ES2638737B1 - Electrode device and measurement procedure for the detection of lactic acid - Google Patents

Abstract

Electrode device for the detection of simple and portable lactic acid comprising a quadrangular plastic support with a first face and a second face, the first face comprises three screen-printed electrodes, in the active area of the working electrode a platinum layer is deposited and a solution containing the enzyme lactate oxidase, which oxidizes originates pyruvate and hydrogen peroxide, and a potentiostat is fixed on the second side. Measurement procedure with the aforementioned device comprising the following steps: sweat deposition containing lactic acid in concentration between 4 mM and 40 mM in the active area of the working electrode; application by the potentiostat of a potential between 0.4 V and 0.9 V between the working electrode and the reference electrode; reading of the current generated between 2 mA and 20 mA by the potentiostat.

Description

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ELECTRICAL DEVICE AND MEASUREMENT PROCEDURE FOR DETECTION

OF LACTIC ACID

DESCRIPTION

OBJECT OF THE INVENTION

The present invention is framed in the field of electrochemistry applied to chemical analysis. Specifically, it refers to the detection of lactic acid by means of an electrode device with screen-printed electrodes.

BACKGROUND OF THE INVENTION

Electrochemical cells are of great interest in the analysis of substances due to their robustness, simple and economical manufacturing.

One of the most used techniques for the manufacture of electrodes used in electrochemical cells is screen printing. This technique allows to build chemical sensors with reproducibility and minimal infrastructure.

By means of the technology of screen-printed electrodes it is possible to miniaturize the sensors offering the advantage of being versatile, they can be manufactured with different electrode configurations and with different inks, and have a low cost and can thus be used for mass production of disposable electrodes. In addition, they can be used for in situ analysis.

However, these advantages of screen-printed electrodes are penalized by the need to connect at least one potentiostat that applies the difference in electrical potential between the electrodes to be measured, in addition to other electrical and electronic devices such as a power supply, a interface, etc.

Currently the standard potentiostats are external devices to the electrodes, of a certain size and weight that make them difficult to portable desktops.

L-lactic acid (AL) plays an important role in sports medicine (Palleschi, G., Mascini, M., & Bomardi, L., 1990. "Lactate and glucose electrochemical biosensor for the

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evaluation of the aerobic and anaerobic threshold in runners. ”Medical & Biological Engineering & Computing, 28, B25-B28.), where its determination is useful for monitoring respiratory failure, crashes, heart failure and metabolic disorders (Gomes, SP, Odlozilikova, M., Almeida, MG, Araujo, AN, Couto, CM, Montenegro, MC, 2007. “Application of lactate amperometric sol-gel biosensor to sequential injection determination of L-lactate.” Journal of Pharmaceutical and Biomedical Analysis, 43 , 1376-1381.). Any change or increase in blood lactate is an indication of survivability (Cowan, BN, Burns, HJ, Boyle, P., Ledingham, IM, 1984. “The relative prognostic value of lactate and haemodynamic measurements in early shock. Anaesthesia ”, 39, 750-755.). In addition, the importance of using the existing AL in sweat as a source of energy production to recharge small batteries such as wristwatch or mobile phones has recently been published. Also in the food industry the level of lactate is an indicator of the fermentation process and is related to the stability, quality and freshness of storage in products such as dairy products, wines, tomato sauces, fruits and juices, where they can be a marker for the process of fermentation by lactic acid bacteria (Gomes, SP, Odlozilikova, M., Almeida, MG, Araujo, AN, Couto, CM, Montenegro, MC, 2007. “Application of lactate amperometric sol-gel biosensor to sequential injection determination of L-lactate. ”Journal of Pharmaceutical and Biomedical Analysis, 43, 13761381.) (Monosík, R., Stredansky, M., Greif, G., Strudík, E., 2012.“ A rapid method for determination of L-lactic acid in real samples by amperometric biosensor utilizing nanocomposite. ”Food Control, 23, 238-244.)

Patents ES2484665, ES2524991 and ES2370733, respectively, relating to a biosensor for the detection of gluconic acid, sulphites and chromium are known. All these patents are specific to the substances mentioned, none is suitable for the detection of lactic acid, in addition none of them include the potentiostat in the electrode device, but is external to it.

DESCRIPTION OF THE INVENTION

The present invention is established and characterized in the independent claims, while the dependent claims describe other features thereof.

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The object of the invention is an electrode device for the detection of lactic acid that is simple and portable in the sense of including all the electrical and electronic elements necessary to carry out the measurement without connecting to external equipment. The problem to solve is to configure the device in its components and in the way of acting among them to reach the mentioned object.

A microcontroller with load architecture refers to the also named Computer with Reduced Instruction Set ("RISC" for "Reduced Instruction Set Computer" according to its English name) which generally has the following characteristics: fixed size instructions and presented in a reduced number of formats; Only the loading and storage instructions access the data memory. This results in high memory access speed for temporarily saved data.

An advantage related to claim 1 is that the device is autonomous in that it can measure itself without the need for an external device, therefore portable; also disposable in the part of the electrodes without replacing them with others when necessary. Platinum allows better electrochemical signals and improves sensitivity for the detection of lactic acid

Another advantage related to claim 2 is that chitosan also helps to obtain better electrochemical signals.

Another advantage related to claim 3 is that the enzyme is fixed in the working electrode stably.

Another advantage related to claim 4 is that it avoids unwanted electrical contacts with parts that do not require direct contact with other parts or elements, thereby avoiding measurement errors.

Another advantage related to claim 5 is that the shape is suitable for handling and handling as well as for easy transport within a wallet, case or the like.

Another advantage related to claim 6 is that it coincides with the circumferential perimeter of a drop of sample to be analyzed.

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Another advantage related to claim 7 is that the size of the active areas is optimized for the available space.

Another advantage related to claims 8 to 11 is that it is simple as it is available in the usual market, as well as that it contributes to its miniaturization due to the structure of said components.

Another advantage related to claim 12 is that the measurement is protected against electromagnetic interference and noise, thereby avoiding possible errors, as well as that the integrated circuit board and the components disposed thereon are physically protected against external agents.

Another advantage related to claim 13 is that the measurement is suitable for measuring in sweat, with a small amount thereof, a drop normally, performed quickly and at low voltage, which implies low consumption.

Another advantage related to claim 14 is that the measurement obtained is transformed into a digital signal and transmitted to the user, which can use a computer equipment of the known for its handling at its convenience.

DESCRIPTION OF THE FIGURES

The present specification is complemented by a set of figures, illustrative of the preferred example and never limiting of the invention.

Figure 1 represents a perspective view of the electrode device.

Figure 2 represents a diagram of the components of the electrode device. The arrows indicate the direction of flow of the electrical signals between the components.

Figure 3 represents the integrated circuit board and the components arranged therein.

DETAILED EXHIBITION OF THE INVENTION

An embodiment of the invention is shown below with support in the figures.

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Figure 1 shows the electrode device for the detection of lactic acid comprising a plastic support (1), normally a rectangular, quadrangular PVC sheet, preferably rectangular, with a first face (1.1) and a second face (1.2), the first face comprises three electrodes (1.11,1.12,1.13) screen-printed, each electrode consists of a contact or terminal (1.14,1.15,1.16) continued by a section (1.17,1.18,1.19) to an active area (1.20,1.21, 1.22), being a reference electrode (1.11), a working electrode (1.12), and another auxiliary or counter electrode (1.13).

Normally the support (1) does not exceed an area of 16 cm2, dimensions of 3cm x 2 cm are advantageous.

In the active area (1.21) of the working electrode (1.12) a platinum layer and a solution containing the enzyme lactate oxidase are deposited, which oxidizes originates pyruvate and hydrogen peroxide. A potentiostat (2) is attached to the second side (1.2) of the support (1.1), see figure 3, which will allow the measurement with the electrodes (1.11,1.12,1.13).

Specifically, on the platinum layer, which favors the catalysis of hydrogen peroxide, a chitosan layer can be arranged.

Thus, the solution containing the enzyme lactate oxidase is deposited on the chitosan layer, which optionally comprises the following reagents: glutaraldehyde (GA), bovine serum albumin (BSA) and a copper-centered metal network - organic framework ”according to English nomenclature) (Cu-MOF) These reagents guarantee the stability of the enzyme and its correct immobilization in the active area (1.21) of the working electrode (1.12), specifically, the GA as a bi-functional agent It allows the entrapment of the enzyme, the BSA protects it from excessive ligation to the GA, the Cu-MOF provides greater stability to the enzyme, improving its activity and sensitivity.

Optionally, the electrode device includes an insulator (3) that covers the device, leaving free the contacts (1.14,1.15,1.16) and active areas (1.20,1.21,1.22) of the electrodes (1.11,1.12,1.13), of this In this way, unwanted electrical contacts are avoided in the parts that do not intervene in the measure.

Preferably, the contacts (1.14,1.15,1.16), the sections (1.17,1.18,1.19) and the active area (1.20) of the reference electrode (1.11) are rectangular in shape, thus achieving an elongated configuration that adapts well to the support (1). It is also advantageous that the active area (1.21) of the working electrode (1.12) is a circle, since the

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Sample to analyze is usually a drop whose contour when deposited on a surface is circular. It is also advantageous that the active area (1.22) of the counter electrode (1.13) consists of a straight section (1.23) that connects with an arc section (1.24), as this achieves a large area in the available space.

For its part, as shown in Figure 3, the potentiostat (2) is arranged on an integrated circuit board (4) ("PCB"), on which a power supply is also provided ( 5) connected to it to provide electric power as well as to a digital analog converter (6), a microcontroller (7) and a communication interface (8), the three electrodes (1.11,1.12,1.13) being electrically connected to the potentiostat ( 2), this to the digital analog converter (6), this to the microcontroller (7) and this to the communication interface (8), as shown in the scheme in Figure 2.

The potentiostat (2) thus arranged fulfills the following functions:

-provides enough current to circulate between the working electrode (1.12) and the reference electrode (1.11);

- stabilizes the reference voltage, between 0.4 V and 0.9 V, preferably 0.6 V, between said electrodes (1.11,1.12) avoiding the polarization of the reference electrode (1.11) so that no current flows through it and Therefore, the voltage applied to it is constant so that the current measurements are reliable;

-provides the rest of the components with a real-time measurement for constant monitoring of the reference voltage, so that its correct operation is verified; -provides a high impedance isolation of the potentiostat (2) with the parts that connect it to the other components, specifically, the reference voltage output to be measured and the bias voltage that must be provided to the working electrode (1.12 ).

Specifically, the potentiostat (2) may comprise:

- a first operational amplifier that provides voltage between 0.4 V and 0.9 V, with a gain of 100 dB in open loop, a bandwidth of 7 MHz, a non-linear effect of 5 V / ^ s, a rejection to the common mode of 90 dB current;

- a second operational amplifier that in turn combines two amplifiers, third and fourth, in a surface-mounted encapsulation that isolates the impedance of voltage input to the working electrode (1.12) and the reference electrode (1.11) and output electrode their current, with a bandwidth of 1 MHz, a non-linear effect of 0.6 V / ^ s, an input bias current of 1 pA at 25 ° C and an input impedance of 100 MOhm.

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Optionally, the potentiostat (2) is also connected to a third-order Butterworth filter (9) with Sallen-Key structure and unit gain.

Advantageously, it is verified that one option is that the microcontroller (7) has 8-bit storage load architecture.

The main functions of the microcontroller (7) are the following:

-load the preprogrammed parameters or program the parameters for the potentiostat (2) and for the digital analog converter (6) for calculating the signal obtained from the working electrode (1.12) and the reference electrode (1.11);

- monitors in real time the voltage of the reference electrode (1.11) to check the correct operation of the potentiostat (2);

-control the on and off of the potentiostat (2);

- acquires from the digital analog converter (6) the values of the signal of the working electrode (1.12) and of the reference electrode (1.11);

-applies programmed algorithms for the conversion and interpretation of the values obtained from the signal and supply voltage of the working electrode (1.12) and the reference electrode (1.11);

-manage communications with the communication interface (8).

Optionally, the integrated circuit board (4) and all the components that are arranged on it (2,5,6,7,8,9) are arranged inside an aluminum enclosure (10) for protection against electromagnetic interference and noises

The measurement procedure for the detection of lactic acid with an electrode device as described comprises the following steps:

- deposition of sweat containing lactic acid in concentration between 3 mM and 50 mM in the active area (1.21) of the working electrode (1.12);

- application by the potentiostat (2) of a potential between 0.4 V and 0.9 V between the working electrode (1.12) and the reference electrode (1.11);

- reading of the current generated between 2 mA and 20 mA by the potentiostat (2).

After reading the generated current, the following stages take place:

- transmission of said reading to the digital analog converter (6) to convert the analog signal into digital;

- transmission of the digital signal to the microcontroller (7) for recording, analysis and monitoring;

- transmission of signal data related to its registration, analysis and monitoring to the communication interface (8) for interaction with a user, for example to a computer

5 staff where you can view, save and manage at your convenience.

Claims (13)

5 10 fifteen twenty 25 30 35 1. -Electronic device for the detection of lactic acid comprising a quadrangular plastic support (1) with a first face (1.1) and a second face (1.2), the first face comprises three electrodes (1.11,1.12,1.13) screen-printed, each electrode consists of a contact or terminal ( 1.14,1.15,1.16) continued along a section (1.17,1.18,1.19) to an active area (1.20,1.21,1.22), being a reference electrode (1.11), a working electrode (1.12), and another auxiliary or counter electrode (1.13), characterized in that in the active area (1.21) of the working electrode (1.12) a layer of platinum and a solution containing the enzyme lactate oxidase is deposited, which oxidizes originates pyruvate and hydrogen peroxide, and the second face (1.2) a potentiostat (2) is fixed. 2. Electrode device according to claim 1 wherein a chitosan layer is arranged on the platinum layer. 3. -Electronic device according to claim 3 wherein the solution containing the enzyme lactate oxidase comprises the following reagents: glutaraldehyde, bovine serum albumin and a copper metal network. 4. -Electronic device according to any of the preceding claims that includes an insulator (3) covering the sections (1.17,1.18,1.19), leaving the contacts free (1.14,1.15,1.16) and active areas (1.20,1.21,1.22 ) of the electrodes (1.11,1.12,1.13). 5. -Electronic device according to any of the preceding claims wherein the contacts (1.14,1.15,1.16), the sections (1.17,1.18,1.19) and the active area (1.20) of the reference electrode (1.11) are rectangular in shape. . 6. Electrode device according to claim 5 wherein the active area (1.21) of the working electrode (1.12) is a circle. 7. Electrode device according to claim 6 wherein the active area (1.22) of the counter electrode (1.13) consists of a straight section (1.23) that connects with an arc section (1.24). 5 10 fifteen twenty 25 30 35 8. -Electronic device according to any of claims 1 to 7 wherein the potentiostat (2) is arranged on an integrated circuit board (4) on which a power supply (5) connected to it is also provided to provide it electric power as well as a digital analog converter (6), a microcontroller (7) and a communication interface (8), the three electrodes (1.11,1.12,1.13) being electrically connected to the potentiostat (2), this to the analog converter digital (6), this one to the microcontroller (7) and this one to the communication interface (8). 9. Device according to claim 8 wherein the potentiostat (2) comprises: - a first operational amplifier that provides voltage between 0.4 V and 0.9 V, with a gain of 100 dB in open loop, a bandwidth of 7 MHz, a non-linear effect of 5 V / ^ s, a rejection to the common mode of 90 dB current; - a second operational amplifier that in turn combines two amplifiers, third and fourth, in a surface-mounted encapsulation that isolates the impedance of voltage input to the working electrode (1.12) and the reference electrode (1.11) and output electrode their current, with a bandwidth of 1 MHz, a non-linear effect of 0.6 V / ^ s, an input bias current of 1 pA at 25 ° C and an input impedance of 100 MOhm. 10. - Device according to claim 9 wherein the potentiostat (2) is also connected to a third-order Butterworth filter (9) with Sallen-Key structure and unit gain. 11. - Device according to claim 10 wherein the microcontroller (7) has 8-bit storage load architecture. 12. -Device according to any of claims 8 to 11 wherein the integrated circuit board (4) and all components thereon (2,5,6,7,8,9) are arranged within a Aluminum enclosure (10) for protection against electromagnetic interference and noise. 13. Measurement procedure for the detection of lactic acid with an electrode device according to any of the preceding claims characterized in that it comprises the following steps: - deposition of sweat containing lactic acid in concentration between 3 mM and 50 mM in the active area (1.21) of the working electrode (1.12); eleven - application by the potentiostat (2) of a potential between 0.4 V and 0.9 V between the working electrode (1.12) and the reference electrode (1.11); - reading of the current generated between 2 mA and 20 mA by the potentiostat (2). 14. Method according to claim 13 with an electrode device according to Any of claims 8 to 12, wherein after the reading of the generated current the following steps take place: - transmission of said reading to the digital analog converter (6) to convert the analog signal into digital; 10 - transmission of the digital signal to the microcontroller (7) for recording, analysis and tracing; transmission of signal data related to its registration, analysis and monitoring to the communication interface (8) for its interaction with a user.