred embodiment, the light output is correlated to the amount of peroxidase used.

[0080] In an assay for detecting an analyte of interest (qualitative or quantitative), the peroxidase is in the form of a conjugate with a detection reagent for the analyte to be detected. Depending on the type of analyte to be detected, the detection reagent may be selected from antibodies, nucleotides, oligonucleotides or nucleic acid molecules. The detection reagent conjugated to the peroxidase may be a detection reagent capable of specifically binding to the analyte, or a second detection reagent capable of binding to the detection reagent that specifically binds to the analyte.

[0081] The chemiluminescent reaction of the present invention is suitable for detecting and quantifying analytes, for example, using bond formation between an analyte (e.g., a protein or nucleic acid molecule) and a solid support (e.g., a membrane or microtiter plate), and using peroxidase as a tracer. The luminescent reaction is initiated by adding a chemiluminescent substrate as disclosed herein to a solid support (previously reacted with a sample containing the analyte), and then adding a solution containing peroxidase to the solid support. The light emission is long-term and can be measured by film, a camera, or other instruments.

[0082] Chemiluminescent assays based on chemiluminescent substrates according to the present invention include dot blot and western blot assays for proteins and Southern and Northern blot assays for nucleic acids.

[0083] The chemiluminescent substrate-based blotting assay according to the present disclosure uses gel electrophoresis to separate the target analyte (protein or nucleic acid molecule) from other components (other proteins or other nucleic acid molecules) present in the sample to be tested. The analyte and other components are then transferred to a membrane where they are probed (detected) using a chemiluminescent substrate as disclosed herein and a detection reagent that can specifically bind to the target analyte, wherein the detection reagent is conjugated to a peroxidase.

[0084] A comparison between Western blots obtained using the L-012-based substrate of the invention (B3 / Example 10) and a commercially available luminol-based substrate (Westar Supernova from Cyanagen) is described in Example 12. Fig.13 As shown in , the L-012 based substrate of the present invention allows for the detection of approximately three additional bands relative to the luminol based substrate.

[0085] Chemiluminescent assays based on the chemiluminescent substrates of the present invention also include enzyme immunoassays (EIA), such as ELISA assays. Enzyme immunoassays are particularly suitable for detecting very small amounts of analytes in samples, such as tumor markers, thyroid hormones, viral proteins (such as HIV, HCV, HPV proteins) or steroid hormones (such as estradiol, aldosterone). The ELISA analysis performed for detecting analytes involves at least one detection reagent that is specific to the target analyte. The analyte in the sample is fixed on a solid support (usually a polystyrene microtiter plate) nonspecifically (by adsorption to a solid support surface) or specifically (by being captured to a solid support surface by another detection reagent specific for the same analyte, in a "sandwich ELISA"). After the analyte is fixed on the solid support surface, a detection reagent is added to form a complex with the analyte. The detection reagent can be covalently linked to a peroxidase as a tracer, or it can itself be detected by a second detection reagent that is linked to a peroxidase by (biological) conjugation (such as via biotin or streptavidin). Between each step, the plate is typically washed with a mild detergent solution to remove any proteins or antibodies that are not specifically bound. After the final wash step, the plate is developed by adding a chemiluminescent substrate as described herein, and, in the case where the detection reagent is not directly conjugated to the peroxidase, the solution contains the peroxidase conjugated to a second detection reagent to produce a light signal that indicates the amount of analyte in the sample.

[0086] Example 13 describes a human HDAC-1 ELISA assay using the L-012-based substrate of the invention (B3 / Example 10) and a commercially available luminol-based substrate (SuperSignal Femto ELISA from Thermo Scientific) for detection. Fig.14 As can be seen in Figure 5, the L-012 / B3 substrate of the present invention is about five times better than the luminol-based substrate in terms of sensitivity.

[0087] According to the present invention, the chemiluminescent signal generated by the SPTZ-enhanced L-012 chemiluminescent substrate is about three times higher than that of the optimized Luminol / SPTZ substrate, as shown in Examples 1, Figure 2 Examples 3 to 11 illustrate the effect of adding a co-enhancer to an L-012 chemiluminescent substrate enhanced with anionic N-alkylphenothiazines in the presence of a co-enhancer selected from 4-dialkylaminopyridines and N-oxazoles.

[0088] L-012 / peroxide / peroxidase enhanced by 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) pH dependence of chemiluminescent reaction (Example 1)

[0089] The optimum pH of HRP is in the range of 6.0 to 6.5. The activity at 7.5 is 84% ​​of the maximum activity. The enzyme is most stable in the pH range of 5.0 to 9.0 [Schomberg et al., Enzyme Handbook 7, EC 1.11.1.7: 1–6 (1993)]. On the other hand, the non-enzymatic chemiluminescent oxidation of luminol by peroxide reaches its maximum efficiency at about pH 11.0 for millimolar peroxide concentrations [Merenyi G. and Lind Johan S. "Role of a peroxide intermediate in the chemiluminescence of luminol. A mechanistic study." Journal of the American Chemical Society 102.18 (1980): 5830-5835].Peroxidase-catalyzed enhanced chemiluminescent oxidation of luminol is well known for its ability to be detected by typical enhancers such as p-iodophenol [Thorpe et al., "Phenols as enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen peroxide reaction: application in luminescence-monitored enzyme immunoassays." Clinical chemistry 31.8 (1985): 1335-1341], p-iodophenylboronic acid [Kricka et al., "Synthesis and characterization of 4-iodophenylboronic acid: a new enhancer for the horseradish peroxidase-catalyzed chemiluminescent oxidation of luminol." Analytical biochemistry 240.1 (1996): 119-125] and more recently 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) [Marzocchi et al., "Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts." Analytical biochemistry 377.2 (2008): 189-194] is most effective at pH 8.4-8.6. The optimal pH of 8.4-8.6 is a compromise between the optimal conversion of HRP (6.0-6.5) and the peroxide oxidation of luminol (pH 11.0).

[0090] In contrast to the luminol system, the L-012 / peroxide / peroxidase chemiluminescent reaction enhanced by 3-(10'-phenothiazinyl)propane-1-sulfonate (SPTZ) reaches its optimum at or below pH 7.2. Figure 2 .

[0091] pH dependence of the chemiluminescent signal in the L-012 / peroxide / SPTZ substrate in the presence of a co-enhancer (Example Examples 7 and 8)

[0092] Addition of co-enhancers (MORP, Example 7; imidazole, Example 8) to the L-012 / peroxide / SPTZ substrate had no effect on the pH optimum of the chemiluminescent signal, which remained centered around pH 7.0 (MORP, Fig. 9 ; Imidazole, Fig.10 Again, this is in stark contrast to the corresponding luminol / peroxide / SPTZ / co-enhancer substrates (MORP, pH optimum: pH 9.0[ ]; imidazole, pH optimum: 8.6[ ].

[0093] Dependence of the chemiluminescent signal on the concentration of L-012 in the L-012 / perborate / SPTZ / MORP substrate (Example 3)

[0094] Another significant difference between luminol and L-012 substrates is that much less chemiluminescent material is required to achieve optimal signal levels. For example, in the L-012 / perborate / SPTZ / MORP substrate of Example 3 (Example 3), only about 0.15 mM L-012 concentration is sufficient to achieve a plateau level of signal output. Figure 4 Similar behavior was observed in other formulations with L-012 (e.g., L-012 / perborate / SPTZ / imidazole substrate). On the other hand, the optimized luminol substrate (luminol / A1-A3 substrate Example 10) required a much higher concentration of luminol, 5.0 mM. Therefore, the L-012 substrate required 33 times less chemiluminescent material than the luminol substrate.

[0095] Dependence of chemiluminescent signal on SPTZ concentration in L-012 / perborate / SPTZ / MORP substrate (Example 4)

[0096] In the L-012 / perborate substrate with MORP as the co-enhancer, the signal level reached a peak when between 2 and 3 mmol of SPTZ enhancer was included. Figure 5 Similar results were obtained using imidazole as a co-enhancer.

[0097] Chemiluminescent signal for L-012 / perborate / SPTZ / co-enhancer (MORP; imidazole) substrate (Example 5, MORP; Example 9, imidazole) co-enhancer concentration dependence

[0098] Co-enhancers such as MORP and imidazole had very strong effects on the L-012 / perborate substrate enhanced with SPTZ. Both co-enhancers increased light output by more than an order of magnitude, 18-fold (MORP, Figure 6 ; Imidazole, Fig.10 Compared to imidazole, much less MORP was required to achieve a plateau in signal output (3 mM versus 50 mM), but imidazole had a much stronger effect on light output.

[0099] Dependence of the chemiluminescent signal on the perborate concentration in the L-012 / perborate / SPTZ / MORP substrate (Implementation Example 6)

[0100] In Example 6, Figure 7In the experiment, it was observed that the amount of perborate required to achieve maximum signal in the L-012 / perborate / SPTZ / MORP substrate was approximately 6 mM. At perborate values ​​above 7 mM, the signal output began to decrease. Similar results were recorded for the L-012 / perborate / SPTZ / imidazole substrate.

[0101] Comparison between L-012 and luminol chemiluminescent peroxidase substrates (Example 10)

[0102] When comparing the L-012-based chemiluminescent substrates of the present invention to the optimized luminol substrates, several major differences emerge. In particular: (a) the optimal pH of the L-012 substrate is between 1.5 and 2 pH units lower than that of the luminol substrate; (b) the effective concentration of L-012 is more than 30 times lower than that of luminol; (c) the effect of the co-enhancer in the SPTZ-enhanced L-012 substrate is much stronger than that in the SPTZ-enhanced substrate ( Fig.11 ); (d) the chemiluminescent light output intensity of the L-012 substrate of the present invention is one order of magnitude higher than that of the available nested luminol substrate.

[0103] in conclusion

[0104] Unexpectedly, the effect of the co-enhancer was much higher in the L-012 chemiluminescent substrate than in the luminol chemiluminescent substrate. After adding imidazole as a co-enhancer, the chemiluminescent signal of the optimized luminol / SPTZ substrate increased by 8 times, while the signal generated by MORP increased by 10 times. In contrast, as shown in Examples 10 and Fig.11 As shown in , inclusion of these co-enhancers in the L-012 / SPTZ chemiluminescent substrate increased the signal output by approximately 40-fold (imidazole) and 23-fold (MORP).

[0105] Thus, the L-012 chemiluminescent substrate according to the present disclosure achieves a signal level that is an order of magnitude (7-12x) higher than the most potent known luminol substrate.

[0106] The following examples are provided to illustrate specific aspects of the present invention. However, they are not intended to limit the present invention. Example

[0107] All reagents used in this application were purchased from Sigma-Aldrich, TCI Europe and Panreac.

[0108] SPTZ and CPTZ enhancers were synthesized according to the common knowledge of those skilled in the art. SPTZ: Marzocchi E, Grilli S, Della Ciana L, Prodi L, Mirasoli M and Roda A (2008) Anal biochem, 377: 189-194; CPTZ: Han, F, Chi, L, Wu, W, Liang, X, Fu, M and Zhao, J. Photochem. Photobiol. A: Chemistry 196.1 (2008): 10-23.

[0109] Using microplate, multi-label spectrometer Victor 3 Chemiluminescence measurements were performed in luminescence mode (without emission filter) using a black 96-well microplate (Optiplate-96F).

[0110] To prepare the HRP test solution, dilute 15 μL of horseradish peroxidase (HRP) stock solution (20 mg / L) to 50 mL with buffer (concentration: 6 ng / mL). Add 28 μL of HRP to each well (final well volume 228 μL). Therefore, the final amount of HRP in each well is 168 pg.

[0111] Example 1 - pH dependence of the L-012 / peroxide / peroxidase chemiluminescent reaction at different levels of sodium 3-(phenothiazin-10-yl)propane-1-sulfonate (SPTZ) enhancer

[0112] A series of chemiluminescent substrates with the following compositions were prepared:

[0113] [L-012] = 0.15 mM

[0114] [Sodium perborate] = 4 mM

[0115] [SPTZ]=0.1mM

[0116] In 50 mM Tris / 30 mM phosphate buffer, pH 7.2-8.0.

[0117] As reference substrate the following solutions were prepared:

[0118] [Luminol] = 5mM

[0119] [Sodium perborate] = 4 mM

[0120] [SPTZ]=1.5mM

[0121] In 150 mM Tris buffer, pH 9.0.

[0122] The initial signal level was plotted against pH, Figure 2 (The signal of the reference substrate was set to 1).

[0123] Example 2 - Screening for co-enhancers of L-012 / peroxide / peroxidase chemiluminescent reaction

[0124] A chemiluminescent substrate with the following composition was prepared:

[0125] [L-012] = 0.15 mM

[0126] [Sodium perborate] = 4 mM

[0127] [SPTZ] = 1 mM; [CPTZ] = 1 mM (enhancer)

[0128] In 50 mM Tris / 30 mM phosphate buffer, pH 7.9.

[0129] [Co-enhancer] = 1 mM

[0130] like Figure 3 As shown in , the initial signal level of each co-enhancer: imidazole, 1-methylimidazole, 1,2,3-triazole, 4-morpholinopyridine (MORP), 4-dimethylaminopyridine (DMAP), 4-pyrrolidinylpyridine (PPY) is plotted as a bar graph.

[0131] Example 3 - Dependence of the chemiluminescent signal on the concentration of L-012 in the L-012 / perborate / SPTZ / MORP substrate

[0132] A series of chemiluminescent substrates with the following compositions were prepared:

[0133] [L-012] = 0.15 mM

[0134] [Sodium perborate] = 4 mM

[0135] [SPTZ] = 3 mM (enhancer)

[0136] [MORP] = 3 mM (co-enhancer)

[0137] In 50 mM Tris / 30 mM phosphate buffer, pH 7.9.

[0138] like Figure 4 As shown in , the initial signal level was plotted against the L-012 concentration.

[0139] Example 4 - Dependence of the chemiluminescent signal on the SPTZ concentration in the L-012 / perborate / SPTZ / MORP substrate

[0140] A series of chemiluminescent substrates with the following compositions were prepared:

[0141] [L-012] = 0.15 mM

[0142] [Sodium perborate] = 4 mM

[0143] [SPTZ] = 1-10 mM (enhancer)

[0144] [MORP] = 3 mM (co-enhancer)

[0145] In 50 mM Tris / 30 mM phosphate buffer, pH 7.9.

[0146] like Figure 5 Initial signal levels were plotted versus SPTZ concentration as shown in .

[0147] Example 5 - Dependence of the chemiluminescent signal on the MORP concentration in the L-012 / perborate / SPTZ / MORP substrate

[0148] A series of chemiluminescent substrates with the following compositions were prepared:

[0149] [L-012] = 0.15 mM

[0150] [Sodium perborate] = 4 mM

[0151] [SPTZ] = 3 mM (enhancer)

[0152] [MORP] = 0-6 mM (co-enhancer)

[0153] In 50 mM Tris / 30 mM phosphate buffer, pH 7.9.

[0154] like Figure 6 Initial signal levels were plotted versus MORP concentration as shown in FIG.

[0155] Example 6 - Dependence of the chemiluminescent signal on the perborate concentration in the L-012 / perborate / SPTZ / MORP substrate

[0156] A series of chemiluminescent substrates with the following compositions were prepared:

[0157] [L-012] = 0.15 mM

[0158] [Sodium perborate] = 0.5-8.0 mM

[0159] [SPTZ] = 3 mM (enhancer)

[0160] [MORP] = 3 mM (co-enhancer)

[0161] In 50 mM Tris / 30 mM phosphate buffer, pH 7.9.

[0162] like Figure 7 Initial signal levels were plotted versus perborate concentration as shown in .

[0163] Example 7 - pH dependence of chemiluminescent signal in L-012 / perborate / SPTZ / MORP substrate

[0164] A series of chemiluminescent substrates with the following compositions were prepared:

[0165] [L-012] = 0.15 mM

[0166] [Sodium perborate] = 4 mM

[0167] [SPTZ] = 3 mM (enhancer)

[0168] [MORP] = 3 mM (co-enhancer)

[0169] pH 5-9 (Tris buffer)

[0170] like Figure 8 Initial signal levels were plotted versus perborate concentration as shown in .

[0171] Example 8 - pH dependence of chemiluminescent signal in L-012 / perborate / SPTZ / imidazole (IMDZ) substrate

[0172] A series of chemiluminescent substrates with the following compositions were prepared:

[0173] [L-012] = 0.15 mM

[0174] [Sodium perborate] = 4 mM

[0175] [SPTZ] = 3 mM (enhancer)

[0176] [IMDZ] = 50 mM (co-enhancer)

[0177] pH range 6.3-8.1

[0178] like Fig. 9 As shown in , the initial signal level was plotted against pH.

[0179] Example 9 - Dependence of the chemiluminescent signal on the concentration of imidazole (IMDZ) in the L-012 / perborate / SPTZ / IMDZ substrate

[0180] A series of chemiluminescent substrates with the following compositions were prepared:

[0181] [L-012] = 0.15 mM

[0182] [Sodium perborate] = 4 mM

[0183] [SPTZ] = 3 mM (enhancer)

[0184] [IMDZ] = 0-100 mM (co-enhancer)

[0185] In 50 mM Tris buffer, pH 7.1.

[0186] like Fig.10 As shown in , the initial signal level is plotted against the imidazole (IMDZ) concentration.

[0187] Example 10 - Comparison between Luminol and L-012 Chemiluminescent Substrates for Peroxidase

[0188] The following chemiluminescent substrates were prepared:

[0189] Luminol substrate

[0190] Luminol / A1 (NO: no co-enhancer; signal set to 1)

[0191] [Luminol] = 5mM

[0192] [Perborate] = 4 mM

[0193] [SPTZ]=3mM

[0194] Tris buffer 150 mM, pH 9.0

[0195] Luminol / A2 (IMDZ: imidazole as co-enhancer)

[0196] [Luminol] = 5mM

[0197] [Perborate] = 4 mM

[0198] [SPTZ]=3mM

[0199] [IMDZ]=35mM

[0200] Tris buffer 150 mM, pH 9.0

[0201] Luminol / A3 (MORP: 4-morpholinopyridine as co-enhancer)

[0202] [Luminol] = 5mM

[0203] [Perborate] = 4 mM

[0204] [SPTZ]=3mM

[0205] [MORP]=3mM

[0206] Tris buffer 150 mM, pH 9.0

[0207] L-012 substrate

[0208] L012 / B1(NO: no co-enhancer)

[0209] [L-012] = 0.15 mM

[0210] [Perborate] = 4 mM

[0211] [SPTZ]=3mM

[0212] Tris-phosphate buffer 50 / 30 mM, pH 7.1

[0213] L012 / B2 (IMDZ: imidazole as co-enhancer)

[0214] [L-012] = 0.15 mM

[0215] [Perborate] = 7 mM

[0216] [SPTZ]=2mM

[0217] [IMDZ]=100mM

[0218] Tris buffer 50 mM, pH 7.1

[0219] L012 / B3 (MORP: 4-morpholinopyridine as co-enhancer)

[0220] [L-012] = 0.15 mM

[0221] [Perborate] = 4 mM

[0222] [SPTZ]=3mM

[0223] [MORP]=3mM

[0224] 50 mM Tris / 30 mM phosphate buffer, pH 7.9.

[0225] The initial chemiluminescent signal level for each substrate was recorded. The signal generated by luminol perborate / SPTZ without co-enhancer was used as the reference signal (REF), and its value was set to 1 (unity).

[0226] Luminol substrate: NO (A1) = REF set to 1; IMDZ (A2) = 8; MORP (A3) = 10

[0227] L-012 substrate: NO (B1) = 3; IMDZ (B2) = 115; MORP (B3) = 70

[0228] like Fig.11 The results are plotted as a bar graph as shown in .

[0229] Example 11 - Chemiluminescent signal of chemiluminescent substrate L-012 / B3 and chemiluminescent substrate luminol / A3 vs. HRP concentration

[0230] Chemiluminescent substrates L-012 / B3 and luminol / A3 were prepared as described in Example 11. 200 μL of these substrates were dispensed into each well of a 96-well plate. Horseradish peroxidase (HRP) (20 μg / mL) was diluted to obtain a 1600 ng / mL solution and then stained using Victor 3 The automated pump system of the (Perkin-Elmer) multi-label plate reader dispenses various aliquots into each well to achieve a series of dilutions spanning the range of 30-300 ng / L. Column 1 in the plate is used as a blank (substrate only, no enzyme added). Fig.12 As shown in , the initial signals of the two substrates are plotted against the HRP concentration. A regression line was calculated for each substrate. The regression slope of the L-012 / B3 substrate is approximately 7 times higher than that of the luminol / A3 substrate, while the measured background signals of the two substrates are similar.

[0231] Example 12 - Human IKBα Western Blot Assay

[0232] Purified human IKBα (nuclear factor of kappa light polypeptide gene enhancer of B-cell inhibitory protein, alpha; Abcam) was diluted to 1.5 ng / mL in electrophoresis reducing sample buffer and diluted 1.5-fold. 5 μL of each dilution was separated by SDS-PAGE and Turbo TM Transfer protein using the BIO-RAD transfer system. Wash the membrane with 1X PBS. 2% Amersham in 20 buffer TM ECL TM Blocking agent (GE Healthcare), followed by incubation with rabbit anti-IKBα (Abcam) at 1:1000, and then incubation with anti-rabbit IgG HRP (Abcam) at 1:500000. ImageQuant was performed using the chemiluminescent substrate L012 / B3 (Example 11) and the luminol-based substrate Westar Supernova (Cyanagen) according to the manufacturer's instructions. TM The results were shown in Fig.13 middle.

[0233] Example 13 - Human HDAC-1 ELISA Assay

[0234] Rabbit anti-human HDAC-1 (Abcam) was diluted in 50mM carbonate / bicarbonate buffer at pH 9.6, and two-fold dilutions starting from 20ng / mL to 19.9pg / mL were prepared. A 96 black microplate with transparent flat-bottomed polystyrene (Corning) was coated overnight at 50μl / well at 4°C. The coating solution was removed and the plate was washed 6 times with 200μl / well PBS-Tween 0.01%. 100μl of 3% ECL blocking agent (GE Healthcare) in PBS-Tween 0.01% was added to each well, and the plate was incubated at room temperature for 1 hour. Wash briefly in PBS-Tween 0.01%, remove the blocking buffer, and add 100μl of goat anti-rabbit-HRP (Abcam) diluted to a concentration of 0.4μg / mL to each well at room temperature for 30 minutes. The goat anti-rabbit-HRP solution was removed and the plate was washed 6 times with 200 μl / well of PBS-Tween 0.01%. 200 μl of chemiluminescent substrate (L012 / B3, Example 11; SuperSignal ELISA Femto, a luminol-based substrate produced by Thermo Scientific) was added to each well and the plate was stained with a Perkin Elmer Victor 3 The analyzer analyzes the plate. Fig.14 The signal-to-noise ratio (S / N) relative to human HDAC-1 (pg) is shown in FIG.

[0235] Naturally, while the principle of the invention remains the same, the details of construction and the embodiments may vary widely with respect to what has been described and shown by way of example, without departing from the scope of the invention as defined in the appended claims.

Claims

1. A method for increasing the light emission produced by the chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, a peroxidase or a conjugate of a peroxidase, an enhancer, a co-enhancer, and a peroxide oxidant, wherein the enhancer is an anionic N-alkylphenothiazine, and The method comprises the following steps: i. preparing a chemiluminescent substrate by mixing together 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, the enhancer, the co-enhancer and the peroxide oxidant, and ii. adding the peroxidase or peroxidase conjugate to the chemiluminescent substrate, wherein the co-enhancer is selected from the group consisting of imidazole, 1-methylimidazole, 4-morpholinopyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinylpyridine (PPY), wherein the concentration of the 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione in the chemiluminescent substrate is comprised between 0.01 mM and 1 mM and wherein the pH of the chemiluminescent substrate is comprised between 6.0 and 7.9; wherein the anionic N-alkylphenothiazine is selected from 3-(10H-phenothiazin-10-yl)propane-1-sulfonic acid, 4-(10H-phenothiazin-10-yl)butane-1-sulfonic acid, 3-(10H-phenothiazin-10-yl)propionic acid and 4-(10H-phenothiazin-10-yl)butanoic acid, and salts thereof; wherein the peroxidase is selected from horseradish peroxidase, soybean peroxidase and sweet potato peroxidase; The peroxide oxidant is selected from hydrogen peroxide, a complex of urea and hydrogen peroxide in a molar ratio of 1:1, perborate, and percarbonate.

2. The method according to claim 1, wherein the concentration of the anionic N-alkylphenothiazine enhancer in the chemiluminescent substrate is comprised between 0.1 and 10 mM.

3. The method according to claim 1 or 2, wherein the concentration of the co-enhancer in the chemiluminescent substrate is comprised between 0.1 and 100 mM.

4. The method according to claim 1 or 2, wherein the concentration of the peroxide oxidant in the chemiluminescent substrate is comprised between 0.1 and 10 mM.

5. The method according to claim 1 or 2, wherein the chemiluminescent substrate contains: a. The anionic N- alkyl phenothiazine enhancer at a concentration between 0.1 and 10 mM; b. the co-enhancer at a concentration between 0.1 and 100 mM; c. the peroxide oxidant at a concentration between 0.1 and 10 mM.

6. The method of claim 1 or 2, wherein the method further comprises detecting the analyte in the sample by measuring light emission produced by the chemiluminescent reaction of 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, peroxidase or a conjugate of peroxidase and a detection reagent for the analyte, an enhancer, a co-enhancer, and a peroxide oxidant.

7. A kit for performing the method according to any one of claims 1 to 6, wherein the kit comprises 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione, a peroxide oxidant, an enhancer and a co-enhancer, wherein the enhancer is an anionic N-alkylphenothiazine and the co-enhancer is selected from imidazole, 1-methylimidazole, 4-morpholinopyridine (MORP), 4-dimethylaminopyridine (DMAP) and 4-pyrrolidinylpyridine (PPY); wherein the anionic N-alkylphenothiazine is selected from 3-(1 The kit comprises a reagent comprising: a 10H-phenothiazine-10-yl) propane-1-sulfonic acid, a 4-(10H-phenothiazine-10-yl) butane-1-sulfonic acid, a 3-(10H-phenothiazine-10-yl) propionic acid and a 4-(10H-phenothiazine-10-yl) butanoic acid, and salts thereof; wherein the peroxide oxidant is selected from hydrogen peroxide, a complex containing urea and hydrogen peroxide in a molar ratio of 1:1, perborate, and percarbonate; wherein the kit further comprises a peroxidase or a conjugate of a peroxidase; wherein the peroxidase is selected from horseradish peroxidase, soybean peroxidase, and sweet potato peroxidase.

8. The kit of claim 7, wherein 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4(2H,3H)-dione is present in a first vial and the oxide oxidant is present in a second vial, and wherein the enhancer and the co-enhancer are present in the first vial or the second vial or both vials.

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