Methods and devices for measuring antioxidant activity in a fluid sample

Abstract

The present invention relates to methods and devices for measuring antioxidant levels in a fluid sample.

Description

METHODS AND DEVICES FOR MEASURING ANTIOXIDANT ACTIVITY IN A FLUID SAMPLEFIELD OF THE INVENTION

[0001] The present invention relates to methods and devices for measuring antioxidant activity in a fluid sample. However, it will be appreciated that the invention is not limited to this field of use.

[0002] The present application claims priority from Australian Provisional Patent Application No. 2023904171 (filed 21 December 2023), the contents of which are incorporated in their entirety herein.BACKGROUND OF THE INVENTION

[0003] Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

[0004] A wide range of human and animal pathologies are thought to be related to the development of oxidative stress including cancer, neurodegenerative conditions, depression, multiple sclerosis, amyotrophic lateral sclerosis, cataracts, diabetes, cardiovascular disease and infertility. For example, oxidative stress is a key factor in the aetiology of male infertility and is thought to arise because of deficiencies in the levels of antioxidant protection afforded to the spermatozoa and / or the exposure of these cells to excess reactive oxygen species (ROS). Spermatozoa are particularly vulnerable to oxidative stress as these highly specialized cells are characterized by limited volume and restricted distribution of cytoplasmic space in which to house antioxidant factors that protect most somatic cell types from oxidative attack.

[0005] There is a role for antioxidants as a potential avenue of remediation for oxidative stress. However, one of the problems with calibrating the dose and duration of antioxidant administration in any clinical situation is knowing when to initiate and when to cease treatment so that the redox dynamics in the tissue of interest are balanced and the system does not revert to a state of reductive stress - which can be just as damaging as its oxidative counterpart.

[0006] Consequently, there is a need for effective assays for rapidly measuring antioxidant activity. Moreover, the successful introduction of a point-of-care device for measuring antioxidant activity would be of value in a wide range of additional clinical situations.

[0007] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.SUMMARY OF THE INVENTION

[0008] According to a first aspect the present invention relates to a method of measuring antioxidant activity in a fluid sample, the method comprising the steps of: a) adding a solution comprising an oxidisable reporter molecule to first and second chambers, wherein the chambers are separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated; b) generating a current across the first and second chambers via an anode in the first chamber and a cathode in the second chamber to activate the reporter molecule; c) adding the fluid sample to the solution in the first and second chambers; and d) measuring absorbance of the solution in the first and second chambers independently, wherein high absorbance in the first chamber compared to the second chamber indicates low antioxidant activity in the fluid sample.

[0009] In one embodiment, the antioxidant activity is free radical scavenging activity and / or reductive activity.

[0010] In one embodiment, step c) is performed before step b)

[0011] In one embodiment, the antioxidant activity is inhibition of free radical formation.

[0012] According to a second aspect, the present invention provides a method of measuring free radical scavenging activity and / or reductive activity in a fluid sample, the method comprising the steps of: a) adding a solution comprising an oxidisable reporter molecule to first and second chambers, wherein the chambers are separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated; b) generating a current across the first and second chambers via an anode in the first chamber and a cathode in the second chamber to activate the reporter molecule in the first chamber; c) adding the fluid sample to the solution in the first and second chambers; and d) measuring absorbance of the solution in the first and second chambers independently, wherein high absorbance in the first chamber compared to the second chamber indicates low free radical scavenging activity and / or reductive activity in the fluid sample.

[0013] According to a third aspect, the present invention provides a method of measuring inhibition of free radical formation in a fluid sample, the method comprising the steps of: a) adding a solution comprising an oxidisable reporter molecule to first and second chambers, wherein the chambers are separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated; b) adding the fluid sample to the solution in the first and second chambers; c) generating a current across the first and second chambers via an anode in the first chamber and a cathode in the second chamber to activate the reporter molecule in the first chamber; and d) measuring absorbance of the solution in the first and second chambers independently, wherein high absorbance in the first chamber compared to the second chamber indicates low inhibition of free radical formation in the fluid sample.

[0014] According to a fourth aspect, the present invention provides a device for measuring antioxidant activity in a fluid sample, the device comprising first and second chambers separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated, wherein an anode is positioned in the first chamber and a cathode is positioned in the second chamber; the first and second chambers both comprise a solution comprising an oxidisable reporter molecule and an aperture that allows the addition of the fluid sample to the solution; and the walls of the first and second chambers adjacent to the membrane allow the passage of light.

[0015] According to a fifth aspect, the present invention provides a method of measuring antioxidant activity in a fluid sample, the method comprising the steps of: a) generating a current across the first and second chambers of the device of the fourth aspect via the anode and cathode to activate the reporter molecule; b) adding the fluid sample to the solution in the first and second chambers of the device; and c) measuring absorbance of the solution in the first and second chambers independently, wherein high absorbance in the first chamber compared to the second chamber indicates low antioxidant levels in the fluid sample.

[0016] In one embodiment, the antioxidant activity is free radical scavenging activity and / or reductive activity.

[0017] In one embodiment, step b) is performed before step a)

[0018] In one embodiment, the antioxidant activity is inhibition of free radical formation.

[0019] According to a sixth aspect, the present invention provides a method of measuring free radical scavenging activity and / or reductive activity in a fluid sample, the method comprising the steps of: a) generating a current across the first and second chambers of the device of the fourth aspect via the anode and cathode to activate the reporter molecule; b) adding the fluid sample to the solution in the first and second chambers of the device; and c) measuring absorbance of the solution in the first and second chambers independently, d) wherein high absorbance in the first chamber compared to the second chamber indicates low free radical scavenging activity and / or reductive activity in the fluid sample.

[0020] According to a seventh aspect, the present invention provides a method of measuring inhibition of free radical formation in a fluid sample, the method comprising the steps of: a) adding the fluid sample to the solution in the first and second chambers of the device of the fourth aspect; b) generating a current across the first and second chambers of the device of the third aspect via the anode and cathode to activate the reporter molecule; and c) measuring absorbance of the solution in the first and second chambers independently, d) wherein high absorbance in the first chamber compared to the second chamber indicates low inhibition of free radical formation in the fluid sample.

[0021] According to an eighth aspect, the present invention provides a method of measuring hydrogen peroxide scavenging in a fluid sample, the method comprising the steps of: a) adding a solution comprising an oxidisable reporter molecule and horseradish peroxidase to first and second chambers; b) adding the fluid sample to the solution in the first chamber and an equal volume of water to the solution in the second chamber;c) adding hydrogen peroxide to the solution in the first and second chambers to activate the reporter molecule; and d) measuring absorbance of the solution in the first and second chambers independently, wherein high absorbance in the first chamber compared to the second chamber indicates low hydrogen peroxide scavenging in the fluid sample.

[0022] In one embodiment, absorbance is measured in the first and second chambers prior to step c) to measure turbidity in the fluid sample.

[0023] According to a ninth aspect, the present invention provides a method of measuring organic peroxide scavenging in a fluid sample, the method comprising the steps of: a) adding a solution comprising an oxidisable reporter molecule and hematin to first and second chambers; b) adding the fluid sample to the solution in the first chamber and an equal volume of water to the solution in the second chamber; c) adding cumene hydroperoxide to the solution in the first and second chambers to activate the reporter molecule; and d) measuring absorbance of the solution in the first and second chambers independently, wherein high absorbance in the first chamber compared to the second chamber indicates low organic peroxide scavenging in the fluid sample.

[0024] In one embodiment, absorbance is measured in the first and second chambers prior to step c) to measure turbidity in the fluid sample.

[0025] According to a tenth aspect, the present invention provides a method of activating an oxidisable reporter molecule, the method comprising: adding a solution comprising the oxidisable reporter molecule to first and second chambers separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated; and generating a current across the first and second chambers via an anode in the first chamber and a cathode in the second chamber to activate the reporter molecule in the first chamber.

[0026] According to an eleventh aspect, the present invention provides a method of measuring antioxidant activity in a fluid sample, the method comprising the method of theany one of the first to third or fifth to seventh aspects and further comprising the method of the eighth aspect.

[0027] According to a twelfth aspect, the present invention provides a method of measuring antioxidant activity in a fluid sample, the method comprising the method of the any one of the first to third or fifth to seventh aspects and further comprising the method of the ninth aspect.

[0028] According to a thirteenth aspect, the present invention provides a method of measuring antioxidant activity in a fluid sample, the method comprising the method of the any one of the first to third or fifth to seventh aspects and further comprising the method of the eighth aspect and the method of the ninth aspect.

[0029] According to a fourteenth aspect, the present invention provides a method of measuring antioxidant activity in a fluid sample, the method comprising the method of the any one of the first to third or fifth to seventh aspects, and further comprising the steps of: a1) adding a solution comprising an oxidisable reporter molecule and horseradish peroxidase to third and fourth chambers; b1) adding the fluid sample to the solution in the third chamber and an equal volume of water to the solution in the fourth chamber; c1) adding hydrogen peroxide to the solution in the third and fourth chambers to activate the reporter molecule; and d1) measuring absorbance of the solution in the third and fourth chambers independently, wherein high absorbance in the third chamber compared to the fourth chamber indicates low hydrogen peroxide scavenging in the fluid sample.

[0030] According to a fourteenth aspect, the present invention provides a method of measuring antioxidant activity in a fluid sample, the method comprising the method of the any one of the first to third or fifth to seventh aspects, and further comprising the steps of: a1) adding a solution comprising an oxidisable reporter molecule and hematin to third and fourth chambers; b1) adding the fluid sample to the solution in the third chamber and an equal volume of water to the solution in the fourth chamber; c1) adding cumene hydroperoxide to the solution in the third and fourth chambers to activate the reporter molecule; and d1) measuring absorbance of the solution in the third and fourth chambers independently,wherein high absorbance in the third chamber compared to the fourth chamber indicates low organic peroxide scavenging in the fluid sample.

[0031] In one embodiment, absorbance is measured in the first and second chambers prior to step c1) to measure turbidity in the fluid sample.

[0032] In one embodiment, the reporter molecule is 2,2'-azino-bis(3-ethylbenzothiazoline- 6-sulfonic acid) (ABTS) or leukomethylene blue (LMB).

[0033] In one embodiment, the reporter molecule is ABTS and the absorbance is measured at about 415 nm, about 645 nm, about 734 nm or about 815 nm.

[0034] In one embodiment, the reporter molecule is ABTS and the absorbance is measured at about 734 nm.

[0035] In one embodiment, the reporter molecule is LMB and the absorbance is measured at about 664 nm.

[0036] In one embodiment, the fluid sample is selected from the group consisting of biological fluids, plant extracts, food constituents, cosmetic preparations, wastewater and industrial fluids.

[0037] In one embodiment, the fluid sample is a biological fluid.

[0038] In one embodiment, the biological fluid is selected from the group consisting of semen, blood, urine, saliva and ovarian follicular fluid.

[0039] In one embodiment, the biological fluid is semen.

[0040] In one embodiment, the biological fluid is serum or plasma.

[0041] In one embodiment, the membrane comprises cellulose, nylon or polyester.

[0042] In one embodiment, the membrane is a polyester track etch (PETE) membrane.

[0043] In one embodiment, the membrane is a 0.4pm PETE membrane.

[0044] In one embodiment, the first chamber and second chamber both comprise 10OpI to10ml of the solution.

[0045] In one embodiment, the first chamber and second chamber both comprise about 2ml of the solution.

[0046] In one embodiment, the device is a cartridge that is insertable into a receiving device comprising means for generating a current across the chambers via the anode and cathode.

[0047] In one embodiment, the receiving device further comprises means for measuring the absorbance of the solution in the first and second chambers independently.

[0048] In one embodiment, the receiving device can process the data from the first and second chamber and provide a readout expressed in vitamin C or Trolox equivalents.

[0049] In one embodiment, the receiving device can control for any non-specific changes in turbidity.

[0050] In one embodiment, the readout incorporates a traffic light display that tells the user whether the readout is high, average or low compared with archived sample data.

[0051] In one embodiment, the current is applied for about 20 seconds at 10 to 40 mAmps.

[0052] In one embodiment, 1 l to 1ml of the fluid sample is added to the solution in the first and second chambers.

[0053] In one embodiment, about 50 pl, about 10OpI, about 150pl, about 200pl, about 250pl, about 300pl, about 350pl, about 400pl, about 450pl, about 500pl, about 550pl, about 600pl, about 650pl, about 700pl, about 850pl, about 900pl, about 950pl or about 1 ml of the fluid sample is added to the solution in the first and second chambers.

[0054] In one embodiment, the absorbance is measured 5 to 30 minutes after the reporter molecule is activated.

[0055] In one embodiment, the absorbance is measured about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes or about 30 minutes after the reporter molecule is activated.BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1. Schematic representation of the detection device of the invention.

[0057] Figure 2. Electrochemical activation of ABTS and its application in the assessment of free radical scavenging activity. (A) Electrochemical activation of ABTS for 20 sec generated the coloured ABTS*+ radical, which absorbed intensely at 734 nm but could besuppressed by post-activation addition of the antioxidant Trolox, in a manner that was not significantly changed by ABTS concentrations ranging from 60-140 pM. (B) The absorption spectrum generated by ABTS*+ showing the peak at 734 nm and the dose-dependent suppression achieved by Trolox. (C) A similar dose-dependent suppression of ABTS*+ absorption at 734 nm achieved with resveratrol. (D) Using unfractionated human semen as a rich biological source of antioxidant activity, a dose-dependent decrease in absorption at 734 nm was achieved. (E) Repeated analysis of the same human semen sample generated an inter-assay coefficient of variation of 11.2% with this post-activation, electrochemical ABTS assay (n = 8). (F) A detailed dose-response analysis with Trolox demonstrated that the suppression of ABTS*+ absorption was linear over the final concentration range, 0.31 - 20 pM.

[0058] Figure 3. Use of the post-activation electrochemical ABTS assay for free radical scavenging activity to determine the ability of candidate antioxidants to suppress the ABTS*+ signal. Dose dependent analyses were conducted for (A) vitamin C, (B) glutathione, (C) N- acetyl cysteine, (D) hypotaurine, (E) BSA, (F) melatonin, (G) carnitine (H) myoinositol, (I) coenzyme Q10, (J) taurine, (K) lipoic acid (L) spermine. All results are presented as the concentration of reagent in the original 15 pL inoculant into the medium. To determine the final concentration following dilution with assay reagents the values on the X-axis should be divided by 55 (10 pM-25 pM Trolox equivalents). All data points represent the mean ± SE of 3 independent replicates.

[0059] Figure 4. Assessment of antioxidant activity, reflecting the inhibition of free radical formation, using the pre-activation ABTS assay. Log dose response analyses for (A) myoinositol (B) carnitine and (C) spermine failed to reveal any free radical scavenging activity. In contrast, both polyphenols, (D) epigallocatechin gallate (EGCG) and (E) resveratrol exhibited such powerful antioxidant activity that they reached a plateau at concentrations above 0.676 mM (100 pM final concentration). All data points represent the mean ± SE of 3 independent replicates.

[0060] Figure 5. Comparison of the pre-activation electrochemical free scavenging assay with a commercial kit manufactured by Cayman Chemicals. (A) Using the electrochemical assay, the standards provided by Cayman (closed circles) or constructed in the laboratory (open circles) were superimposable and linear. (B) When the commercial TEAC (Trolox Equivalent Antioxidant Capacity) kit was used for these assessments, the standard curves were again linear and superimposable at the lower doses, but the Cayman assay lost sensitivity above 0.5 mM Trolox. (C) A comparison of the ability of the post-activationelectrochemical and Cayman assays to measure the antioxidant content of human semen revealed a highly significant correlation (P <0.001) between these methods of assessment. (D) However, the Cayman assay detected almost twice as much antioxidant activity in semen as the electrochemical equivalent ((P <0.001; n = 20), possibly because the former is a preactivation assay whereby test sample is added prior to ABTS oxidation, thereby enabling the assay to reflect the inhibition of free radical formation as well as free radical scavenging. (E) A pre-activation version of the electrochemical assay was therefore developed and shown to generate a linear standard curve with vitamin C. X-axis presents the concentration of vitamin C in the stock solution, final concentration following dilution with assay reagents was 20- 40 pM. All data points represent the mean ± SE of 3 independent replicates. (F) This preactivation electrochemical assay was extremely stable generating an inter-assay coefficient of replication of 0.001% on 5 independent replicate analyses of a solution containing 35 pM vitamin C.

[0061] Figure 6. Use of the pre-activation electrochemical ABTS assay to determine the ability of candidate antioxidants to suppress the ABTS*+ signal. Dose dependent analyses were conducted for (A) vitamin C, (B) glutathione, (C) N-acetyl cysteine, (D) hypotaurine, (E) melatonin, (F) BSA, (G) resveratrol (H) EGCG, (I) lipoic acid, (J) DMSO, high dose, (K) DMSO low dose, (L) DMF. All results are presented as the concentration of reagent in the original solution; to determine final concentration in the reaction mixture the values on the X- axis should be divided by 55. All data points represent the mean ± SE of 3 independent replicates.

[0062] Figure 7. Development of ABTS assays for hydrogen- and organic- peroxides. (A) The hydrogen peroxide assay was completely unresponsive to 0.15-0.45 mM cumene hydroperoxide over a time scale of 20 min. However, the assay responded extremely rapidly to the presence of the same doses of hydrogen peroxide, such that a clear dose-response was evident at the earliest time point assessed (T 1 , which essentially represented the time taken to transfer the reaction mixture to the spectrophotometer (~1 min). Once generated the ABTS*+ radical was relatively stable although some deterioration on the signal was evident over 20 min with the highest standard assessed. (B) Conversely, the cumene hydroperoxide assay was extremely sensitive to 0.5-2.0 mM organic peroxide but did not respond at all to hydrogen peroxide over the 40 min incubation period. Unlike the hydrogen peroxide assay, the generation of an ABTS*+ signal following exposure to cumene hydroperoxide and hematin was not instantaneous but evolved gradually. The results represent the mean ± SE of 3 independent experiments. The doses quoted represent the initial concentration prior todilution in the reaction mixture. To arrive at the final concentration, these values must be divided by 10.

[0063] Figure 8. Use of ABTS and HRP to monitor hydrogen peroxide scavenging activity of putative antioxidants. Dose-dependent analyses were conducted for (A) vitamin C, (B) glutathione, (C) N-acetyl cysteine, (D) hypotaurine, (E) melatonin, (F) resveratrol (G) EGCG, (H) BSA, (I) DMSO, (J) lipoic acid, (K) carnitine, (L) co-enzyme Q10. All results are presented as the concentration of reagent in the original solution; to determine final concentration in the reaction mixture the values on the X-axis should be divided by 10. All data points represent the mean ± SE of 3 independent replicates.

[0064] Figure 9. Use of the ABTS and hematin to monitor the scavenging activity of putative antioxidants towards organic peroxide, peroxyl and alkoxyl radicals. Dosedependent analyses were conducted for (A) vitamin C, (B) glutathione, (C) N-acetyl cysteine, (D) hypotaurine, (E) melatonin, (F) BSA (G) resveratrol (H) EGCG, (I) spermine, (J) carnitine, (K) lipoic acid, (L) co-enzyme Q10. All results are presented as the concentration of reagent in the original solution; to determine final concentration in the reaction mixture the values on the X-axis should be divided by 10. All data points represent the mean ± SE of 3 independent replicates

[0065] Figure 10. Performance of the antioxidant assays in the assessment of human semen. (A) Analysis of the antioxidant levels recorded in semen samples donated by 27 randomly selected males. All groups are significantly different from each other by ANOVA (*** p < 0.001). Note the extremely high-level activity recoded by the pre-activation free radical formation assay equivalent to a mean of 25 mM vitamin C equivalents. (B) Correlation between the hydrogen peroxide scavenging assay and post-activation free radical scavenging assay (p < 0.001). (C) Correlation between post-activation, free radical scavenging assay and the pre-activation assessment of free radical formation (p < 0.01). (D) Correlation between the hydrogen peroxide scavenging assay and the pre-activation assessment of free radical formation (p < 0.001). (E) Relationship between the assessment of organic peroxide scavenging activity and the pre-activation assessment of free radical formation (NS). (F) Multiple regression analysis demonstrating how the integrated data from 4 antioxidant assays can be used to predict sperm count. (G) Multiple regression analysis demonstrating how the integrated data from 4 antioxidant assays can be used to predict seminal MDA concentration. (H) Multiple regression analysis demonstrating how the integrated data from 4 antioxidant assays can be used to predict sperm DNA damage. (I)Multiple regression analysis demonstrating how the integrated data from 4 antioxidant assays can be used to predict mitochondrial ROS generation by spermatozoa.

[0066] Figure 11. Impact of sperm presence and freezing on the antioxidant activity of human semen. (A) Organic peroxide scavenging activity. (B) Hydrogen peroxide scavenging activity. (C) Inhibition of ABTS,+radical formation. (D) ABTS,+radical scavenging activity. All results are expressed as vitamin C equivalents. Statistical analysis by ANOVA and data presented as means ± SEM (n = 3). Where a statistically significant impact of treatment on antioxidant activity was observed, differences between groups were characterised by a connecting letters report generated by JMP, whereby all columns not connected by the same letter are significantly different.

[0067] Figure 12. Antioxidant activity in the seminal plasma in different species. (A) Organic peroxide scavenging activity. (B) Hydrogen peroxide scavenging activity. (C) Inhibition of ABTS,+radical formation. (D) ABTS,+radical scavenging activity. All results are expressed as vitamin C equivalents. Statistical analysis by ANOVA on n = 3 biological replicates and data presented as means ± SEM. Statistical analysis by ANOVA following Box-Cox transformation; data presented as means ± SEM (n = 3). Where a statistically significant impact of treatment on antioxidant activity was observed, differences between groups were characterised by a connecting letters report generated by JMP, whereby all columns not connected by the same letter are significantly different.

[0068] Figure 13. Dose-dependent impact of cumene hydroperoxide on sperm motility in different species. Both total motility and progressive motility were assessed using a CASA system and two time points were examined: 10 min and 2h. (A) Human spermatozoa after 10 min exposure to cumene hydroperoxide. (B) Human spermatozoa after 2 h exposure to cumene hydroperoxide. (C) Bovine spermatozoa after 10 min exposure to cumene hydroperoxide. (D) Bovine spermatozoa after 2 h exposure to cumene hydroperoxide. (E) Equine spermatozoa after 10 min exposure to cumene hydroperoxide. (F) Equine spermatozoa after 2 h exposure to cumene hydroperoxide. Statistical analysis by ANOVA and data presented as means ± SEM (n = 3). Where a statistically significant impact of treatment on antioxidant activity was observed, differences between groups were characterised by a connecting letters report generated by JMP, whereby all columns not connected by the same letter are significantly different.

[0069] Figure 14. Analysis of antioxidant activity in a range of biofluids. (A) Organic peroxide scavenging activity. (B) Hydrogen peroxide scavenging activity. (C) Inhibition of ABTS,+radical formation. (D) ABTS,+radical scavenging activity. All results are expressedas vitamin C equivalents. Statistical analysis by ANOVA following Box-Cox transformation; data presented as means ± SEM (n = 3). Where a statistically significant impact of treatment on antioxidant activity was observed, differences between groups were characterised by a connecting letters report generated by JMP, whereby all columns not connected by the same letter are significantly different.

[0070] Figure 15. Analysis of antioxidant activity in a range of fruit juices. (A) Organic peroxide scavenging activity. (B) Hydrogen peroxide scavenging activity. (C) Inhibition of ABTS,+radical formation. (D) ABTS,+radical scavenging activity. All results are expressed as vitamin C equivalents. Statistical analysis by ANOVA following Box-Cox transformation; data presented as means ± SEM (n = 3). Where a statistically significant impact of treatment on antioxidant activity was observed, differences between groups were characterised by a connecting letters report generated by JMP, whereby all columns not connected by the same letter are significantly different.

[0071] Figure 16. Analysis of antioxidant activity in a range of cosmetic skin sera. (A) Organic peroxide scavenging activity. (B) Hydrogen peroxide scavenging activity. (C) Inhibition of ABTS,+radical formation. (D) ABTS,+radical scavenging activity. All results are expressed as vitamin C equivalents. Statistical analysis by ANOVA following Box-Cox transformation; data presented as means ± SEM (n = 3). Where a statistically significant impact of treatment on antioxidant activity was observed, differences between groups were characterised by a connecting letters report generated by JMP, whereby all columns not connected by the same letter are significantly different.

[0072] Figure 17. Evaluation of leukomethylene blue (LMB) as an alternative probe to ABTS in conducting assessments of antioxidant activity. In the presence of a hematin catalyst, cumene hydroperoxide induced the oxidation of both probes. (A) Absorption spectra for ABTS oxidized with different doses (25-200 pM) of cumene hydroperoxide showing characteristic absorbance peaks at 415, 645 and 734 nm. (B) Absorption spectra for LMB oxidized with different doses (25-200 pM) of cumene hydroperoxide showing a major peak at 664 nm and a shoulder at around 614 nm. (C) Time-dependent analysis of ABTS oxidation using different doses of cumene hydroperoxide (25-200 pM) in the presence of hematin monitored at 734 nm. (D) Time-dependent analysis of LMB oxidation using different doses of cumene hydroperoxide (25-200 pM) in the presence of hematin monitored at 664 nm. (E) Across this entire dataset an extremely close correlation was noted between ABTS and LMB oxidation.

[0073] Figure 18. Comparison of LMB and ABTS for their relative ability to respond to oxidation by hydrogen peroxide or cumene hydroperoxide in the presence of a hematin catalyst. (A) Time - and dose- dependent analysis of the ability of cumene hydroperoxide to oxidize LMB in the presence of hematin, monitored at 664 nm. (B) Time - and dosedependent analysis of the ability of hydrogen peroxide to oxidize LMB in the presence of hematin, monitored at 664 nm. (C) Time - and dose- dependent analysis of the ability of cumene hydroperoxide to oxidize ABTS in the presence of hematin, monitored at 734 nm. (D) Time - and dose- dependent analysis showing the inability of hydrogen peroxide to oxidize ABTS in the presence of hematin, monitored at 734 nm. These data demonstrate that LMB is a broad-spectrum probe that can be oxidized by both hydrogen peroxide and cumene hydroperoxide in the presence of hematin, whereas ABTS can only be oxidized by cumene hydroperoxide under these circumstances.

[0074] Figure 19. Comparison of LMB and ABTS for their relative ability to respond to oxidation by hydrogen peroxide or cumene hydroperoxide in the presence of horse radish peroxidase (HRP), as a catalyst. (A) Time - and dose- dependent analysis of the ability of hydrogen peroxide to oxidize LMB in the presence of HRP, monitored at 664 nm. (B) Time - and dose- dependent analysis showing the inability of cumene hydroperoxide to oxidize LMB in the presence of HRP, monitored at 664 nm. (C) Time- and dose- dependent analysis of the ability of hydrogen peroxide to oxidize ABTS in the presence of HRP, monitored at 734 nm. (D) Time - and dose- dependent analysis demonstrating the inability of cumene hydroperoxide to oxidize ABTS in the presence of HRP, monitored at 734 nm. (E) and (F) Detailed comparison of the ability of ABTS and LMB to detect low doses of hydrogen peroxide in the presence of HRP. These data demonstrate the specificity of HRP in the mediation hydrogen peroxide-induced oxidation and the relative responsiveness of LMB compared with ABTS as a probe for detecting this oxidant.

[0075] Figure 20. Analysis of the ability of LMB to reflect the antioxidant properties of vitamin C. (A) Using a post-electrochemical activation strategy, the reductive power of vitamin C is evident, with vitamin C doses as low as 5 pM being effective in reducing the oxidation product of LMB, methylene blue (MB), back to its reduced colourless form. (B) Using a pre-electrochemical activation strategy, vitamin C could also be shown to suppress the formation of MB when LMB was electrochemically oxidized. (C) Dose-dependent analysis of the ability of vitamin C to suppress the ability of cumene hydroperoxide to oxidize LMB in the presence of a hematin catalyst. (D) Dose-dependent analysis of the ability of vitamin C to inhibit hydrogen peroxide induced oxidation of LMB in the presence of HRP. (E) Use of the LMB pre-activation assay to demonstrate the powerful ability of DMSO to inhibit oxidation ofLMB to MB; this contrasts with cyrene, an aprotic dipolar replacement solvent, that possesses little inherent antioxidant activity in this assay. *** P < 0.001.DEFINITIONS

[0076] In describing and claiming the present invention, the following terminology has been used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.

[0077] As used herein the term “about” can mean within 1 or more standard deviation per the practice in the art. Alternatively, “about” can mean a range of up to 20%. When particular values are provided in the specification and claims the meaning of “about” should be assumed to be within an acceptable error range for that particular value.

[0078] In the context of the present invention, the words “comprise”, “comprising” and the like are to be construed in their inclusive, as opposed to their exclusive, sense, that is in the sense of “including, but not limited to”.

[0079] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.

[0080] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term ‘about’.

[0081] The recitation of a numerical range using endpoints includes all numbers subsumed within that range (e.g., 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0082] As used herein, the term “fluid sample” refers to a small amount of liquid taken from a larger volume for the purpose of analysis or testing. Examples of fluid samples include biological samples, food constituents, plant extracts, cosmetic preparations, wastewater and industrial fluids.

[0083] As used herein, the term “biological fluid” encompasses intravascular biological fluids, interstitial biological fluids and intracellular biological fluids. Examples of biological fluids include, but are not limited to, blood (including plasma and serum), saliva, semen, vaginal fluids, mucus and urine.

[0084] As used herein, the term “antioxidant activity” is synonymous with terms such as “antioxidant levels, “antioxidant content”, “antioxidant concentration” and the like.PREFERRED EMBODIMENT OF THE INVENTION

[0085] ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is a chemical compound that is used to measure the antioxidant potential of biological fluids. In use, ABTS is activated to the green-blue stable radical cationic chromophore, 2,2-azinobis-(3- ethylbenzothiazoline-6-sulfonate) (ABTS*+) by oxidation. ABTS*+ is blue / green in colour and absorbs light at 734 nm. In the presence of a wide range of antioxidant materials, ABTS*+ is converted back to its colourless neutral form. This system is traditionally calibrated using a water-soluble vitamin E formulation, Trolox, with the results expressed in Trolox equivalents. The ABTS assay has become the laboratory standard for the measurement of total antioxidant activity in a wide range of situations, particularly in diagnostic medicine and the food industry. However, the use of ABTS is hampered by the difficulty in activating it to ABTS*+, which requires long incubations (e.g., 6 to 12h incubation with sodium persulphate), complex chemistry (e.g., oxidation of metmyoglobin with H2O2 to generate the ferrylmyoglobin radical, which is then reacted with ABTS to form ABTS*+) or high temperatures.

[0086] The present invention relates to the finding that ABTS can be rapidly converted to ABTS*+ by electrolysis in a static electrochemical cell with anodic and cathodic chambers separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated. One advantage of the present invention is the activation of ABTS to ABTS*+ only takes place in the anodic compartment which turns dark blue on the application of current and occurs within seconds. In the cathodic compartment, the ABTS remains unoxidized and the solution is colourless. This arrangement allows the cathodic compartment to be used as a control chamber where any differences in absorption due to any interfering properties (turbidity, acellular debris, cellular composition, etc.) of the sample (semen, blood, urine, follicular fluid, cell suspension, etc.) can be controlled for. Using this arrangement a sensitive, accurate and robust measurement of antioxidant activity can been achieved with low amounts of a sample. In contrast to the 16-20 hours required forchemical activation of ABTS, electrochemical activation can be achieved in 20 seconds, making this system ideal for a rapid point-of-care diagnostic device.

[0087] The present invention also relates to the finding that ABTS can be activated by hydrogen peroxide in the presence of horse radish peroxidase or by cumene hydroperoxide in the presence of hematin.

[0088] The present invention further relates to the finding that ABTS can be replaced with leukomethylene blue.

[0089] Although the invention has been described with reference to certain embodiments detailed herein, other embodiments can achieve the same or similar results. Variations and modifications of the invention will be obvious to those skilled in the art and the invention is intended to cover all such modifications and equivalents.

[0090] The present invention is further described by the following non-limiting examples.EXAMPLE 1

[0091] Several assays were developed to measure antioxidant activity in semen.Clinical materials

[0092] The major clinical material used in the development of the antioxidant assays was human semen. Ethical approval from the University of Newcastle human research ethics committee and State Government was secured for the use of human semen samples in this study (H-2013-0319 and 200621). Donor samples were provided to the laboratory following 48 h of sexual abstinence. After allowing at least 30 min for liquefaction to occur, the spermatozoa were separated by 3 cycles of centrifugation and resuspension at 500 x g for 5 min using HEPES-buffered Biggers-Whitten-Whittingham medium (BWW) supplemented with 1 mg / mL polyvinyl alcohol. The spermatozoa were finally resuspended at BWW at a concentration of 10 x 106 / mL.Chemical materials

[0093] All chemicals were purchased from Sigma-Aldrich (St Louis, MO). For the free radical scavenging assay, the antioxidants assessed in this study were dissolved in 0.85% saline (vitamin C, glutathione, N-acetyl cysteine, hypotaurine, carnitine, taurine, spermine, myoinositol and epigallocatechin gallate). Additional compounds were dissolved in DMSO (Trolox, melatonin, lipoic Acid, coenzyme Q10 and resveratrol) or ethanol (Trolox). In general, the stock solutions were made up at a concentration of 5 mM, although 70 mMstocks were used for log dose-response studies. In the pre-activation version of the ABTS assay where the focus was on the inhibition of free radical creation, DMSO could not be used because this compound was extremely active in suppressing ABTS,+formation. As alternatives, some compounds were dissolved in ethanol (melatonin, lipoic acid, resveratrol, Trolox) while co-enzyme Q10 was dissolved in N,N-dimethylformamide (DMF). For the hydrogen peroxide and lipid peroxide scavenging assays, the compounds were solubilized as described for the free radical scavenging assay.Statistics

[0094] All data were analysed using JMP Pro software (Version 17, SAS Institute, Cary, NC) employing simple or multiple linear regression analysis and ANOVA, while comparisons between group means were conducted using the Tukey-Kramer HSD post hoc test. The normality of the data distribution was assessed using the Shapiro-Wilk procedure and, where necessary, data were normalized by square root transformation as indicated.Sperm counts and sperm motility

[0095] Sperm counts in semen were determined using a Nucleocounter (ChemoMetec, Allerod, Denmark) while the quality of sperm movement was assessed with a CASA system, (Hamilton Thorne, IVOS II, Beverly, MA, USA) using the following settings: minimum total count 200; kinematics: progressive STR (%) 80, progressive VAP (pm / s) 25, static VAP (pm / s) 0, static VSL (pm / s) 0.Sperm DNA fragmentation

[0096] DNA Integrity was studied using the Halo assay on spermatozoa that had been resuspended in BWW and snap frozen in liquid nitrogen. This laboratory assay involved setting the spermatozoa in 0.65% agarose on slides which were then immersed in 0.08 M HCI for 7 min, followed by 100 mM DTT in Tris buffer 1 (4.84 g of Tris, 10 mL of 10% SDS, 10 mL of 0.5 M EDTA made up to 100 mL with MilliQ; pH 7.5) for 10 min. The slides were then immersed in Tris buffer 2 (4.84 g Tris, 11.69 g NaCI, 10 mL 10% SDS made up to 100 mL with MilliQ; pH 7.5) for 5 min, followed by immersion in Tris-Boric Acid-EDTA Buffer (TBE: 5.4 g Tris, 2.75 g boric acid, 2 mL of 0.5 M EDTA made up to 100 mL with MilliQ; pH 7.5) for 2 min. The slides were then transferred through increasing strengths of ethanol (70%, 90% and 100% ethanol) allowing 2 min for each step. They were then air dried and stained with 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI, Sigma-Aldrich, NSW Australia) solution for 10 min (1 pL of DAPI per 2 mL of phosphate-buffered saline [PBS: pH 7.4, 137 mM NaCI, 2.7 mM KCI, 8 mM Na2HPO4, and 2 mM KH2PO4] generating a final DAPI concentration of 1.8 pg / mL). The slides were finally rinsed with PBS, 30 pL of Mowiol(Sigma-Aldrich, NSW Australia) added and coverslips applied. For scoring purposes, the cells were classified into five categories: large halo, medium halo, small halo, no halo, or degraded sperm. The percentage of DNA damaged spermatozoa was given by the percentage of cells falling into the small halo, no halo and degraded categories, as recommended (Gallegos et al. 2008, Fertil. Steril. 90:328-334).Lipid aldehyde formation in semen

[0097] The measurement of malondialdehyde levels in unfractionated human semen samples was undertaken using 1-methyl-2-phenylindone (Gerard-Monnier et al. 1998, Chem. Res. Toxicol. 11 :1176-1183). For this assay 15.4 mM 1-methyl-2-phenylindone was made up in acetonitrile: methanol (3:1). Iron (64 mM ferrous sulfate) and ascorbate (1M) were combined 1:1 and 2 pL of this promoter added to 98 pL of unfractionated semen and the mixture diluted with 100 pL acetonitrile: methanol (3:1). Either 650 pL 1-methyl-2- phenylindone or the same volume of acetonitrile: methanol (as a blank) was then added to this mixture. The solution was subsequently acidified with the addition of 150 pL 32% HCI and incubated for 1 h at 45°C. Following incubation, the samples were clarified by centrifugation at 1500 x g for 5 min and finally read at 586nm in a plate reader (SPECTROstar Nano, BMG Labtech, Ortenberg, Germany). The standard used for this assay was 1,1,3,3-tetramethoxypropane (Sigma-Aldrich, NSW Australia).Mitochondrial reactive oxygen species

[0098] Reactive Oxygen Species generation by the mitochondria of isolated spermatozoa was detected by flow cytometry using MitoSOX™ Red dye (MSR; Molecular Probes). This reagent was prepared at a dilution of 1 pL of 5 mM MSR in 249 pL BWW. Sytox® Green dye (SyG, Molecular Probes) was then added at a dilution of 1 pL 12.5 mM SyG in 249 pL BWW to differentiate live and dead cells (required because trace amounts of ethidium in the MSR probe can bind directly to the nuclei of dead cells with compromised plasma membrane integrity). As a positive control, arachidonic acid (AA; Sigma Aldrich, St Louis, USA) was added at a concentration of 50 pM. Once stained with MSR and SyG solutions, the spermatozoa were incubated at 37°C for 15 min before centrifugation and resuspension in BWW. The results were ultimately expressed as the percentage of live MSR positive cells I all live cells (Koppers et al. 2008, J. Clin. Endocrinol. Metab. 93:3199-3207).Electrophoresis device

[0099] The device of the present invention (Figure 1) comprises two chambers each with a capacity of around 2.5 ml separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated, such as a 0.4 pmPETE (hydrophilic polyester track etch) membrane (Sterlitech, WA, USA). On the lateral walls of these chambers, electrodes (MVCF-4S50K carbon electrodes from CAPLINQ, Ml, USA or stainless-steel electrodes) are attached that create an anodic (oxidizing) chamber and a cathodic (reducing) chamber with the passage of current. The adjacent faces of the cassette are made of optical quality material, such as optical quality polystyrene, that allow the undistorted passage of light so that light absorption at a specific wavelength can be measured.

[0100] The device may be a cassette that is insertable into a receiving device that (i) delivers a current across the cassette for a defined period to activate the ABTS probe in the anodic chamber, and (ii) read the subsequent level of absorbance in both the anodic chamber (sample) and cathodic chamber (control). The unit may then display the outcome of the assay in terms of both net absorbance and antioxidant level expressed with reference to an archived standard curve.Free radical scavenging activity assay

[0101] A parent ABTS solution was made up in 50 mM phosphate buffer at pH 4.8 (20.1 mg sodium phosphate dibasic heptahydrate, 1.71 g sodium dihydrogen phosphate monohydrate and 4.5 g sodium chloride in 500 mL MilliQ water). An acid pH was chosen for this assay because it helped stabilize the ABTS*+ radical and generated maximal levels of absorbance. Where indicated, the pH of the phosphate buffer was raised to 7.5 in order to see whether an elevated pH might enhance the antioxidant activity of some compounds by promoting deprotonation of hydroxyl groups and facilitating electron transfer reactions. For the routine assay, 4 mL of buffer containing ABTS (100 pM) was pipetted into the chambers of the electrochemical cell and activated by passing current (45V and 10 mA) for 20 seconds; at the end of this time the fluid in the anodic chamber had turned blue green while the cathodic chamber remained colourless. After 3 min, 1 mL aliquots of the cathodic and anodic ABTS solutions were then pipetted into cuvettes for spectrophotometry (SPECTROstar Nano, BMG Labtech). After an initial reading (TO) at 734 nm, 15 pL aliquots of semen or antioxidant were added to each cuvette, mixed and allowed to stand for 5 min at room temperature. At the end of this period the absorbance was measured again. The corrected absorbance was then calculated as follows:[(TOAnode) - (T5Anode)] + [(T5Cathode) - (TOCathode)]

[0102] Using T rolox as a model antioxidant, a brief 20 sec application of current was found to generate the coloured ABTS,+cation radical within the ensuing 5 min. This radical could be returned to its non-coloured parent state by Trolox in a dose-dependent manner that waslinear over the 5-25 pM dose range (final concentration) and was only modestly impacted by ABTS concentrations extending from 60-140 pM (Figure 2A). The absorption spectrum (Figure 2B) exhibited peaks at 415, 645, 734 and 815 nm, and 734 nm was selected as the optimum wavelength to monitor the assay since it minimises interference due to other absorbing materials and turbidity. The ability of this system to rapidly assess antioxidant activity was subsequently confirmed using the known antioxidants, Trolox and resveratrol which induced the anticipated dose-dependent suppression of absorption at 734 nm as depicted in Figure 2B and C. Using this approach, we then assessed the ability of human semen for its ability to suppress the electrochemically generated ABTS,+radical signal and observed a clear dose-dependent suppression (Figure 2D). The robustness of the assay was indicated by multiple (10 x) measurements of a single standard containing 10 pM Trolox, which generated an intra-assay coefficient of variation of 4.36%. The inter-assay coefficient of variation was determined by analysing aliquots of a single human semen sample on 8 independent occasions; this analysis generated an inter-assay coefficient of variation of 11.2% (Figure 2E). Using this electrochemical system, a Trolox standard curve was created which indicated that the response was linear over the range 0.31 - 20 pM and that the system could readily detect 5 pM Trolox in the final reaction mixture (Figure 2F).

[0103] To determine whether the electrochemical activation of ABTS generated results that are representative of antioxidant activity, we examined the performance of this postactivation assay in the presence of a number of compounds that are reputed to possess such properties. The results of this analysis are presented in Figure 3 in terms of the actual concentration of reagent in the 15 pL inoculated into the assay; the final concentration following dilution with the assay reagents requires division by 67.6 (equivalent to 10 - 25 pM). In addition to Trolox, compounds known to possess free radical scavenging activity such as vitamin C, glutathione, N-acetyl cysteine, bovine serum albumin (BSA) and hypotaurine, all exhibited clear dose-dependent activity in this assay (Figure 3A-E). Melatonin was also active as an antioxidant, but to a much lesser degree (Figure 3F). However, no dosedependent activity could be detected for carnitine, myoinositol, coenzyme Q10, taurine, lipoic acid or spermine (Figure 3G-L). To ensure that this was a qualitative rather than a quantitative difference, log dose responses were conducted for myoinositol, carnitine and spermine (Figure 4A-C) and still no free radical-scavenging activity was detected at doses of up to 67.6 mM (equivalent to a final concentration of 1 mM once the stock had been diluted into the reaction buffer). In addition, this experiment was repeated at a pH of 7.4 to determine whether enhanced deprotonation of the hydroxyl groups in the case of carnitine and myoinositol would facilitate electron transfer. However, even at this pH, none of theseputative antioxidants exhibited significant free radical scavenging activity (Supplemental Figure 1). In complete contrast, the polyphenols, epigallocatechin gallate (EGCG) and resveratrol were such efficient free radical scavengers at pH 4.8 that they saturated the assay at low concentrations (final concentrations of 0.01 mM and 0.025 mM for EGCG and resveratrol respectively) (Figure 4D,E).

[0104] To determine how this rapid antioxidant assay compared with the conventional ABTS assay marketed by the Cayman Chemical Company (Ann Arbor, Ml), we took the Trolox standards from each assay and analysed them using either the electrochemical activation protocols described in this study or the Cayman kit. This analysis indicated that while the results for Trolox standards were superimposable regardless of which assay was used, the electrochemical activation assay was clearly linear over a much greater range of concentrations (Figure 5A,B)

[0105] Despite their apparent equivalence, an interesting difference between the Cayman procedure and the post-addition electrochemical system is the order in which reagents are added. With the latter, the passage of electric current is used to oxidize ABTS to the ABTS+radical in the anodic chamber and then the assay looks at the ability of a given test sample to scavenge the radical species so generated. With the Cayman assay, the test sample is added first and then the ABTS is oxidized using hydrogen peroxide in concert with metmyoglobin. It therefore measures the ability of a test substance to interfere with free radical formation as well as scavenge any radicals subsequently formed. This procedural difference may account for the fact that when the electrochemical assay and Cayman kit were compared with a group of 20 semen samples, the levels of antioxidant activity recorded were highly correlated with each other (R2= 0.68; p < 0.001 Figure 5C) however the values generated by the Cayman assay were significantly higher (p < 0.001) than those secured with the post-activation, electrochemical assay (Figure 5D). To determine whether this difference reflected the ability of the Cayman assay to capture the suppression of radical formation as well as free radical scavenging we revised the electrochemical assay to incorporate a pre-activation strategy, whereby the test sample would be added prior to the application of electric current.Inhibition of free radical formation assay

[0106] A solution was prepared by mixing 5.4 mL of 100 pM ABTS in phosphate buffer with 100 pL of sample and pipetted into the anodic and cathodic chambers of the electrochemical cell (55-fold dilution). When the assay was used for assessing human semen samples, 16.5 pL of the latter were mixed with 183.5 pL of 50 mM phosphate buffer (pH 4.8)to create an 8.25% dilution and 100 pL of this mixture was added to 5.4 mL of 100 pM ABTS as indicated above (667-fold final dilution). When assessing constituents of the antioxidant panel, the dilution factor was 100 pL into 5.4 mL ABTS (55-fold dilution). After exactly 1.5 min, electric current was run through the cell (20 sec at 45V and 10 mA) to achieve activation of the ABTS in anodic chamber, the cathodic chamber serving as a passive control for sample turbidity. Immediately after the passage of current the contents of the anodic chamber were agitated by pipetting up and down 10 x and the cell left for 5 min to allow the activation process to complete. The contents of the anodic and cathodic chambers were then transferred to a spectrophotometer and the absorbance measured at 734 nm (SPECTROstar Nano, BMG Labtech). The corrected absorbance was then calculated as described above. In running this assay, it was found that the ability of T rolox to inhibit ABTS,+radical formation in this assay was less effective and less consistent than vitamin C, so the latter was used to create the corresponding standard curves.

[0107] Both vitamin C and Trolox were tested as standards for this assay and vitamin C found to be both more sensitive and more reliable than Trolox, which was inconsistently active in this system. Vitamin C however, generated a clear dose-dependent suppression of ABTS,+radical formation and an extremely low inter-assay coefficient of variation (0.001%) on 5 independent assays conducted over a period of 3 days (Figure 5E,F). Application of this assay to the same range of antioxidants that had been tested in the post-activation assay described above generated very similar results. Antioxidants such as vitamin C, glutathione, N-acetyl cysteine, hypotaurine, melatonin, BSA, resveratrol and EGCG were highly active in this version of the assay (Figure 6A-H). Interestingly, lipoic acid which had been inactive in the post-activation electrochemical assay, was intensely active in this pre-activation system, suggesting an important role for this compound in the suppression of free radical formation (Figure 6I). Another major surprise with the pre-activation system was the activity displayed by DMSO, which had been inactive in the post-activation model. Concentrations of DMSO in excess of 10% completely saturated this version of the assay, preventing all formation of the ABTS,+radical. A more restricted standard curve revealed that as little as 0.0275% DMSO (0.0005% final concentration) could suppress ABTS,+radical formation in this assay, suggesting that this compound is an extremely effective inhibitor of free radical formation. For this reason, we had to use DMF or ethanol as solvents in this assay because they possessed much more limited antioxidant activity (Figure 6L).Hydrogen peroxide scavenging assay

[0108] This assay examined the ability of test samples to inhibit the oxidation of ABTS to the coloured cation radical induced by the action of hydrogen peroxide in the presence ofHRP. For this reaction, the buffer employed was a 50 mM phosphate buffer at pH 6.5 (1.197 g sodium phosphate dibasic heptahydrate, 1.109 g sodium phosphate monobasic monohydrate made up in 250 mL Milli Q water). Using this buffer, the following reagents were prepared: (i) 1 mg / mL HRP, (ii) 10 mM ABTS, (iii) 300 pM hydrogen peroxide, (iv) diluted test sample comprising 7.5 pL of semen in 500 pL phosphate buffer. The final reaction mixture contained 735 pL buffer, 15 pL ABTS (150 pM), 100 pL antioxidant / semen sample (0.15% semen) and 50 pL HRP (0.05 mg / mL). The reaction was activated by the addition of 100 pL hydrogen peroxide (30 pM) and incubated at room temperature for 10 min to allow formation of the coloured ABTS,+radical cation. The absorbance was finally read at 734 nm in a plate reader (SPECTROstar Nano, BMG Labtech). Controls for sample turbidity were created by omitting ABTS, while a no sample control was created by replacing the addition of diluted semen with distilled water. The level of hydrogen peroxide scavenging activity was calculated as follows:[(No Sample control) - (Sample)] + [(Sample Turbidity) - (No Sample Control Turbidity)]

[0109] Trolox was used to calibrate this assay, and the results were expressed in Trolox equivalents. Standards were created from a 5 mM Trolox stock solution so that the final concentrations ranged from 0 to 20 pM. When assessing constituents of the antioxidant panel, the dilution was 100 pL into 900 pL (10-fold dilution), however when assessing semen samples an additional dilution (667-fold) was required, given the extremely high level of antioxidant activity expressed by this fluid.

[0110] Addition of increasing amounts of hydrogen peroxide generated a proportional, dose-dependent increase in ABTS,+radical formation that could be assessed by measuring absorption at 734 nm. The use of HRP made this assay both rapid (maximal activity within 1 min) and specific for hydrogen peroxide; organic peroxides such as cumene hydroperoxide (a lipid peroxide mimetic) were minimally active in this system (Figure 7A). Using this assay, we then determined the hydrogen peroxide scavenging activity of the antioxidants previously analysed with the pre- and post- activation electrochemical assay. The results of this analysis are presented in Figure 8. Dose-dependent antioxidant activity was again observed with vitamin C, glutathione, N-acetyl cysteine, hypotaurine, melatonin, resveratrol, EGCG and BSA (Figure 8A-H). In contrast, DMSO, lipoic acid, carnitine, coenzyme Q10, taurine, spermine, myoinositol and ethanol were all devoid of significant hydrogen peroxide scavenging activity in this assay (Figure 8).Organic peroxide scavenging assay

[0111] In this assay the HRP was replaced by the broad-spectrum peroxidase activity presented by hematin (Cordoba et al. 2015, Chemical Engineering Science, 129:249-259). Hematin was prepared at 0.5 mg / mL in DMSO. The buffer for this assay was the same 50 mM phosphate buffer (pH 6.5) as employed for the hydrogen peroxide scavenging assay. The constituents of the reaction mixture (final concentrations in brackets) were 675 L buffer, 25 L ABTS (250 pM), 100 pL antioxidant or diluted semen sample (0.15% semen) and 100 pL hematin (0.05 mg / mL). The reaction was activated by the addition of 100 pL, 1 mM cumene hydroperoxide (100 pM) and incubated at room temperature for 20 min before the absorbance was determined at 734 nm using a plate reader as described above. ABTS was replaced with phosphate buffer in order to create a turbidity control while a no sample control was created by replacing the test material with distilled water. The calculation of corrected absorbance was achieved as described above and the results were expressed in Trolox equivalents.

[0112] The assay was used to gauge the ability of test compounds and complex biological fluids to scavenge the mixture of hydroperoxides (ROOH), peroxyl (ROO*) and alkoxyl (RO*) radicals created via the interaction between cumene hydroperoxide and hematin (Kalyanaraman et al. 1983, J. Biol. Chem. 258:3855-3858) (Figure 7B). The response of ABTS to hematin / cumene peroxide-induced oxidation was slower and less sensitive than the HRP / hydrogen peroxide reaction, however it was relatively specific for the organic peroxide, generating little response with hydrogen peroxide (Figure 7B). Using this procedure, the same group of antioxidants were scanned for their ability to scavenge the mixture of organic peroxide and free radical species responsible for ABTS oxidation in the presence of hematin and organic peroxide. In this case, we again saw significant dose-dependent antioxidant activity on the part of vitamin C, glutathione, N-acetyl cysteine, hypotaurine, melatonin, BSA, resveratrol and EGCG (Figure 9A-H). Spermine also exhibited some scavenging activity that plateaued at 0.3 mM Trolox equivalents (Figure 9I). The other compounds tested also exhibited a very low level of scavenging activity that was not dose dependent and plateaued at 0.2 mM Trolox equivalents including carnitine, lipoic acid, coenzyme Q10 (Figure 9J-L).Comparison of antioxidant assays

[0113] A comparison of how the above 4 assays antioxidant activity responded to the presence of human semen revealed high levels of correlation between the various assays as well as significant differences. Examination of the total amount of antioxidant activity recorded in the assays, indicated that the pre-activation electrochemical assay, revealed levels of antioxidant activity that were significantly higher than those recorded with the post-activation assay (p < 0.001 ; Figure 10A), even allowing for the fact that different standards (vitamin C and Trolox) were used in these assays. Since the post-activation assay is entirely focused on the scavenging of a pre-formed radical, this difference must reflect the presence of factors in semen with a powerful capacity to impede free radical formation in the preactivation ABTS assay. The next highest levels of antioxidant activity were recorded with the hydrogen peroxide scavenging assay (p < 0.001), suggesting that seminal plasma also contains factors that scavenge this oxidant (such as catalase, peroxiredoxin 6 or the glutathione peroxidase system) that are not captured by the post-activation free radical scavenging assay. The organic peroxide scavenging assay detected significantly less activity that the hydrogen peroxide scavenging assay (p < 0.001 ; Figure 10A) while the postactivation free radical scavenging assay captured the lowest levels of antioxidant activity of all (p < 0.001; Figure 10A).

[0114] All of the assays were therefore recording different facets of the same fundamental protective mechanism and as such, the activities they measured in semen were highly correlated, even though the absolute levels of antioxidant activity they recorded varied greatly (Figure 10B-E). The highest correlation was observed between the post-activation free radical- and hydrogen peroxide- scavenging activities (p = 0.001) while the lowest was between the organic peroxide- and pre-activation electrochemical assay (NS).

[0115] The assay that would be optimal for any given situation presumably varies with the nature of the ROS involved and the vulnerabilities of the target cell type. Thus, if the MDA level in unfractionated semen is used as a marker of oxidative stress in male fertility then the hydrogen peroxide scavenging assay was found to be the most highly correlated with this parameter (r= 0.41 ; p = 0.02). Alternatively, if sperm count or progressive motility of the isolated cells is selected as a variable reflective of semen quality, then the pre-activation electrochemical assay generated the best correlations with the same group of 27 donors (r = 0.48; p = 0.01 and r= 0.41; p = 0.03, respectively). With mitochondrial ROS generation, it was the organic peroxide assay that predominated (r = 0.4; p = 0.03). In order to capture all of the information contained within the 4 antioxidant assays in making predictions of human semen quality, we next undertook a series of multiple regression analyses using semen quality biomarkers as the dependent variable. Using this approach, predictive algorithms could be written using the antioxidant data, that exhibited highly significant correlations with seminal characteristics related to oxidative stress including sperm count (r= 0.58; p = 0.001 ; Figure 10F), seminal MDA (r= 0.65; p = 0.01 ; Figure 10G ), sperm DNA damage (r= 0.56; p = 0.006; Figure 10H) and mitochondrial ROS generation (r = 0.52; p = 0.004; Figure 101).Summary

[0116] The results obtained show that the assays are simple, robust and highly repeatable, and enable the rapid assessment of antioxidant activity in semen. The assays are sensitive enough to be able to detect variation in antioxidant activity in complex biological fluids and are a valuable means of identifying people requiring antioxidant supplementation. The protocol has very few steps and is capable of serving as a point-of-care assay for oxidative stress.EXAMPLE 2

[0117] Application of the assays described in Example 1 for a range of materials including blood plasma, serum, urine, saliva, semen, seminal plasma, follicular fluid, food extracts and cosmetics was examined.Human Sample Preparation

[0118] All human experiments were conducted following the protocols approved by the University of Newcastle Human Research and Ethics Committee and the State Government (H-2013-0319 and 200621). Semen samples obtained from healthy unselected male donors following 2-3 days of sexual abstinence and delivered to the laboratory within 1 hour of ejaculation. Whenever seminal plasma (SP) was required, the semen was centrifuged for 5 min at 500 x g and the plasma removed, either to be analysed fresh, or after freezing in liquid nitrogen and storage at -80°C. Whenever spermatozoa were required, the semen was processed through Percoll gradients as previously described (Aitken et al., Mol. Cell.Endocrinol. 1996, 117 83-93) and the high-density fraction resuspended in HEPES-buffered Biggers-Whitten-Whittingham medium (BWW) supplemented with 1 mg / mL polyvinyl alcohol at a concentration of 10 x106 / mL. In addition to spermatozoa, saliva samples were collected from randomly selected individuals and analysed fresh without freezing.Equine Sample Preparation

[0119] Institutional and New South Wales State Government ethical approval was secured for the use of equine material in this study (ACEC number A-2011-122). Equine semen was collected from normozoospermic Shetland and miniature crossbred pony stallions of proven fertility using a Missouri artificial vagina (AV; Minitube, Ballarat, VIC, Australia). Semen was centrifuged (5 min at 500 x g), and SP was collected. Blood was collected using vacutainers using either a clotting tube for serum formation or a green-topped heparin tube for blood plasma. Tubes were centrifuged (10 min at 1000 x g) either immediately, or 10-15 min after collection for serum, and the resulting supernatants frozen and stored at -80°C.Bovine Sample Preparation

[0120] Institutional and New South Wales State Government ethical approval was secured for the use of bovine material in this study (ACEC number A-2022-223). The bulls utilized in this study were confined to a crush for semen collection. After a rectal examination to clear the anus of any excess faecal matter, a water-based, non-spermicidal lubricant was applied to a rectal probe (75 mm to 90 mm) and inserted into the anal cavity. Pulses from an electroejaculation unit were rhythmically applied (every 2-3 seconds, with 1-second interval breaks) until ejaculation occurred. A silicon funnel with a 1 5 mL Falcon tube attached was used to collect the ejaculate. The raw ejaculate was centrifuged (400 x g) for 20 min, and the SP collected, snap frozen and stored at -80°C. Cow ovaries were obtained from a local abattoir and transported back to the laboratory in prewarmed 0.9% saline solution (35-38°C). Antral follicles were subsequently aspirated using an 18-gauge needle attached to a 10 mL syringe and the resulting follicular fluid snap frozen in liquid nitrogen and stored at -80°C. In similar fashion, an 18-gauge needle was inserted into the bladder to allow the collection of postmortem urine specimens, which were subsequently snap frozen and stored at -80°C.Canine Sample Preparation

[0121] Canine semen was kindly collected and donated by an accredited breeder registered with Dogs NSW (Kerensa Kennels, Dogs NSW registration number 2100076617). Three healthy male dogs, 4-9 years old, were used for the semen collections. The breeds were an Australian Kelpie and two Border Collie dogs. The included ejaculates had > 80% morphologically normal spermatozoa and a motility of >80%. From each dog, the sperm rich fraction of the ejaculate was collected in a calibrated, plastic Falcon tube by digital manipulation. A portion of each ejaculate was subsequently transferred to Eppendorf tubes and centrifuged at 500 x g for 5 min. The SP was then carefully collected from above the sperm pellet and transferred into clean tubes in 500 pL aliquots before being frozen and stored at -80°C.Impact of cumene hydroperoxide on sperm motility

[0122] In order to determine whether the observed inter-species differences in levels of antioxidant protection provided by SP reflected the vulnerability of the spermatozoa to oxidative stress, time- and dose- dependent studies were conducted on the impact of cumene hydroperoxide on sperm motility. For this study spermatozoa from different species were prepared on discontinuous density gradients and the isolated spermatozoa suspended in medium at a concentration of 10 x 106 / mL in BWW. Spermatozoa were then treated with various doses of cumene hydroperoxide (1 , 0.5, 0.25, 0.125, 0.06 and 0 mM) and after two time points (10 min and 2 h) both total motility and progressive motility were assessed usinga CASA (Computer Aided Sperm Analysis) system (Hamilton Thorne, IVOS II, Beverly, MA, USA) and parameter settings that have been optimized for each species assessed.Food and Cosmetics

[0123] Various fruit juices were purchased from a local supermarket to test for antioxidant activity: cranberry (OceanSpray, Lakeville-Middleboro, MA, USA), cherry (Bickfords, Salisbury South, SA, Australia), grape (Bickfords), pomegranate (Bickfords), superberry (an antioxidant blend of red grapes, pomegranates, cranberries, strawberries and raspberries; Bickfords) and orange (Nudie - nothing but orange juice with pulp, Eastgardens, NSW, Australia). Water-based cosmetic serums were also purchased from a retailer for the assessment of antioxidant activity in skincare products and included: (1) Mecca Cosmetica Replenishing Niacinamide Serum (Mecca, Melbourne, VIC, Australia), (2) Estee Lauder Advanced Night Repair, (Estee Lauder Inc, NY, USA) and (3) Dermalogica Dynamic Retinol Serum (Dermatological Carson, CA, USA).Assays and statistics

[0124] Antioxidant assays and statistical analysis was conducted as described in Example 1. Briefly:

[0125] Free radical scavenging activity was assessed by determining the ability of a given fluid to scavenge the ABTS + cation radical prepared by the electrolytic oxidation of ABTS (100 pM) in phosphate buffer (pH 4.8) within the anodic chamber of an electrochemical cell. For this post-activation assay, 1 mL of activated ABTS + was removed to a 1.5 mL Eppendorf tube and the suppression of absorbance measured at 734 nm after 5 min.

[0126] Inhibition of free radical formation in a preactivation assay in which 100 pL of diluted sample was added to 5.4 mL 100 pM ABTS at pH 4.8. A 2 mL aliquot of the diluted sample was then added to the anodic chamber of the electrochemical cell. Current (20 sec at 45V and 10 mA) was subsequently applied resulting in the appearance of ABTS + at the anode, the cathodic chamber serving as a passive control for sample turbidity. Following the passage of current, the anodic and cathodic chambers were agitated and then left for 5 min to allow the activation process to complete and the suppression of ABTS + formation to be determined.

[0127] Hydrogen peroxide scavenging activity, assessed by determining the ability of a given fluid to suppress formation of the ABTS + radical in a reaction mixture comprising 735 pL buffer, 15 pL ABTS (150 pM), 100 pL diluted sample and 50 pL HRP (0.05 mg / mL). The reaction was activated by the addition of 100 pL hydrogen peroxide (30 pM finalconcentration) and incubated at room temperature for 10 min to allow formation of the coloured ABTS-+ radical cation which was then read at 734 nm.

[0128] Organic peroxide scavenging activity, assessed by determining the ability of a given fluid to suppress the formation of the ABTS + radical using a reaction mixture containing 675 pL phosphate buffer, 25 pL ABTS (250 pM), 100 pL diluted sample and 100 pL hematin (0.05 mg / mL) at pH 6.5. The reaction was activated by the addition of 100 pL, 1 mM cumene hydroperoxide (100 pM final concentration) and incubated at room temperature for 20 min before the absorbance was determined at 734 nm in a spectrophotometer (SPECTROstar Nano, BMG Labtech).

[0129] For all these antioxidant assays vitamin C (ascorbic acid) was used as a positive control and the results are expressed as mM vitamin C equivalents. Three repeat analyses were conducted on 3 independent biological replicates for each of the materials assessed in this study.Impact of spermatozoa and freezing on the antioxidant activity of human semen

[0130] This analysis was initiated by an assessment of human semen samples to determine the levels of antioxidant activity expressed, and the extent to which they would be influenced by the presence or absence of spermatozoa and / or being frozen in liquid nitrogen (Fig. 11 A-D). The results indicated that for three of the antioxidant activities assessed (hydrogen peroxide scavenging, free radical scavenging and the suppression of free radical formation), the performance of human semen was neither impacted by the presence of spermatozoa nor being snap frozen in liquid nitrogen (Fig. 11B-D). However, the organic peroxide scavenging assay revealed a modest decrease (p < 0.01) when semen was frozen and a slight reduction when spermatozoa were removed, that bordered statistical significance (p = 0.055) (Fig. 11A). Together, these results suggest that live spermatozoa make a minor contribution to the scavenging of organic peroxides. However overall, a vast majority of the antioxidant activity was contained within the SP fraction and did not change significantly following freezing (Fig. 11 A). The dominant form of antioxidant activity in semen involved the capacity of this fluid to suppress free radical formation (-30-35 mM vitamin C equivalents), followed by organic peroxide scavenging (-13-15 mM vitamin C equivalents), hydrogen peroxide scavenging (-12-14 mM vitamin C equivalents) and free radical scavenging (0.75-1.35 mM vitamin C equivalents) activities (Fig. 11 A-D).Seminal plasma in different species

[0131] Following on from these conclusions, a comparative analysis was undertaken profiling the antioxidant activity in SP collected from different species. The results of thisanalysis indicated that the ability of human plasma to scavenge organic peroxides was significantly greater than that of any of the other species, while equine and bovine SP possessed significantly (p < 0.001) more activity than their canine counterpart (Fig. 12A). Similarly, in terms of hydrogen peroxide scavenging activity, human SP possessed significantly (p < 0.001) greater activity than any of the other species examined, while equine SP was significantly greater than bull, and both equine and bovine SP was more active than the canine material (p < 0.001) (Fig. 12A). The ability of human SP to suppress free radical formation was also significantly greater than either bovine or equine plasma (p < 0001). However, with this assay canine SP was just as active in the suppression of free radical formation as human SP and significantly greater than that observed with bovine or equine material (p < 0.001) (Fig. 12C). The ability of human seminal plasma to scavenge free radicals was also higher (p < 0.001) than all of the other species examined, while both equine and bovine SP possessed more such activity than the canine equivalent (Fig. 12C). In general, the hierarchy of antioxidant protection observed in this comparative study was human > equine> bovine > canine, although the latter was surprisingly active in the suppression of free radical formation.Impact of oxidative stress on sperm motility in different species

[0132] A possible explanation for the relatively high level of antioxidant protection afforded by human SP, particularly with respect to organic peroxide scavenging (Fig. 12A) would be that human spermatozoa are particularly vulnerable to oxidative stress created by lipid peroxides. In order to test this hypothesis spermatozoa from three different species were exposed to cumene hydroperoxide and the impact on sperm motility assessed using a CASA system. As hypothesised, human spermatozoa were found to be very vulnerable to oxidative attack. Thus, an analysis of sperm movement within 10 min of adding peroxide revealed that both total and progressive motility were significantly (p < 0.001) reduced relative to control levels at doses of cumene hydroperoxide above 0.25 mM (Fig. 13A). In contrast, no significant impact on bovine or equine spermatozoa was recorded under identical conditions (Fig. 13C, E). When the exposure time to cumene hydroperoxide was extended to 2 h, the total and progressive motility of human spermatozoa was significantly suppressed at doses above 0.06 mM (p < 0.001), while at doses greater than 0.25 mM, these cells were completely motionless (Fig. 13B). In the case of bovine spermatozoa however, neither the total nor the progressive motility of these cells was impacted by a 2h exposure to cumene hydroperoxide except at the highest dose examined of 1 mM (p < 0.05; Fig. 13D). Similarly, a 2 h exposure to cumene hydroperoxide did not impact the total motility of equine spermatozoa until doses of 0.5 mM (p < 0.01) and 1 mM (p < 0.001) were reached (Fig.13F), while progressive motility was not compromised until the cumene hydroperoxide doseexceeded 0.25 mM (p < 0.05; Fig. 13F). Taken together these data indicate a hierarchy of vulnerability such that human > equine > bovine, in keeping with the above analyses of SP antioxidant activity.Additional biological fluids

[0133] To determine whether seminal plasma has evolved to provide spermatozoa with particularly high levels of antioxidant protection in relation to other biological fluids, the assay system was used to undertake a comparative analysis of the antioxidant profiles expressed by blood, urine, saliva, ovarian follicular fluid and semen. The results of this analysis revealed interesting differences between fluids in the levels of antioxidant protection afforded, as well as the types of antioxidant activity expressed (Fig. 14A-D). With all four measures of antioxidant activity human semen was significantly more active than any other fluid examined (p < 0.001) apart from urine, which was more active in scavenging free radicals and in suppressing the latter’s formation (Fig 14C, D). Both of these fluids were significantly (p < 0.001) more active than any of the other biological fluids tested, which all exhibited low levels of antioxidant protection, including follicular fluid which evidently does not confer the same protection to oocytes as seminal plasma affords to spermatozoa (Fig. 14A-D). Both blood serum and plasma exhibited low levels of antioxidant protection relative to urine and semen in all assays (p < 0001). However, in terms of the suppression of free radical formation (Fig. 14C), blood was more active than either saliva or follicular fluid and plasma was significantly more effective than serum (p < 0.001). Overall, saliva exhibited lower levels of antioxidant protection than any other biofluid with all assays except organic peroxide scavenging. With this version of the assay, saliva was significantly more active than blood serum and plasma as well as follicular fluid (p < 0.001) (Fig. 14A).Fruit juices

[0134] The fruit juices examined in this study, revealed extremely high levels of antioxidant activity. With all 4 antioxidant assays pomegranate juice exhibited the highest antioxidant content while orange juice contained the lowest (Fig. 15A-D). In terms of organic peroxide scavenging activity, cherry, cranberry and superberry juice were as active as pomegranate and significantly (p < 0.05 - p < 0.001) more active in this respect than both orange and grape juice (Fig. 15A). With respect to hydrogen peroxide scavenging, pomegranate juice was significantly (p < 0.05 - p < 0.001) more active than all of the other fruit juices examined, followed by cherry, cranberry and superberry juices, while orange juice was the least active (p < 0.001) (Fig. 15B). Pomegranate juice was also the most active in suppressing free radical formation (p < 0.001) while orange juice was significantly (p < 0.05 - p < 0.001) less active than any other juices examined (Fig. 15C). Finally, the free radicalscavenging activity of pomegranate juice was significantly greater than all of the other juices examined (p < 0.01 - p < 0.001) while grape and orange juice were the least active in expressing this form of antioxidant action (Fig. 15D).Cosmetics

[0135] To determine whether the assay system could detect differences in the antioxidant properties of commercial cosmetic preparations, 3 separate face sera were examined. The results of this analysis (Fig. 16) demonstrated that the antioxidant profile of different serum formulations varied significantly. Thus, while all three sera were equally active as scavengers of organic peroxides (Fig. 16A), Samples 2 and 3 were significantly (p < 0.01) more active in scavenging hydrogen peroxide than Sample 1 (Fig. 16B). However, in terms of the suppression of free radical formation, Sample 2 was the most active (p < 0.01) (Fig. 16C), whereas Sample 3 was the most effective free radical scavenger (p < 0.05) (Fig. 16D).Summary

[0136] The results of this study demonstrate that the assays are capable of profiling antioxidant activity in a wide range of different materials, highlighting the general applicability of this system for high throughput, point-of-care assessments of antioxidant potential. One of the key features of this system is that it can be configured to measure different types of antioxidant activity.EXAMPLE 3

[0137] Application of the assays described in Examples 1 and 2 using a different oxidisable reporter molecule was examined.Electrochemical assays and vitamin C scavenging

[0138] A 10 mM Leucomethylene Blue (LMB) stock was created in 1ml of dimethylformamide. This stock was then used to establish a solution containing 100 pM LMB in phosphate buffer (pH 4.8) including 0.1% Triton X-100 and 10% ethanol as an aid to solubilization.

[0139] A post-activation electrochemical procedure was used to oxidize LMB to methylene blue (MB) and then test the ability of this compound to respond to a model reductant in the form of vitamin C. In this assay of reductive activity, _1mL of the LMB solution was oxidized to MB in the anodic chamber of the electrochemical cell using a 20 second pulse of electrical current at 45V and 10 mA. Post activation reductive activity was then assessed by adding 15 pl of vitamin C (0,2.5, 5. 10, 15, 20 and 25 pM), and the suppression of absorbance measured at 664nm after 5 min.

[0140] In similar fashion, the electrochemical cell was used in a pre-activation strategy to determine whether an antioxidant such as vitamin C could be used to suppress the formation of MB from LMB if added prior to the application of current. For this assay, 100 pL of vitamin C solution (0, 2.5, 5, 10 and 20 pM) was added to 5.4 mL of solution. 2 mL of this solution was then placed into both the cathodic and anodic chamber of the electrochemical cell and current applied (20 seconds, 45V, 10mA). Following the passage of current, both chambers were mixed thoroughly 10 times and absorbance recorded after 5 min at 664nm. For both the pre- and post-activation assays performed with the electrochemical cell, the cathodic chamber was used to control for any changes in turbidity.ABTS or LMB for measuring hydrogen peroxide scavenging activity

[0141] The following assay was used to determine the relative ability of either LMB or ABTS to detect hydrogen peroxide scavenging activity, only on this occasion the oxidation of these probes was achieved using a combination of horse radish peroxidase and hydrogen peroxide. The final reaction mixture contained 735 pL phosphate buffer (50 mM at pH 6.5), 15 pL ABTS or LMB (150 pM), 100 pL sample and 50 pL HRP (0.05 mg / mL). The reaction was activated by the addition of 100 pL hydrogen peroxide (15 pM - 45 pM) and incubated at room temperature for 10 min to allow formation of the colored ABTS,+radical cation and methylene blue respectively. The comparative ability of these probes to measure cumene hydroperoxide using HRP as the catalyst was also assessed using 15 - 45 pM cumene peroxide and hydrogen peroxide respectively.Measurement of lipid peroxide scavenging activity

[0142] For this assay, LMB was made up as a 10 mM stock solution in dimethyl sulfoxide (DMSO) and subsequently diluted with 50 mM phosphate buffer (pH 6.5) to achieve a final concentration of 250 pM and ABTS was prepared in exactly the same manner. A stock solution of hematin was prepared at 0.5 mg / mL in DMSO and diluted to a final concentration of 0.05 mg / ml with phosphate buffer. Similarly, a dose range of cumene hydroperoxide (25- 200 pM) was prepared in the same phosphate buffer. The constituents of the reaction mixture (final concentrations in brackets) were 675 pL buffer, 25 pL ABTS (250 pM), 100 pL sample and 100 pL hematin (0.05 mg / mL). The reaction was activated by the addition of 100 pL, cumene hydroperoxide (25 - 200 pM) and incubated at room temperature for 20 min before being read in a spectrophotometer (SPECTROstar Nano, BMG Labtech). Initially absorption spectra were secured (400-800 nm) for the two probes and then oxidation of ABTS and LMB was measured at preselected wavelengths of 734 nm and 664 nm respectively over a 30 min period. The comparative ability of this hematin-based assay tomeasure organic - and hydrogen- peroxide scavenging activity was assessed using 50 -200 pM cumene peroxide and hydrogen peroxide respectively.Hematin, ABTS or LMB for measuring lipid peroxide activity

[0143] In the presence of a hematin catalyst, cumene hydroperoxide was able to oxidize both ABTS and LMB. In the case of ABTS, the oxidation product exhibited characteristic peaks at 415, 645, 734 and 815 nm while LMB oxidation to methylene blue (MB) resulted in a major peak at 664 nm and a shoulder at around 614 nm (Fig. 17A,B). Dose response analyses with 20-200 pM cumene hydrogen peroxide revealed the evolution of a dosedependent response over 30 min with either ABTS and LMB as the probe Fig. 17B,C). Across the entire date set, the absorbances generated with ABTS and LMB were highly correlated R2= ; P < 0.001), although slightly higher absorbances were observed with LMB (Fig, 17E).Hematin, ABTS or LMB for measuring hydrogen peroxide activity

[0144] Using hematin as the catalyst LMB generated a dose-dependent signal with both cumene hydroperoxide and hydrogen peroxide (Fig 18 A, B). However, the combination of ABTS and hematin only generated a signal with cumene hydroperoxide; hydrogen peroxide was incapable of oxidizing ABTS when hematin was used as the catalyst. When HRP was used as catalyst, both LMB and ABTS were able to detect hydrogen peroxide in a dosedependent manner (Fig 19 A,C,E,F) however cumene hydroperoxide was unable to oxidize these probes when HRP was used as the catalyst, thereby demonstrating the latter’s specificity for hydrogen peroxide (Fig. 19 B,D). A comparison of the LMB and ABTS assays for hydrogen peroxide demonstrated that both assays generate stable signals after the initial 5 min of incubation but that higher levels of absorbance were achievable with the LMB assay (Fig. 19 E,F).Sensitivity to vitamin C inhibition

[0145] When current was applied for 20 sec to an electrochemical cell containing LMB in both the anodic and cathodic chambers, the oxidation of LMB to MB was observed in the anodic but not the cathodic chamber, which served as a turbidity control. Such electrochemical oxidation could be suppressed by vitamin C if added either after (postactivation assay) or before (pre-activation assay) the application of current (Fig. 20A,B). In the case of the post -activation assay, which is essentially testing the ability of vitamin C to reduce electrochemically generated MB, a rapid suppression was observed with 5 pM vitamin C but thereafter the suppression of MB absorbance was very gradual (Fig. 20A). With the pre-activation assay which examined the ability of vitamin C to inhibit the electrochemicaloxidation of LMB, a similar dose response was observed with vitamin C, with a rapid reduction in oxidation recorded with up to 5 pM vitamin C, but a slower reduction thereafter (Fig. 20B) The other LMB oxidation strategies examined in this study (cumene hydroperoxide / hematin or hydrogen peroxide / HRP) were also suppressible with vitamin C. With cumene hydroperoxide / hematin the inhibition was rapid and linear up to 20 pM vitamin C and was essentially complete at doses above 30 pM (Fig. 20C). Similarly, with hydrogen peroxide / HRP induced LMB oxidation, vitamin C -mediated inhibition was rapid and linear with doses up to 30 pM and complete thereafter (Fig. 20D).Application of LMB to measure the antioxidant activity of solvents DMSO and cyrene

[0146] In order to validate the use of LMB as a probe for measuring antioxidant activity we turned to a comparison between DMSO (dimethyl sulfoxide- a powerful solvent that we have previously shown is a very powerful inhibitor of free radical formation in a pre-activation assay utilizing ABTS) and cyrene. The latter has recently been introduced as a ‘green’ alternative solvent that might substitute for toxic regents such as DMSO or DMF, however the antioxidant potential of cyrene is uncertain. Using LMB as the probe in a pre-activation assay we were able clearly confirm that DMSO is a powerful inhibitor of free radical formation, while cyrene is not. These data resonate with previous studies showing that DMSO can protect bacteria from ROS (reactive oxygen species)- mediated cell death whereas cyrene is ineffective in this regard (Camp et al., RSC Med Chem. 2020, 11, H I- 117). This information will be useful in developing assays to assess the antioxidant potential of poorly soluble reagents, where cyrene can be used to solubilise the material without compromising any subsequent assessment of antioxidant activity.Summary

[0147] The results of this study demonstrate that the measurement of antioxidant activity in the assays can be achieved using oxidisable reporter molecules other than ABTS, such as LMB.

Claims

CLAIMS1. A method of measuring antioxidant activity in a fluid sample, the method comprising the steps of: a) adding a solution comprising an oxidisable reporter molecule to first and second chambers, wherein the chambers are separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated; b) generating a current across the first and second chambers via an anode in the first chamber and a cathode in the second chamber to activate the reporter molecule; c) adding the fluid sample to the solution in the first and second chambers; and d) measuring absorbance of the solution in the first and second chambers independently, wherein high absorbance in the first chamber compared to the second chamber indicates low antioxidant activity in the fluid sample.

2. The method according to claim 1, wherein the antioxidant activity is free radical scavenging activity and / or reductive activity.

3. The method of claim 1 , wherein step c) is performed before step b).

4. The method of claim 3, wherein the antioxidant activity is inhibition of free radical formation.

5. A device for measuring antioxidant activity in a fluid sample, the device comprising first and second chambers separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated, wherein an anode is positioned in the first chamber and a cathode is positioned in the second chamber; the first and second chambers both comprise a solution comprising an oxidisable reporter molecule and an aperture that allows the addition of the fluid sample to the solution; and the walls of the first and second chambers adjacent to the membrane allow the passage of light.

6. A method of measuring antioxidant activity in a fluid sample, the method comprising the steps of:a) generating a current across the first and second chambers of the device of claim 5 via the anode and cathode to activate the reporter molecule; b) adding the fluid sample to the solution in the first and second chambers of the device; and c) measuring absorbance of the solution in the first and second chambers independently, wherein high absorbance in the first chamber compared to the second chamber indicates low antioxidant activity in the fluid sample.

7. The method according to claim 6, wherein the antioxidant activity is free radical scavenging activity and / or reductive activity.

8. The method of claim 6, wherein step b) is performed before step a).

9. The method of claim 8, wherein the antioxidant activity is inhibition of free radical formation.

10. A method of activating an oxidisable reporter molecule, the method comprising: adding a solution comprising the oxidisable reporter molecule to first and second chambers separated by a membrane that permits the flow of electrons but confines reaction products to the chamber in which they were generated; and generating a current across the first and second chambers via an anode in the first chamber and a cathode in the second chamber to activate the reporter molecule in the first chamber11. A method of measuring antioxidant activity in a fluid sample, the method comprising the method of any one of claims 1 to 4 or 6 to 9, and further comprising the steps of: a1) adding a solution comprising an oxidisable reporter molecule and horseradish peroxidase to third and fourth chambers; b1) adding the fluid sample to the solution in the third chamber and an equal volume of water to the solution in the fourth chamber; c1) adding hydrogen peroxide to the solution in the third and fourth chambers to activate the reporter molecule; and d1) measuring absorbance of the solution in the third and fourth chambers independently,wherein high absorbance in the third chamber compared to the fourth chamber indicates low hydrogen peroxide scavenging in the fluid sample.

12. A method of measuring antioxidant activity in a fluid sample, the method comprising the method of any one of claims 1 to 4 or 6 to 9, and further comprising the steps of: a1) adding a solution comprising an oxidisable reporter molecule and hematin to third and fourth chambers; b1) adding the fluid sample to the solution in the third chamber and an equal volume of water to the solution in the fourth chamber; c1) adding cumene hydroperoxide to the solution in the third and fourth chambers to activate the reporter molecule; and d1) measuring absorbance of the solution in the third and fourth chambers independently, wherein high absorbance in the third chamber compared to the fourth chamber indicates low organic peroxide scavenging in the fluid sample.

13. The method of claim 11 or 12, wherein absorbance is measured in the first and second chambers prior to step c1) to measure turbidity in the fluid sample14. The method according to any one of claims 1 to 4 or 6 to 13, or the device according to claim 5, wherein the reporter molecule is 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) or leukomethylene blue (LMB).

15. The method according to any one of claims 1 to 4, 6 to 9 or 11 to 13, wherein the reporter molecule is ABTS and the absorbance is measured at about 734 nm.

16. The method according to any one of claims 1 to 4, 6 to 9 or 11 to 13, wherein the reporter molecule is LMB and the absorbance is measured at about 664 nm.

17. The method according to any one of claims 1 to 4, 6 to 9 or 11 to 13, or the device according to claim 5 or 14, wherein the fluid sample is a biological fluid.

18. The method or device according to claim 17, wherein the biological fluid is selected from the group consisting of semen, blood, urine, saliva and ovarian follicular fluid.

19. The method according to any one of claims 1 to 4 or 6 to 18, or the device according to any one of claims 5, 14, 17 or 18, wherein the membrane is a polyester track etch (PETE) membrane.

20. The device according to any one of claims 5, 14 or 17 to 19, wherein the device is a cartridge that is insertable into a receiving device comprising means for generating a current across the chambers via the anode and cathode.

Images