WO2007039775A1 - Measurement of the total antioxidant capacity in liquids and solutions using tmb - Google Patents

Measurement of the total antioxidant capacity in liquids and solutions using tmb Download PDF

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Publication number
WO2007039775A1
WO2007039775A1 PCT/GR2006/000053 GR2006000053W WO2007039775A1 WO 2007039775 A1 WO2007039775 A1 WO 2007039775A1 GR 2006000053 W GR2006000053 W GR 2006000053W WO 2007039775 A1 WO2007039775 A1 WO 2007039775A1
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Prior art keywords
antioxidant capacity
tmb
total antioxidant
sample
antioxidants
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PCT/GR2006/000053
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French (fr)
Inventor
George Koliakos
Daryoush Hamidi Alamdari
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George Koliakos
Daryoush Hamidi Alamdari
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Publication of WO2007039775A1 publication Critical patent/WO2007039775A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N31/00—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods
    • G01N31/22—Investigating or analysing non-biological materials by the use of the chemical methods specified in the subgroup; Apparatus specially adapted for such methods using chemical indicators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances

Definitions

  • the present invention concerns a method of determining the total antioxidant capacity of a sample by using TMB cation solution.
  • the total antioxidant capacity of a sample is determined by the reduction (decolourization) of TMB cation.
  • the results compared with the reducing action of uric acid with known concentration give values expressed in ⁇ mol/litre.
  • ROS reactive oxygen species
  • ROS reactive oxygen species
  • OH hydroxyl radical
  • O 2 superoxide radical
  • NO nitric oxide radical
  • LOO lipid peroxyl radical
  • ROS can readily react with and damage other molecules that in some cases, the body uses this to fight infections, or in other cases, the damage may be to the body's own molecules such as DNA, lipid, proteins and carbohydrates (Stadtman, E. R. Annu. Rev. Biochem. (1993), 62:797- 821).
  • the antioxidants were defined as enzymes or molecules that can retard or prevent the damaging effects of ROS in tissues.
  • Antioxidants have thus become a topic of increasing interest recently and the number of publications has nearly quadrupled in the past decade (Based on the Web of Knowledge search on articles containing the word antioxidant or antioxidants).
  • Plasma (or serum) concentrations of different antioxidants can be measured in laboratories separately, but the measurements are time-consuming, labor-intensive, costly and they require complicated techniques. Because the measurement of different antioxidant molecules separately, is not practical and antioxidant effects of them are additive, total antioxidant response (TAR) of a sample is measured and this is named as total antioxidant capacity Miller NJ, et al., Clin Sci (Lond). (1993), Apr; 84(4):407-12); (Re R, et al. Free Radic Biol Med.
  • the invention seeks to provide a method of determining the total antioxidant capacity of a liquid and/or solution by using a marker consisting of a stable substance and by providing values expressed in ⁇ mol/litre.
  • redox indicator is sued hereafter to denote the oxidant in such redox reactions.
  • Redox indicators are known to undergo physiochemical change, for example a colour change, when reduced by antioxidants in a sample. This physicochemical change can be easily monitored by known methods, for example by monitoring a change in absorption, or emission, of electromagnetic radiation in a sample or by other methods that are known to monitor redox reactions.
  • the method of the invention utilises the ability of antioxidants to reduce TMB cation which causes decolourization of TMB cation and hence results in a decrease in absorbance at 450 nm.
  • the amount of the redox indicator reduced by the antioxidant is a reflection of the total antioxidant capacity of the antioxidant. Therefore, the total antioxidant capacity of the sample can easily be measured by recording the absorbance signal at a specific wavelength (450 nm) with reference wavelength (620 nm or 570 nm), after redox indicator/sample mixing. This absorbance is then compared with the absorbance given by a known concentration of a standard solution at the same wavelength.
  • the assay used in the present invention is of use with many different types of samples. Thus it is applicable to biological fluids including mammalian serum or EDTA plasma, cerebrospinal fluid, synovial fluid or saliva.
  • the assay is of further use in assaying the antioxidant capacity of tissues, cells or other materials such as foodstuffs and oils.
  • Synovial fluid will provide useful information in the diagnosis and/or prognosis of arthritic or rheumatic disorders whereas CSF will provide possible implications of free radical induced nerve degeneration in the brain and spinal cord, and any possible role they may have in neural inflammation in diseased states or after injury.
  • the invention can be used to determine the contribution of specific or particular classes of antioxidants known or suspected to be present in a sample, by comparing the antioxidant capacity as measured by the method of the invention before and after the specific or particular classes of antioxidants have been removed or extracted.
  • the antioxidant contribution of the proteins contained within a sample may be determined by comparing the antioxidant capacity of the sample before and after the effect of proteins have been eliminated. Elimination of protein effect is achieved by mixing TMB solution with hydrochloride acid solution before adding to sample.
  • the invention has application in monitoring the progress of patients suffering from the large number of diseases and infections that cause a change in the balance of pro-oxidants and antioxidants in body fluids.
  • a further application of the invention is in monitoring the effectiveness of antioxidants drugs.
  • the total antioxidant capacity can be monitored to provide information on the rate of recovery.
  • the relative activity of different reducing agents is calculated.
  • the relative activity of vitamin C, Trolox, GSH, albumin and bilirubin in compare with uric acid were about 1, 1, 1, 0.6, 2.
  • GSH is an antioxidant whose concentration is low in human serum.
  • the functional part of GSH as an antioxidant is the SH group, which is also present in non- protein antioxidants such as lipoic acid and some amino acids. Therefore, GSH was used here to represent the SH-group-containing non-protein compounds.
  • FIG. 1 shows the rate of increase for standards solutions (the subtraction of the blank absorbance from the standards solutions absorbance at 450 nm (reference wavelength 620 or 570 nm), uric acid (filled triangles), , ascorbic acid (cross), trolox (filled circles), GSH (cross square) and bilirubin (open squares), all measured in parallel.
  • FIG. 2 shows the rate of increase for standard solutions of Albumin (the subtraction of the blank absorbance from the standard solutions absorbance at 450 nm (reference wavelength 620 or 570 nm).
  • the determination of the total antioxidant capacity of a sample provides the following steps:
  • substrate buffer phosphate citrate buffer
  • TMB.2HC1 tablet (Sigma, contain 1 mg 3,3',5,5'-Tetramethylbenzidine.2HCl) is dissolved in 10ml substrate buffer (unused TMB solution can be stored for 4 days at 4°C); 15
  • hydrochloride acid solution (HC1:2N): 40ml HCl 37% is added to 150 dH 2 O and volume is adjusted up to 200ml with dH 2 O;
  • ammonium persulfate 0.04 gr ammonium persulfate is dissolved in 20 10 ml distilled water, is dispensed in aliquots (80 ⁇ l) and stored at -20 0 C for six months.
  • TMB cation buffer freshly prepared before using and in case that all wells of a microplate is used: in a dark bottle, to 5.5 ml of TMB solution is added 50 ⁇ l of ammonium persulfate (0.4%), incubate at room temperature for 3-5 min, 20 ml of substrate buffer is added, mixed well gently and is used immediately;
  • the method described above has the advantage of using TMB cation with a typical spectrum of absorption (450 nm, reference 620 or 570 nm), thus facilitating the work of the analyst. Moreover, it allows the values to be quantified by simple comparison with a standard curve obtained by samples with a known concentration of a typical reducing agent. It can be done for a lot of samples rapidly and cost effective and ELISA reader exist in every laboratory.

Abstract

A method that can be used for the measurement of the total antioxidant capacity of an organic or inorganic liquid. The antioxidant capacity is determined by the decolourization of 3,3',5,5'-Tetramethylbenzidine (TMB) cation exerted by the antioxidants of the sample .TMB cation will be decolourized by reduction in a redox reaction. The quantitative analysis of the total antioxidant capacity of the sample can be easily made by spectrophotometry at 450 nm (reference wavelength 620 or 570 nm) both by macro- and micromethods (ELISA reader) with the use of a number of reducing standard compositions with known concentrations.

Description

Measurement of the total antioxidant capacity in liquids and solutions using TMB.
Description of the invention
FIELD OF THE INVENTION
The present invention concerns a method of determining the total antioxidant capacity of a sample by using TMB cation solution. According to the invention, the total antioxidant capacity of a sample is determined by the reduction (decolourization) of TMB cation. The results compared with the reducing action of uric acid with known concentration give values expressed in μmol/litre.
BACKGROUND OF THE INVENTION In human body, reactive oxygen species (ROS, as pro-oxidants) such as the hydroxyl radical (OH), the superoxide radical (O2), the nitric oxide radical (NO ) and the lipid peroxyl radical (LOO ) are derived either from normal essential metabolic processes or from external sources such as exposure to X-rays, ozone, cigarette smoking, air pollutants and industrial chemicals (Dean, R. T., et al, Biochem. J. (1997), 324:1-18). ROS can readily react with and damage other molecules that in some cases, the body uses this to fight infections, or in other cases, the damage may be to the body's own molecules such as DNA, lipid, proteins and carbohydrates (Stadtman, E. R. Annu. Rev. Biochem. (1993), 62:797- 821). In body, there is always a balance between pro-oxidants and antioxidants and the antioxidants mop up ROS before they damage other essential molecules. The antioxidants were defined as enzymes or molecules that can retard or prevent the damaging effects of ROS in tissues. Clinical trials and epidemiological studies have established an inverse correlation between the intake of antioxidants and the occurrence of disease such as inflammation, cardiovascular disease, cancer, and aging-related disorders (Halliwell, B.; Gutteridge, J. Oxford Univ. Press, NY (1999)); (Willet,W.C, The Harvard Medical School Guide to Healthy Eating; Simon and Schuster: New York (2001)).
Antioxidants have thus become a topic of increasing interest recently and the number of publications has nearly quadrupled in the past decade (Based on the Web of Knowledge search on articles containing the word antioxidant or antioxidants). Plasma (or serum) concentrations of different antioxidants can be measured in laboratories separately, but the measurements are time-consuming, labor-intensive, costly and they require complicated techniques. Because the measurement of different antioxidant molecules separately, is not practical and antioxidant effects of them are additive, total antioxidant response (TAR) of a sample is measured and this is named as total antioxidant capacity Miller NJ, et al., Clin Sci (Lond). (1993), Apr; 84(4):407-12); (Re R, et al. Free Radic Biol Med. (1999), May; 26(9- 10): 1231-7), total antioxidant activity , total antioxidant power, total antioxidant status , or other synonyms (Prior RL, Cao G5 Free Radic Biol Med. (1999); Dec;27(ll-12):l 173-81. Review).
To date, various methods have been developed to measure the total antioxidant capacity and each of them has advantages and disadvantages (Cao G, Prior RL. Clin Chem. (1998), Jun;44(6 Pt 1): 1309-15); (Frankel EN, Meyer AS. J Sci Food Agr (2000), 80 (13): 1925-1941 OCT. Review); (Prior RL, et al, J Agric Food Chem. (2005), May 18;53(10):4290-302. Review); (Benzie IF, Strain JJ. Anal Biochem. (1996), JuI 15;239(l):70-6). Advantage of the present invention include the following: 1) the assay can be carried out in a single step for each sample, i.e. there is no series of separate reactions required to obtain the result; 2) the apparatus required is relatively simple, easy to maintain and 3) permits several assays to be carried out simultaneously for a numbers of samples.
THE INVENTION
The invention seeks to provide a method of determining the total antioxidant capacity of a liquid and/or solution by using a marker consisting of a stable substance and by providing values expressed in μmol/litre.
It is an object of the invention to provide a simple and efficient method for measuring the total antioxidant capacity of a sample.
The term redox indicator is sued hereafter to denote the oxidant in such redox reactions. Redox indicators are known to undergo physiochemical change, for example a colour change, when reduced by antioxidants in a sample. This physicochemical change can be easily monitored by known methods, for example by monitoring a change in absorption, or emission, of electromagnetic radiation in a sample or by other methods that are known to monitor redox reactions. The method of the invention utilises the ability of antioxidants to reduce TMB cation which causes decolourization of TMB cation and hence results in a decrease in absorbance at 450 nm.
The amount of the redox indicator reduced by the antioxidant is a reflection of the total antioxidant capacity of the antioxidant. Therefore, the total antioxidant capacity of the sample can easily be measured by recording the absorbance signal at a specific wavelength (450 nm) with reference wavelength (620 nm or 570 nm), after redox indicator/sample mixing. This absorbance is then compared with the absorbance given by a known concentration of a standard solution at the same wavelength.
The assay used in the present invention is of use with many different types of samples. Thus it is applicable to biological fluids including mammalian serum or EDTA plasma, cerebrospinal fluid, synovial fluid or saliva. The assay is of further use in assaying the antioxidant capacity of tissues, cells or other materials such as foodstuffs and oils. Synovial fluid will provide useful information in the diagnosis and/or prognosis of arthritic or rheumatic disorders whereas CSF will provide possible implications of free radical induced nerve degeneration in the brain and spinal cord, and any possible role they may have in neural inflammation in diseased states or after injury.
In a further embodiment the invention can be used to determine the contribution of specific or particular classes of antioxidants known or suspected to be present in a sample, by comparing the antioxidant capacity as measured by the method of the invention before and after the specific or particular classes of antioxidants have been removed or extracted. Thus, the antioxidant contribution of the proteins contained within a sample may be determined by comparing the antioxidant capacity of the sample before and after the effect of proteins have been eliminated. Elimination of protein effect is achieved by mixing TMB solution with hydrochloride acid solution before adding to sample. Similarly, the invention has application in monitoring the progress of patients suffering from the large number of diseases and infections that cause a change in the balance of pro-oxidants and antioxidants in body fluids.
A further application of the invention is in monitoring the effectiveness of antioxidants drugs. Thus, after administration of such a drug, the total antioxidant capacity can be monitored to provide information on the rate of recovery.
By comparing the change in absorbance for an equivalent molar concentration of different reducing agents with an equivalent molar concentration of uric acid, the relative activity of different reducing agents is calculated. The relative activity of vitamin C, Trolox, GSH, albumin and bilirubin in compare with uric acid were about 1, 1, 1, 0.6, 2.
GSH is an antioxidant whose concentration is low in human serum. However, the functional part of GSH as an antioxidant is the SH group, which is also present in non- protein antioxidants such as lipoic acid and some amino acids. Therefore, GSH was used here to represent the SH-group-containing non-protein compounds.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
FIG. 1 shows the rate of increase for standards solutions (the subtraction of the blank absorbance from the standards solutions absorbance at 450 nm (reference wavelength 620 or 570 nm), uric acid (filled triangles), , ascorbic acid (cross), trolox (filled circles), GSH (cross square) and bilirubin (open squares), all measured in parallel.
FIG. 2 shows the rate of increase for standard solutions of Albumin (the subtraction of the blank absorbance from the standard solutions absorbance at 450 nm (reference wavelength 620 or 570 nm). DETAILED DESCRIPTION OF THE INVENTION
Further features and advantages of the invention will be more readily apparent from the following description of a preferred embodiment of the method. According to the invention, the determination of the total antioxidant capacity of a sample provides the following steps:
1. preparing a serial dilution of uric standard (μM ):
6000, 3000, 1500, 750, 375, 187.5, 93.75, 46.9, 23.4, 0 (blank); 5
2. preparing substrate buffer (phosphate citrate buffer):
1.455gr di-sodium hydrogen phosphate anhydrous (Na2HPO4) , 1.91gr citric acid anhydrous (C6HsO7) is dissolved in 150 ml dH2O, pH is adjusted at 5, the volume is adjusted up to 200 ml with dH2O and is stored at 4 °C; 10
3. preparing TMB solution(freshly prepared before using):
One TMB.2HC1 tablet (Sigma, contain 1 mg 3,3',5,5'-Tetramethylbenzidine.2HCl) is dissolved in 10ml substrate buffer (unused TMB solution can be stored for 4 days at 4°C); 15
4. preparing hydrochloride acid solution (HC1:2N): 40ml HCl 37% is added to 150 dH2O and volume is adjusted up to 200ml with dH2O;
5. preparing ammonium persulfate (0.4%): 0.04 gr ammonium persulfate is dissolved in 20 10 ml distilled water, is dispensed in aliquots (80 μl) and stored at -20 0C for six months.
6. pouring a 10 μl quantity of each sample, standard and blank (distilled water) into the wells of a multiwell plate;
25 7. preparing TMB cation buffer (freshly prepared before using and in case that all wells of a microplate is used): in a dark bottle, to 5.5 ml of TMB solution is added 50 μl of ammonium persulfate (0.4%), incubate at room temperature for 3-5 min, 20 ml of substrate buffer is added, mixed well gently and is used immediately;
30 8. adding 200 μl of TMB cation buffer to each well and incubating it at room temperature in dark place for 3-4 minutes;
9. adding 50 μl hydrochloride acid solution (2N) to each well; 35 10. incubate the plate for 30-45 minutes in dark place;
11. carrying out a spectrophotometry of the samples at 450 nm with reference wavelength 620 or 570 nm by an ELISA reader;
40 12. subtracting the absorbance of samples and standards from the absorbance of blank;
13. providing a hyperbolic standard curve from the values relative to the standard samples by Microsoft Excel program and obtain a equation curve for it, i.e. the sample with known concentration of uric acid used as a typical reducing agent;
45
14. calculating the values of the unknown samples on the base of the values obtained from the above standard curve that the values are expressed as reducing equivalents in μmol/litre.
15. total antioxidant power of fresh and aged (one month at -80 0C or one week at -20 0C) 50 plasma of healthy Greece adults were 900- 2000 μmol/litre (mean 1210; SD, 286; n=50).
It is self-evident from the foregoing that the method described above has the advantage of using TMB cation with a typical spectrum of absorption (450 nm, reference 620 or 570 nm), thus facilitating the work of the analyst. Moreover, it allows the values to be quantified by simple comparison with a standard curve obtained by samples with a known concentration of a typical reducing agent. It can be done for a lot of samples rapidly and cost effective and ELISA reader exist in every laboratory.
References
Dean, R. T.; Fu, S.; Stoker, R.; Davies, M. J. Biochemistry and pathology of radical- mediated protein oxidation. Biochem. J. 324:1-18; 1997.
Stadtman, E. R. Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu. Rev. Biochem. 62:797- 821; 1993.
Halliwell, B.; Gutteridge, J. Free radicals in biology and medicine. Oxford Univ. Press,
NY; 1999.
Willet,W.C. Eat, Drink, and be Health- The Harvard Medical School Guide to Healthy
Eating; Simon and Schuster: New York, 2001. Based on the Web of Knowledge search on articles containing the word antioxidant or antioxidants.
Miller NJ, Rice-Evans C5 Davies MJ, Gopinathan V, Milner A. A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clin Sci (Lond). 1993 Apr;84(4):407-12. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans. Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radic Biol Med.
1999 May;26(9-10): 1231-7.
Prior RL, Cao G. In vivo total antioxidant capacity: comparison of different analytical methods.Free Radic Biol Med. 1999 Dec;27(l 1-12):1173-81. Review. Cao G, Prior RL. Comparison of different analytical methods for assessing total antioxidant capacity of human serum. Clin Chem. 1998 Jun;44(6 Pt l):1309-15.
Frankel EN, Meyer AS. The problems of using one-dimensional methods to evaluate multifunctional food and biological antioxidants. J Sci Food Agr 80 (13): 1925-1941
OCT 2000. Review. Prior RL, Wu X, Schaich K. Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. J Agric Food Chem. 2005 May 18;53(10):4290-302. Review
Benzie IF, Strain JJ. The ferric reducing ability of plasma (FRAP) as a measure of "antioxidant power": the FRAP assay. Anal Biochem. 1996 JuI 15;239(l):70-6.

Claims

ClaimsWhat is claimed is:
1. A method for determining the total antioxidant capacity of a liquid sample by using
TMB cation, which comprises: mixing a sample of said liquid with TMB solution; adding a hydrochloride acid solution to the obtained mixture; determining the quantity of TMB cation decolourization exerted by the antioxidants of the sample and correlating the determined quantity of the decolourization with the total antioxidant capacity of the liquid.
2. A method for determining the total antioxidant capacity of a liquid sample according to claim 1, wherein the liquid is EDTA plasma, serum, and other biological liquid.
■Ψ
PCT/GR2006/000053 2005-10-03 2006-10-03 Measurement of the total antioxidant capacity in liquids and solutions using tmb WO2007039775A1 (en)

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US8317997B2 (en) 2011-02-28 2012-11-27 Institute For Molecular Medicine, Inc. Method and apparatus for measuring oxidation-reduction potential
US8512548B2 (en) 2007-05-18 2013-08-20 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US9063070B2 (en) 2007-05-18 2015-06-23 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US9360446B2 (en) 2012-10-23 2016-06-07 Aytu Bioscience, Inc. Methods and systems for measuring and using the oxidation-reduction potential of a biological sample
US9372167B2 (en) 2012-04-19 2016-06-21 Aytu Bioscience, Inc. Oxidation-reduction potential test device including a multiple layer gel
IT201800003475A1 (en) * 2018-03-13 2019-09-13 Fondazione St Italiano Tecnologia Procedure for determining the antioxidant capacity of a biological sample and relative kit.

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TATZBER FRANZ ET AL: "Dual method for the determination of peroxidase activity and total peroxides-iodide leads to a significant increase of peroxidase activity in human sera.", ANALYTICAL BIOCHEMISTRY. 15 MAY 2003, vol. 316, no. 2, 15 May 2003 (2003-05-15), pages 147 - 153, XP002397815, ISSN: 0003-2697 *

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US9063070B2 (en) 2007-05-18 2015-06-23 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US10036723B2 (en) 2007-05-18 2018-07-31 Aytu Bioscience, Inc. Measurement and uses of oxidative status
US8512548B2 (en) 2007-05-18 2013-08-20 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US9423372B2 (en) 2007-05-18 2016-08-23 Aytu Bioscience, Inc. Measurement and uses of oxidative status
US8709709B2 (en) 2007-05-18 2014-04-29 Luoxis Diagnostics, Inc. Measurement and uses of oxidative status
US9383331B2 (en) 2007-05-18 2016-07-05 Aytu Bioscience, Inc. Measurement and uses of oxidative status
US9528959B2 (en) 2011-02-28 2016-12-27 Aytu Bioscience, Inc. Method and apparatus for measuring oxidation-reduction potential
US9034159B2 (en) 2011-02-28 2015-05-19 Luoxis Diagnostics, Inc. Method and apparatus for measuring oxidation-reduction potential
US8641888B2 (en) 2011-02-28 2014-02-04 Luoxis Diagnostics, Inc. Method and apparatus for measuring oxidation-reduction potential
US8317997B2 (en) 2011-02-28 2012-11-27 Institute For Molecular Medicine, Inc. Method and apparatus for measuring oxidation-reduction potential
US8329012B2 (en) 2011-02-28 2012-12-11 Institute For Molecular Medicine, Inc. Method and apparatus for measuring oxidation-reduction potential
US9372167B2 (en) 2012-04-19 2016-06-21 Aytu Bioscience, Inc. Oxidation-reduction potential test device including a multiple layer gel
US10281425B2 (en) 2012-04-19 2019-05-07 Aytu Bioscience, Inc. Multiple layer gel
US9360446B2 (en) 2012-10-23 2016-06-07 Aytu Bioscience, Inc. Methods and systems for measuring and using the oxidation-reduction potential of a biological sample
US9410913B2 (en) 2012-10-23 2016-08-09 Aytu Bioscience, Inc. Methods and systems for measuring and using the oxidation-reduction potential of a biological sample
US10184931B2 (en) 2012-10-23 2019-01-22 Aytu Bioscience, Inc. Methods and systems for measuring and using the oxidation-reduction potential of a biological sample
IT201800003475A1 (en) * 2018-03-13 2019-09-13 Fondazione St Italiano Tecnologia Procedure for determining the antioxidant capacity of a biological sample and relative kit.
WO2019175749A1 (en) * 2018-03-13 2019-09-19 Fondazione Istituto Italiano Di Tecnologia Method for determining the antioxidant capacity of a biological sample and related kit

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