METHOD FOR SELECTIVELY MEASURING A CONCENTRATION OF A COMPOUND FOR ANALYSIS OR ENZYME ACTIVITY IN A COMPLEX SAMPLE BY MEANS OF HYDROGEN PEROXIDE QUANTIFICATION

Abstract

A method for measuring the concentration of a compound for analysis in an original sample includes placing a first compound in a first electrochemical transducer, measuring a first concentration by applying a potential in the electrochemical transducer, mixing the first compound with a second compound, placing the modified sample in a second electrochemical transducer, and measuring a second concentration by applying a potential in the electrochemical transducer. The first or the second compound is a part of the original sample. Lastly, an operation is performed between the first and the second concentration to obtain the concentration of the compound for analysis in the original sample.

Claims

1. A method for measuring a concentration of a hydrogen peroxide in an original sample, the method comprising the steps of placing the original sample in a first fungible strip containing a working electrode, a pseudo-reference electrode, and an auxiliary electrode made of carbon modified with Co-Phthalocyanine; measuring a first concentration by applying a potential in the electrochemical transducer; mixing the original sample with an enzyme, obtaining a modified sample, where the modified sample has a concentration of hydrogen peroxide different from that of the original sample due to the action between the original sample and the enzyme; placing the modified sample in a second fungible strip containing a working electrode, a pseudo-reference electrode, and an auxiliary electrode made of carbon modified with Co-Phthalocyanine; measuring a second concentration by applying a potential in the electrochemical transducer; performing a subtraction operation between the first and the second concentration to obtain the concentration of hydrogen peroxide in the original sample.

2. The method according to claim 1, wherein the step of measuring the first or the second concentration comprises applying a voltage to the working electrode by a potentiostat according to an amperometry method, said voltage being comprised within the range between 0.35 and 0.45 V; obtaining a mean of several current intensity measurements obtained in a given time range.

3. The method according to claim 2, wherein the time range is comprised in the range between 20 and 40 seconds, specifically between 25 and 30 seconds.

4. The method according to claim 1, wherein the enzyme is catalase.

5. The method according to claim 1, wherein the second transducer is the first transducer after having been cleaned.

6. The method according to claim 1, wherein, after mixing the first compound with the second compound, the method comprises the additional step of leaving the modified sample to incubate for at least 5 seconds.

7. The method according to claim 1, wherein the original sample has a volume comprised between 25 and 55 ?l.

8. A measurement apparatus comprising a first electrochemical transducer containing a working electrode, a reference or pseudo-reference electrode, and an auxiliary electrode and controller for measuring according to the steps of the method of claim 1.

9-13. (canceled)

Description

DESCRIPTION OF THE FIGURES

[0051] A brief description of each of the figures used to complete the following description of the invention is provided below. Said figures relate to the state of the art or to preferred embodiments of the invention, which are presented as non-limiting examples thereof.

[0052] FIG. 1 shows the steps of a first method according to the invention.

[0053] FIG. 2 shows the graphic evolution of current in nanoamperes with respect to time in measurements performed in a method according to the invention.

[0054] FIG. 3 shows the relationship between the differential current and the concentration of hydrogen peroxide in a method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

[0055] An example of preferred embodiment of the present invention, provided for the purpose of illustrating but not limiting the invention, is described below.

[0056] FIG. 1 shows the steps of a first method according to the invention.

[0057] In this first method, an original food sample 1 in respect of which the concentration of hydrogen peroxide is to be obtained is used as the starting material.

[0058] To that end, 50 ?l of the original sample 1 are placed in a fungible strip 2 having three electrodes: a working electrode, a pseudo-reference electrode, and an auxiliary electrode made of carbon modified with Co-Phthalocyanine.

[0059] Once the sample is in place, the fungible strip 2 is introduced in a portable measurement device 3 where a voltage of 0.4 V is applied to the working electrode by means of a potentiometer 4 according to an amperometry method. In this process, the mean of the current obtained is obtained between 25 and 30 seconds.

[0060] Next, another 50 ?l of the original sample 1 are taken and a catalase solution is added thereto. The mixture 5 is left to incubate for one minute and placed on a second fungible strip 2 identical to the first strip, is introduced in the portable measurement device 3 again, and an amperometry is performed again under the same terms as above, obtaining a second value that is the mean of the current obtained between 25 and 30 seconds.

[0061] The difference in nA between the mean current obtained in the first measurement and the mean current obtained in the second measurement is directly proportional to the concentration of hydrogen peroxide in the original sample, because the difference between the first and the second sample is the action of the catalase, which has eliminated the concentration of hydrogen peroxide from the sample.

[0062] Therefore, by performing a second amperometry under the same terms as the first one, the difference will be due only to the difference in the concentration of hydrogen peroxide between both samples, which is precisely what is to be measured. Taking into account that the concentration is zero in the second sample, since it has been eliminated by the catalase, the difference in concentration will provide the concentration of hydrogen peroxide in the unmodified original sample.

[0063] As will be evident to one skilled in the art, this method can be reproduced using another different enzyme for measuring the concentration of another different compound, provided that the enzyme is suitable for selectively and effectively breaking down said compound the concentration of which is to be measured.

[0064] FIG. 2 shows the graphic evolution of current in nanoamperes with respect to time in each of the two measurements.

[0065] At the top line of the graph, represented in a dark line, the evolution of current with respect to time in the first measurement performed on the original sample with the concentration of hydrogen peroxide to be measured is represented.

[0066] In this evolution, several measurements were taken between 25 and 30 seconds, obtaining a mean current value of 0.2736 mA.

[0067] This value alone does not allow the concentration of hydrogen peroxide to be calculated, since the form of the current may be due to the many and very different compounds.

[0068] The bottom line of the graph, represented in a lighter coloured line, shows the evolution of current over time in the second measurement performed on the mixture of the original sample and catalase, once it has been left to stand.

[0069] In this evolution, several measurements were taken between 25 and 30 seconds, obtaining a mean current value of 0.1600 mA.

[0070] The difference between both values means gives a differential current of 0.1136 mA.

[0071] FIG. 3 shows the relationship between this differential current and the concentration of the eliminated hydrogen peroxide. As can be observed, the relationship is virtually linear, so the differential current, obtained as the difference between the mean of the measurements before applying the catalase and the mean of the measurements after applying the catalase, allows the concentration of hydrogen peroxide in the original sample to be deduced in a simple manner.

[0072] There is a variant of this method. For example, if what is to be measured in a sample is the concentration of glucose, the method particularly designed for hydrogen peroxide simply with a prior step of transforming all the glucose into hydrogen peroxide can be used.

[0073] Therefore, once that prior step has been performed, it provides an altered original sample having a concentration of hydrogen peroxide that is equivalent to the concentration of glucose of the original sample. By applying the method described above to the altered original sample, the concentration of hydrogen peroxide of the altered original sample will be obtained and used to readily deduce concentration of glucose of the original sample.