METHOD FOR DETERMINING THE ANTIOXIDANT CAPACITY OF A BIOLOGICAL SAMPLE AND RELATED KIT

Abstract

A method is provided for determining antioxidant power of a sample of a biological fluid or a food. The method essentially consists in contacting the sample to be tested with an aqueous solution of platinum nanoparticles, an oxidizing agent, and a chromogenic peroxidase substrate, and detecting color of the final solution thus obtained. Color intensity of the solution is proportional to the antioxidant power of the sample. A kit suitable for carrying out the method is also provided.

Claims

1. A method for determining antioxidant power of a sample of a biological fluid or a food, comprising the steps of: contacting the sample with an aqueous solution of metal nanoparticles, wherein said metal nanoparticles comprise platinum optionally in combination with gold, palladium and/or silver, an oxidizing agent, and a chromogenic peroxidase substrate, and detecting color intensity of a final solution thereby obtained, the color intensity being proportional to the antioxidant power of the biological sample.

2. The method of claim 1, wherein the chromogenic peroxidase substrate is 3,3,5,5-tetramethylbenzidine (TMB).

3. The method of claim 1, wherein the oxidizing agent is hydrogen peroxide.

4. The method of claim 1, wherein said metal nanoparticles have a diameter varying within the range of from 0.1 nm to 1000 nm.

5. The method of claim 1, wherein the final solution is prepared in a buffer solution having a pH comprised between 1 and 7.

6. The method of claim 1, wherein the color intensity of the final solution is detected with the naked eye.

7. The method of claim 1, wherein the color intensity of the final solution is detected by UV-visible spectroscopy.

8. The method of claim 7, wherein the color intensity of the final solution is detected by measuring absorbance at a wavelength between about 600 and 700 nm.

9. The method of claim 1, wherein the biological fluid comprises saliva, blood, sweat and urine.

10. The method of claim 1, wherein the food is a fruit juice or an oil.

11. A kit for determining antioxidant power of a sample of a biological fluid or a food, comprising a chromogenic peroxidase substrate and an aqueous solution of metal nanoparticles, wherein said metal nanoparticles comprise platinum optionally in combination with gold, palladium and/or silver.

12. The kit of claim 11 comprising: a predetermined amount of an aqueous solution of metal nanoparticles, wherein said metal nanoparticles comprise platinum optionally in combination with gold, palladium and/or silver, at a concentration within the range of from 0.01 ppm to 1000 ppm; a predetermined amount of a 3,3,5,5-tetramethylbenzidine (TMB) solution at a concentration within the range of from 0.001 M to 1 M; and optionally a predetermined amount of hydrogen peroxide at a concentration comprised between 0.1 M and 10 M; and/or a predetermined amount of an acetate buffer solution at a concentration comprised between 0.01 M and 1 M, having a pH value comprised between 1 and 7.

13. An in vitro diagnostic method for assessing oxidative stress in a subject, comprising determining antioxidant power of a biological fluid sample from the subject by the method of claim 1, wherein a decreased antioxidant power of the biological fluid sample from the subject compared to a reference sample or value is indicative of the oxidative stress of the subject.

14. The in vitro diagnostic method of claim 13, wherein the subject is suspected of conducting or conducts a health-damaging lifestyle, or the subject is suffering or is suspected to be suffering from a disease selected from the group comprising kidney damage, gout, endometriosis, diabetes and cancer.

15. The in vitro diagnostic method of claim 14, wherein the health-damaging lifestyle is alcohol abuse.

16. The in vitro diagnostic method of claim 14, wherein the health-damaging lifestyle is unhealthy diet.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0047] FIG. 1 shows results expressed as absorbance values measured at 652 nm obtained by performing the method of the invention on saliva samples from healthy volunteers;

[0048] FIG. 2 shows detection by the naked eye, using a reference color scale;

[0049] FIG. 3 shows results expressed as absorbance values measured at 652 nm obtained by performing the method of the invention on food samples.

DETAILED DESCRIPTION

[0050] In the method of the present invention, the platinum nanoparticles perform a catalytic function. As an alternative to nanoparticles made entirely of platinum, platinum-based nanoparticles can be used in combination with another metal such as gold, palladium and/or silver.

[0051] The substrate of the oxidation reaction is preferably the chromogen TMB (3,3,5,5-tetramethylbenzidine). Hydrogen peroxide is typically used as the oxidizing agent.

[0052] The method of the invention is based on the fact that the antioxidant substances in the sample under examination interact with hydrogen peroxide, causing a partial or total inhibition of the TMB oxidation reaction. The result is a less intense development of the blue color, which, as is known, is formed by oxidation of the TMB chromogenic substrate. The observable decreased oxidation of TMB by hydrogen peroxide is directly proportional to the amount of antioxidant substances and can therefore be used to colorimetrically quantify the concentration of antioxidants in the sample under examination. This reaction scheme is particularly effective and specific since it allows the total antioxidant capacity of a sample to be measured without the need of any preliminary purification step.

[0053] The assay method of the present invention can be applied to several biological fluids such as saliva, blood, sweat, or urine. It can also be applied to liquid food such as fruit juices and edible oils. In the case of oils, it is preferable that the sample is first mixed with a solution of methanol and isopropanol, to which the above listed reagents are subsequently added, without however requiring separation or purification.

[0054] The examples that follow are provided for illustration purposes only and do not limit the scope of the invention as described and claimed herein.

EXAMPLES

[0055] The following assay is performed:

[0056] 400 microlitres of an acetate buffer solution, typically between 0.01 and 1 M, preferably between 0.05 and 0.3 M were added to a test tube; the pH can vary between 1 and 7, preferably between 3 and 5.5;

[0057] 200 microlitres of a TMB 3,3,5,5-tetramethylbenzidine) solution were added at a concentration of between 0 and 1 M, preferably between 0.002 and 0.05 M;

[0058] 100 microlitres of a solution containing platinum nanoparticles were added at a concentration comprised between 0.01 and 1000 ppm platinum, preferably between 0.1 and 10 ppm; the diameter of the nanoparticles may vary between 0.1 nm and 1000 nm, preferably between 1 and 100 nm;

[0059] 100 microlitres of the sample to be tested, previously diluted in an aqueous solution by a factor comprised between 1:2 and 1:500, preferably between 1:2 and 1:100, were added; if the sample is in the form of an oil it is first mixed with a solution of methanol and isopropanol;

[0060] the color development reaction (blue) was initiated by the addition of 200 microliters of a 1 M hydrogen peroxide solution.

[0061] FIG. 1 shows the results obtained by performing the test on 100 saliva samples obtained from healthy volunteers and carrying out the detection by UV-Vis spectroscopy (measurement of the absorbance at 652 nm). Absorbance values lower than 0.1 correspond to saliva samples obtained following the intake of food supplements (Vitamin C).

[0062] As an alternative to the spectroscopic method, it is possible to carry out the detection by the naked eye, using a reference color scale (which shows different color intensity bands and the related antioxidant score (e.g., excellent, medium-high, standard, medium-low, low, cf. FIG. 2).

[0063] As indicated above, the method of the invention can also be used to analyze food samples such as fruit juices. FIG. 3 shows the results obtained by testing food samples of fruit juice and other industrial drinks and performing the detection by UV-Vis spectroscopy (measurement of the absorbance at 652 nm).