Patent Application
20160376630
The present invention relates to a method for determining an amount of a peroxide, particularly hydrogen peroxide, by determining the luminescence of a lanthanide-ligand complex.
Hydrogen peroxide (H2O2) is a highly reactive oxygen species and a strong oxidizer. Hydrogen peroxide is a component of a variety of chemicals industrially applied at large scale, such as suds or disinfectants. Furthermore, hydrogen peroxide is naturally produced as a by-product of several biological processes such as the oxidative metabolization of sugar. Hydrogen peroxide also plays an important role in the immune system, in diseases such as asthma or cancer and as a signalling molecule in the regulation of a variety of biological processes, for example in the regulation of oxidative stress-related states. Therefore, there is a considerable interest in sensitive methods for detection or quantification of hydrogen peroxide, particularly in biological or environmental samples.
A variety of methods for quantifying hydrogen peroxide exist. Among those, luminescence based methods are characterized by high sensitivities. A well-known example for such a luminescence based method is the Europium tetracycline (EuTc) assay, wherein the lanthanide europium is complexed with the antibiotic tetracycline. The luminescence of that complex increases with increasing hydrogen peroxide concentration.
One drawback of this assay is its sensitivity to compounds such as citrate and phosphate in submillimolar and low micromolar concentrations. These compounds increase the fluorescence intensity of the EuTc-complex and interfere with the increase in fluorescence caused by the EuTc—H2O2 complex, rendering the assay inaccurate when applied to biological samples.
Thus, it is the objective of the present invention to provide a sensitive and reliable method for the spectroscopic determination of hydrogen peroxide, which is particularly characterized by an increased stability against interfering compounds occurring in biological samples.
According to a first aspect of the invention, a method for determining of an amount of a peroxide is provided, wherein the method comprises the steps of:
The determination of an amount of a peroxide in the context of the present specification particularly refers to the measurement of a concentration of the peroxide, which can easily be converted to the molar/mass amount in a given volume.
The luminescence of the lanthanide-ligand complex changes in presence of the peroxide.
The luminescence may be measured in terms of luminescence intensity or luminescence decay time. Both the intensity and decay time of the lanthanide ligand complex change in presence of the peroxide.
The term “luminescence” in the context of the present specification refers to the emission of electromagnetic radiation by a substance not resulting from heat, particularly after excitation by electromagnetic radiation. Non-limiting examples for luminescence encompass fluorescence and phosphorescence.
The sample can be any sample, for which the amount of the peroxide needs to be determined. The sample can for example be an environmental sample or a biological sample. In certain embodiments, the sample is a liquid. In certain embodiments, the liquid is aqueous.
Non-limiting examples for an environmental sample are a sample from waters such as rivers, lakes or oceans, a waste sample, a sewage sample, a soil sample or an air sample.
The peroxide in the sample to be determined may of any origin and may for example originate from biological, geological or industrial processes.
An advantage of the method of the invention is an increased sensitivity of the method for the peroxide. The method provided herein particularly allows to decrease the lower limit of detection (LOD) and the lower limit of quantification (LOQ) to the sub-micromolar range.
Another advantage is an increased insensitivity of the method of the invention against compounds known for their interference in luminescence assays, particularly in lanthanide based assays. Examples for such interfering compounds are citrate and phosphate. The method of the invention compares favourably to state of the art lanthanide based assays such as the EuTc-assay.
Thus, the method of the invention is not disturbed by many salts and other serum components that interfere with the methods known in the art, and is compatible to human serum samples.
As hydrogen peroxide is a byproduct of a number of enzymatic reactions, the method of the invention is also suitable for the detection of these enzymes and their underlying substrates.
The determination of the luminescence may be performed in a suitable container that is permeable to the light emitted by the lanthanide-ligand complex of the invention, and particularly permeable for the light with which the lanthanide-ligand complex can be excited. Examples for such containers include, without being restricted to, microtiter plates, cuvettes, specimen slides and microfluidic chips transparent to light between 200 and 700 nm.
In general, the term peroxide in the sense of the present invention particularly refers to a compound comprising a peroxo group (—O—O—) or a peroxide anion (O22−).
Likewise, the amount of a compound that decomposes to a peroxide can be determined by the method of the invention, conducting the reaction for example in a protic solvent such as an aqueous solvent. The amount of the peroxide formed by the decomposing reaction can be quantified. A non-limiting example for such a decomposing compound is a compound comprising a superoxide (O2−).
An aqueous solvent in the context of the present invention refers to a solvent comprising water, particularly at least 50% (v/v), 60% (v/v), 70% (v/v), 80% (v/v), 90% (v/v), 95% (v/v), or 100% (v/v) water.
In some embodiments, the peroxide is characterized
wherein
R1 and R2 are independently from each other hydrogen, a C1-C8 alkyl, a C1-8 cyclic alkyl, a C5-C10 aryl, a C1-C9-heteroaryl, a —C(O)—C1-C8 alkyl, a —C(O)—C1-8 cyclic alkyl, a —C(O)—O5—C10 aryl, a —C(O)—C1-C9-heteroaryl, a transition metal or S, wherein S is an acid moiety or a salt thereof,
A C1-C8 alkyl in the context of the present specification signifies a saturated linear or branched hydrocarbon having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, wherein one carbon-carbon bond may be unsaturated and one CH2 moiety may be exchanged for oxygen (ether bridge). Non-limiting examples for a C1-C8 alkyl are methyl, ethyl, 1-propyl, isopropyl, prop-2-enyl, n-butyl, 2-methylpropyl, tert-butyl, but-3-enyl, prop-2-inyl, C2H5—O—CH3, but-3-inyl, pentyl, hexyl, heptyl or octyl.
The term aryl in the context of the present specification signifies a cyclic aromatic hydrocarbon. Examples of aryl include, without being restricted to, phenyl and naphthyl. A heteroaryl in the context of the present invention is an aryl that comprises one or several nitrogen, oxygen and/or sulphur atoms. Examples for heteroaryl include, without being restricted to, pyrrole, thiophene, furan, imidazole, pyrazole, thiazole, oxazole, pyridine, pyrimidine, thiazin, quinoline, benzofuran and indole. An aryl or a heteroaryl in the context of the invention additionally may be substituted by one or more alkyl groups.
The term C1-8 cyclic alkyl signifies a cyclic, non-aromatic hydrocarbon having 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms, wherein one carbon-carbon bond or two carbon-carbon bonds may be unsaturated. Non-limiting examples for a C1-8 cyclic alkyl include cyclopropyl, cylclopropenyl, cyclobutyl, cyclobutenyl, cyclobutadienyl, cyclopentyl, cylcopentenyl, cyclopentdienyl, cylcohexyl, cyclohexenyl, cyclohexadienyl, cylcoheptyl, cycloheptenyl, cylcoheptadienyl, cyclooctyl, cylcooctenyl and cylcoocetadienyl.
In some embodiments, S is selected from —C(O)OH, —S(O2)OH, —B(OH)2, a chromate (HCrO4)−, —PO(OH)2, —NO3, —N2OH and SeO(OH),
In some embodiments, the transition metal is selected from TiIV, VV, CrVI/V, MnIV, COIII, NiII, ZrIV, NbV, MoVI, RuII/IV, RhIII, PdII, HfIV, TaV, WVI, OsII/IV, IrIII and PtII.
In some embodiments, R1 and R2 is the same transition metal.
Examples for such peroxide include, without being restricted to,
In some embodiments, the peroxide is hydrogen peroxide, a radical or a salt thereof, wherein in particular a radical or a salt of hydrogen peroxide decomposes to hydrogen peroxide in an aqueous solvent. Examples for such salts include, without being restricted to, alkali metal salts such as sodium peroxide, earth alkali metal salts such as barium or magnesium peroxide, and transition metal peroxides such as uranyl peroxide.
In some embodiments, the dicarboxylic acid is phthalic acid.
In some embodiments, the peroxide is hydrogen peroxide, and the benzene dicarboxylic acid is phthalic acid.
In some embodiments, the method according to the invention is performed in presence of an aqueous buffer. In some embodiments, the aqueous buffer comprises HEPES (2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; CAS No. 7365-45-9), tris (tris(hydroxymethyl)aminomethane; CAS No. 77-86-1), imidazole (CAS No. 288-32-4), MOPS (3-(N-morpholino)propanesulfonic acid; CAS No. 1132-61-2), bicine (2-(bis(2-hydroxyethyl)amino)acetic acid; CAS No. 150-24-4), phosphate buffered saline, tricine (N-(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)glycine; CAS No. 5704-04-1), TAPS (3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propane-1-sulfonic acid; CAS No. 29915-38-6), TAPSO (3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]-2-hydroxypropane-1-sulfonic acid; CAS No. 68399-81-5), PIPES (1,4-piperazinediethanesulfonic acid; CAS No. 5625-37-6), TES (2-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]ethanesulfonic acid; CAS No. 7365-44-8), CAPS (3-(cyclohexylamino)-1-propanesulfonic acid; CAS No. 1135-40-6), CHES (2-(cyclohexylamino)ethanesulfonic acid; CAS No. 103-47-9), HEPPS (3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid; CAS No. 16052-06-5) and/or MES (2-(N-morpholino)ethanesulfonic acid; CAS No. 4432-31-9).
In some embodiments, the method of the invention is performed in an aqueous solvent.
In some embodiments, the luminescence is determined at a wavelength above 470 nm.
The luminescence may be determined with a photodiode or a photomultiplier, wherein for example the emitted light is filtered by a monochromator that allows only the light with the desired wavelength to pass. Alternatively, the emitted light can be filtered by a cut-off filter that allows only light above a desired wavelength to pass.
In some embodiments, the luminescence is determined at a wavelength of 550±10 nm.
In some embodiments, the luminescence is determined after excitation of the lanthanide-ligand complex of the invention with light characterized by a wavelength of 200 nm to 300 nm.
The lanthanide-ligand complex of the invention may be excited by suitable means such as a lamp, a diode or a laser.
In some embodiments, the luminescence is determined after excitation of the lanthanide-ligand complex with light characterized by a wavelength of 280 nm.
In some embodiments, determining of the luminescence is performed by measuring the luminescence decay time and/or the luminescence intensity of the lanthanide-ligand complex.
In some embodiments, the lanthanide-ligand complex is characterized by a molar ratio of lanthanide to ligand between 3:1 and 2:1, for example 3:1, 2.75:1, 2.5:1, 2.25:1 or 2:1.
In some embodiments, the luminescence is determined at a pH value above 6.
In some embodiments, the luminescence is determined at a pH value between 6.6 and 11.
In some embodiments, the luminescence is determined at a pH between 7 and 11.
In some embodiments, the luminescence is determined at a pH value between 8 and 11.
In some embodiments, the luminescence is determined at pH 8.0.
In some embodiments, the luminescence is determined at pH 8.5.
In some embodiments, the sample is contacted for 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min or 10 min with the lanthanide-ligand complex before the luminescence is determined.
In some embodiments, the sample is selected from the group comprised of blood, sperm, saliva, and an interstitial fluid. In some embodiments, the sample is a body fluid of mammal, particularly a human being. In some embodiments, the sample is a plant or seed material or an extract thereof. In some embodiments, the sample is an environmental sample, for example a freshwater sample, a salt water sample, a waste water sample, a sewage sample, a soil sample or an air sample. In some embodiments, the sample is a cell culture sample.
In some embodiments, the sample is diluted in a suitable solvent system, particularly water, before addition of the lanthanide-ligand-complex of the invention and/or determining the luminescence. Such dilution may be necessary in case of high peroxide concentrations causing too high intensity due to the sensitivity of the method of the invention.
In some embodiments, the peroxide to be determined is hydrogen peroxide and is enzymatically generated or consumed.
Such enzymes may use hydrogen peroxide as substrate, for example the enzyme catalase. Such enzymes may also generate hydrogen peroxide in their catalysed reaction, for example oxidases, which use elemental oxygen as electron acceptor.
Determining the amount of hydrogen peroxide generated or consumed by enzymes allows for the determination of the enzymatic activity of these enzymes. Likewise, determining the amount of hydrogen peroxide generated or consumed by enzymes allows for the determination, particularly the quantification, of compounds consumed as substrates or formed as products by the aforementioned enzymes.
In some embodiments, the hydrogen peroxide is generated or consumed by an enzyme selected from glucose oxidase, pyruvate oxidase, lactate oxidase, bilirubin oxidase, alcohol oxidase, sarcosine oxidase, galactose oxidase, amino acid oxidase, monoamine oxidase, cholesterol oxidase, choline oxidase, catalase, superoxide dismutase and urate oxidase.
According to another aspect of the invention, a method for determining a compound is provided, wherein the compound is selected from glucose, galactose, an amino acid, a monoamine, lactate, pyruvate, choline, cholesterol, bilirubin, xanthine, urate, sarcosine, and ethanol, wherein the compound is enzymatically converted, thereby producing or consuming hydrogen peroxide, and the hydrogen peroxide is determined by the method of the invention. The term “monoamine” in the context of the present specification refers to compounds characterized by an aromatic ring that is connected to an amino group via an ethylene group, and particularly refers to a neurotransmitter. Such monoamines include, without being restricted to histamine (CAS Nr. 51-45-6), dopamine (CAS Nr. 51-61-6), noradrenaline (CAS Nr. 51-41-2 or 138-65-8), adrenaline (CAS Nr. 51-43-4), serotonine (CAS Nr. 50-67-9), melatonin (CAS Nr. 73-31-4), 3-phenylethylamine (CAS Nr. 64-04-0), tyramine (CAS Nr. 51-67-2), tryptamine (CAS Nr. 61-54-1), octopamine (CAS Nr. 104-14-3), 3-iodothyronamine (CAS No. 712349-95-6), thyronamine (CAS Nr. 500-78-7).
In one embodiment, the amount or the concentration of the compound is determined.
According to yet another aspect of the invention, a method for determining the enzymatic activity of an enzyme is provided, wherein the enzyme is selected from the group comprised of glucose oxidase, pyruvate oxidase, lactate oxidase, bilirubin oxidase, alcohol oxidase, sarcosine oxidase, galactose oxidase, amino acid oxidase, monoamine oxidase, choline oxidase, cholesterol oxidase, catalase, superoxide dismutase and urate oxidase. These enzymes consume or form hydrogen peroxide, and the consumption or the formation of the hydrogen peroxide is determined by the method of the invention.
In some embodiments, the enzyme producing or consuming the hydrogen peroxide is coupled to an antibody. Such enzyme-coupled antibody is particularly useful in an ELISA-assay and may be used as primary antibody for detection of an analyte or as secondary antibody directed against a primary antibody for signal amplification. The amount of the enzyme-coupled antibody can be determined by measurement of the enzymatic activity as described above (yielding an optical signal caused by the luminescence of the lanthanide-ligand-complex of the invention).
According to another aspect of the invention, a method for determining the pH value of a sample is provided, wherein the method comprises the steps of:
The luminescence of the terbium(III) benzene dicarboxylic acid complex changes with the pH value of the sample.
According to an alternative to the above aspect, a method for determining the pH-value of a sample is provided, wherein the method comprises the steps of.
The term sample has the same meaning as described above.
According to another aspect of the invention, a method for determining the amount of an antibody is provided, wherein the antibody is coupled to an enzyme that produces or consumes hydrogen peroxide, the amount of the antibody is determined by the enzymatic activity of the coupled enzymes, and the enzymatic activity is determined by determining the produced or consumed hydrogen peroxide by the method of the invention.
Wherever alternatives for single separable features such as, for example, a certain peroxide or a certain benzene dicarboxylic acid are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein. Thus, any of the alternative embodiments for a dicarboxylic acid may be combined with any of the alternative embodiments of a peroxide.
The invention is further characterized, without limitations, by the following examples, from which further features, advantages and embodiments can be derived. The examples are meant to illustrate but not limit the invention.
The assay of the example detects hydrogen peroxide in fluids such as water and serum samples. The assay is based on a phosphorescence signal of phthalic acid in complex with terbium ions, which decreases with increasing concentration of hydrogen peroxide. A certain ratio of phthalic acid to terbium in a suitable buffer is advantageous for an optimal performance of the assay. Suitable buffers are for example aqueous buffers such as HEPES, Tris- or imidazole buffer with a concentration between 50 and 100 mmol/I. HEPES is a preferred buffer.
This could be demonstrated for the determination of glucose by using glucose oxidase as a converting enzyme and for choline by using choline oxidase, both for water and serum samples. Other suitable enzymes are those belonging to EC (Enzyme Commission) number 1.11.1. Naturally, all substrates for those enzymes are also potential analytes for this assay.
It could be shown that the best terbium/phthalic acid ratio for optimal assay performance in presence or absence of hydrogen peroxide is 3:1 (
The luminescence signal is relatively stable over a prolonged measurement period, whereby at pH 7.5 virtually no decrease of the signal intensity over a broad range of the hydrogen peroxide concentration can be observed (
The signal responses of the assay of the invention at different pH values are shown in
Table 2 shows the recovery rates, intra- and interassay variation coefficient after 3 min incubation.
Further, the assay of the invention is characterized by increased stability against a variety of different substances, which frequently occur in biological sample and are known for interfering luminescence assays. None of them interferes with the assay of the invention when physiological concentrations of these substances were used. Table 3 shows a selection of different substances tested on the assay of the invention, wherein the minimal interfering concentration signifies a threshold, under which no interference of the assay can be observed.
The assay of the invention is suitable for the detection or quantification of hydrogen peroxide in both aqueous samples and biological samples, in particular in human serum samples.
The determination of hydrogen peroxide was performed as following: a water sample or a serum sample was diluted with water (0.5 mL serum plus 9.5 mL water) yielding in solution A. Then, 10 μL of solution A and 90 μl of solution B (lanthanide complex, 2.33 mmol/L terbium, 0.77 mmol/L phthalic acid in 80 mmol/L HEPES buffer, pH 8.0) were added to a microtiter plate, mixed and incubated at room temperature for 3 minutes. After incubation the luminescence (phosphorescence) of the lanthanide ligand complex of the invention was measured at an emission wavelength 550 nm after excitation at 280 nm with a (time resolved) fluorescence plate reader.
The assay of the invention can also be used for the enzymatic determination of substances which are converted with or to hydrogen peroxide, such as glucose that is converted by the glucose oxidase to glucono lactone and hydrogen peroxide.
The determination of glucose was performed as following: 0.5 ml serum sample or water sample was diluted with 9.5 ml assay buffer yielding in solution A. Then, 10 μL of solution A, 85 μl of solution B (lanthanide complex, 2.33 mmol/L terbium, 0.77 mmol/L phthalic acid in 80 mmol/L HEPES buffer, pH 8.0) and 5 μL of glucose oxidase solution (0.1 units in HEPES buffer, pH 8.0, 100 mmol/L, wherein one unit will oxidize 1.0 μmol of β-D-glucose to D-gluconolactone and H2O2 per min at pH 5.1 at 35° C., equivalent to an O2 uptake of 22.4 μL per min) were added to a microtiter plate, mixed and incubated at room temperature for 2 minutes. After incubation the luminescence (phosphorescence) of the lanthanide ligand complex of the invention was measured at an emission wavelength 550 nm after excitation at 280 nm with a (time resolved) fluorescence plate reader. If the reaction mixture is saturated with oxygen, the activity may increase by up to 100%.
A fourfold improvement in sensitivity can be achieved by increasing the incubation time from 2 minutes to 10 minutes (
Another application of the assay of the invention is the enzymatic determination of choline, which is converted by the choline oxidase to glycine betaine aldehyde and hydrogen peroxide.
The determination of choline was performed as following: 0.5 ml serum sample or water sample was diluted with 9.5 ml assay buffer yielding in solution A. Then, 10 μL of solution A, 45 μl of solution B (lanthanide complex, 2.33 mmol/L terbium, 0.77 mmol/L phthalic acid in 80 mmol/L HEPES buffer, pH 8.0) and 45 μL of choline oxidase solution (0.9 units in HEPES buffer, pH 8.0, 100 mmol/L, wherein one unit will form 1 μmol of H2O2 with oxidation of 1 μmol of choline to betaine aldehyde per min at pH 8.0 at 37° C.) were added to a microtiter plate, mixed and incubated at room temperature for 2 minutes. After incubation the luminescence (phosphorescence) of the lanthanide ligand complex of the invention was measured at an emission wavelength 550 nm after excitation at 280 nm with a (time resolved) fluorescence plate reader. Note, that during the conversion of choline to betaine by choline oxidase, 2 μmol of H2O2 are produced for every μmol of choline.
Here, an even twenty-fivefold improvement in sensitivity can be achieved by increasing the incubation time from 2 minutes to 10 minutes (
| Number | Date | Country | Kind |
|---|---|---|---|
| 13174786.7 | Jul 2013 | EP | regional |
| 13180004.7 | Aug 2013 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2014/063981 | 7/1/2014 | WO | 00 |
