WO2010130861A1

WO2010130861A1 - Non-invasive device for detecting metabolites in sweat

Info

Publication number: WO2010130861A1
Application number: PCT/ES2010/070315
Authority: WO
Inventors: Francisco Javier MUÑOZ PASCUAL; Roser Mas Colomina
Original assignee: Consejo Superior De Investigaciones Científicas (Csic)
Priority date: 2009-05-11
Filing date: 2010-05-11
Publication date: 2010-11-18
Also published as: ES2349841B1; ES2349841A1

Abstract

Non-invasive device (1) for measuring metabolites in sweat, which comprises a substrate (2) suitable for attaching to a patient's skin, the inner face of which substrate has at least the following elements: a sweat-generation means (3a, 3b) suitable for causing sweating over a stimulated surface of the patient's skin; and a measuring chip (5), connected to the simulated surface by means of microchannels (4) capable of directing the sweat generated from the stimulated surface towards the measuring chip (5). The measuring chip (5) comprises a working microelectrode (5a), a reference microelectrode (5b) and an auxiliary microelelectrode (5c), the surface thereof being suitable for immobilizing enzymes that generate a current when they enter into contact with the metabolite of which the concentration is to be measured.

Description

NON INVASIVE DEVICE FOR METABOLITE DETECTION IN

THE SWEAT

D E S C R I P C I Ó N

OBJECT OF THE INVENTION

The present invention is directed to a device that serves to detect metabolites, such as glucose or bilirubin, in the sweat of a patient. The detection and / or measurement are carried out non-invasively, and therefore more comfortable for the patient.

BACKGROUND OF THE INVENTION

Some diseases can be detected by an analysis that determines the concentration of certain metabolites in the blood, for example, glucose in the case of patients with diabetes. However, the use of invasive techniques makes many methods used so far unfeasible for periodic monitoring of the concentration of said metabolite. An indirect way of carrying out this control is through the measurement of the concentration of the metabolite in question in some body fluid other than blood whose obtaining does not require invasive techniques, subsequently making a correlation of the concentration in said metabolite body fluid with the blood concentration

For example, it is possible to measure the concentration of glucose in diabetic patients through sweat. In this case, the sweat glands are stimulated to cause sweat, being able to use different methods, such as the use of drugs such as pilocarpine, inducing them to emit a sweat with the same qualities as a physiologically emitted sweat. In order for pilocarpine to pass through the patient's skin and reach the sweat glands, it is also necessary to apply a small current electric in the area. Next, the sweat generated is collected in order to analyze it. The collection must be done without any alteration of its quality: neither stain, nor evaporation, nor loss of liquid. To do this, after stimulation, the skin is usually cleaned, dried and simply coated with a Parafilm, so that a tight coating is obtained. After 2 to 15 minutes of sweating, a measuring electrode is placed on the place that received the pilocarpine.

A drawback of this method is that it is practically impossible to control the amount of sweat emitted, only the operator's experience allows us to see if the sweating is sufficient or not. To solve this problem, some systems propose the collection in a small dome or perculated above, fixed on the skin with an adhesive. In the patent application DE

10113143 describes a system where the generation of sweat is achieved by heating the measurement and subsequent measurement zone.

WO 94/00048 suggests the measurement of chloride and sodium on sweat, generated by the electro-stimulation stage integrated in the same substrate.

DESCRIPTION OF THE INVENTION

The drawbacks described above are solved by the device of the invention, which allows the detection of metabolites in the sweat of a patient in a simple, safe and non-invasive way. In this document, the term "detection" is intended to refer both to determine whether or not there is metabolite present in the sample, and to obtain a measure of the amount of metabolite.

According to one aspect of the invention, the device comprises a substrate suitable for fixation on the skin of a patient, on whose inner face (the face that is in contact with the patient's skin) are elements that cause the patient's sweating and measure the concentration of a certain substrate. The substrate can be made of different materials, both organic, for example polymeric such as SU-8, and inorganic, for example glass. In addition, in a preferred embodiment of the invention, the substrate is self-adhesive, so that it can be fixed on the patient's skin for carrying out the measurement.

In turn, the following elements are arranged on the inner side of the substrate:

- A means of generating sweat

The means of generating sweat can be of any type provided that it obtains a sufficient amount of sweat for the realization of the measurement. For example, it could simply be a heating element.

However, in a preferred embodiment of the invention, pilocarpine is used, which is made to penetrate through the patient's skin to the sweat glands by means of an electric current generated between an anode and a cathode that are disposed on the internal face of the substratum. In preferred embodiments of the invention, the current density through the anode and the cathode is between 0.2 and 0.8 mA / cm ² .

In this case, the pilocarpine can be applied in any way on the stimulation surface, which is the skin of the patient located between the anode and the cathode, although preferably cationic pilocarpine is applied on the cathode. Thus, the difference in potential between the anode and the cathode causes the cationic pilocarpine to migrate from the cathode to the anode, thus crossing the patient's skin until it reaches the sweat glands. The concentration of pilocarpine, the electrical intensity and the stimulation surface condition the duration of the stimulation. - A measurement chip

The measurement chip is connected to the stimulation surface by means of microchannels that serve to direct, by capillarity, the sweat generated in the stimulation surface to some microelectrodes of said measurement chip, preferably a working microelectrode, an auxiliary microelectrode and a reference microelectrode. The surface of the measurement chip must be adequate to immobilize proteins that generate a current after coming into contact with the metabolite whose concentration is to be measured. Finally, the measured current, which will be proportional to the amount of metabolite, is detected by means of microelectrodes.

In a second aspect of the invention, a method of measuring metabolites in the sweat of a patient by means of a device is described, the device comprising a substrate on whose internal face there is a means of generating sweat connected by microchannels to a measurement chip that has microelectrodes, and where the procedure comprises the following steps:

1) Cause sweating on a patient's stimulation surface.

This can be done in any known way, although in preferred embodiments of the invention heat is applied on a stimulation zone of the patient, or pilocarpine is applied together with an electric current by means of an anode and a cathode.

2) Transport the sweat generated through the microchannels to the measurement chip, on which proteins have been previously immobilized that cause the appearance of an electric current after coming into contact with the metabolite to be measured. Proteins can be immobilized in any known way, and will be based on the metabolite that is desired to be detected. For example, if it is desired to detect glucose or bilirubin, the protein to be immobilized could be glucose oxidase or bilirubin oxidase. 3) Detect the metabolite present in sweat from the measurement of the electric current by means of the microelectrodes. In this context, the term "detect" is intended to refer both to determine the presence or not of the metabolite and to measure its concentration or any equivalent parameter.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows a simplified scheme of the device of the invention.

Fig. 2 shows a detail of the measurement chip of the device of the invention.

PREFERRED EMBODIMENT OF THE INVENTION

An example of embodiment of the device (1) of

The invention referring to the attached figures where the different parts that compose it are appreciated. Figs. 1 and 2 show the device (1) of the invention. The substrate (2) on which the anode (3a) and the cathode (3b), which are connected by means of the microchannels (4) to the measurement chip (5), are observed. The measurement chip (5), which is represented in greater detail in Fig. 2, comprises a working microelectrode (5a), a reference microelectrode (5b) and an auxiliary microelectrode (5c). In addition, in this example, the substrate (2) is made of quartz, the anode (3a) and the cathode (3b) have a diameter of 1 mm. In relation to the microchannels (4), to achieve an adequate capillary effect they must be between 50 microns and 250 microns wide, and between 50 microns and 200 microns deep. Specifically, in this example they are 200 microns deep and wide.

The method of using the device (1) of the example for the detection of bilirubin in sweat is now described. In the first place, the device (1) of the invention is prepared for this particular application: the cathode (3b) is covered by a chloride salt-shaped pilocarpine gel, and they adhere Bilirubin oxidase enzymes on the surface of the measurement chip (5) by immobilization on a sensitive membrane. For the formation of this enzymatic membrane on the working electrode, bilirubin oxidase is immobilized in a molecular conductor of polypyrrole by means of a rapid electropolymerization process.

Once the device (1) is prepared, a current density of about 0.6 mA / cm ² is applied between the anode (3a) and the cathode (3b) for a few seconds. Approximately 5 minutes later the sweat generated by the effect of pilocarpine emerges on the surface of the skin. The microchannels (4) direct the sweat towards the measurement chip (5), where the microelectrodes (5a, 5b, 5c) allow measuring the current generated due to the metabolization of the bilirubin by the bilirubin oxidase enzyme.

Finally, Figure 3 shows the response of the bilirubin biosensor that is in the measurement chip (5) when it comes into contact with the bilirubin present in the sweat. The ability to detect and quantify 1 nanogram of bilirubin can be observed.

Claims

1. Non-invasive device (1) for the measurement of metabolites in sweat, characterized in that it comprises a substrate (2) suitable for fixation on the skin of a patient, whose internal face has at least the following elements:

a means of generating sweat (3a, 3b) suitable to cause sweating on a surface stimulating the patient's skin; Y

a measurement chip (5), connected to the stimulation surface by means of microchannels (4) capable of directing the sweat generated from the stimulation surface to the measurement chip (5), which comprises microelectrodes (5a, 5b , 5c), and where the surface of said measurement chip (5) is suitable for immobilizing a protein that generates an electric current when it comes into contact with the metabolite whose concentration is to be measured.

2. Device (1) according to claim 1, wherein the measurement chip (5) comprises a working microelectrode (5a), a reference microelectrode (5b) and an auxiliary microelectrode (5c).

3. Device (1) according to any of the preceding claims, wherein the sweat generating means (3a 3b) comprises an anode (3a) and a cathode (3b) capable of generating a suitable electric current to facilitate the penetration of Pilocarpine through the patient's skin.

4. Device (1) according to claim 3, wherein the electric current generated between the anode (3a) and the cathode (3b) is between 0.2 mA / cm ² and 0.8 mA / cm ² .

5. Device (1) according to any of claims 3-4, wherein the cathode (3b) is impregnated with cationic pilocarpine.

6. Device (1) according to any of the preceding claims, wherein the substrate (2) is self-adhesive.

7. Device (1) according to any of the preceding claims, wherein the microchannels are between 50 microns and 250 microns wide and between 50 microns and 200 microns deep.

8. Device (1) according to any of the preceding claims, wherein the protein is glucose oxidase.

9. Device (1) according to any of the preceding claims, wherein the protein is bilirubin oxidase.

10. Method of measuring metabolites in the sweat of a patient using a device (1) comprising a substrate (2) on whose inner face there is a means (3a, 3b) for generating sweat connected by microchannels (4) to a measurement chip (5) having microelectrodes (5a, 5b, 5c), which comprises the following operations:

cause sweating on a patient's stimulation surface;

transport the sweat generated through the microchannels (4) to the measurement chip (5), on which proteins that cause the appearance of an electric current after coming into contact with the metabolite to be measured have been previously immobilized; Y

Detect the metabolite present in sweat from the measurement of the electric current by means of the microelectrodes (5a, 5b, 5c).

11. Method according to claim 10, wherein the operation of causing the patient's sweating is performed by applying heat to the patient's stimulation surface.

12. Procedure according to claim 10, wherein the operation of causing the patient's sweating in turn comprises the following steps:

- apply pilocarpine to the stimulation surface; Y

- Apply an electric current to the stimulation surface by means of an anode (3a) and a cathode (3b).

13. Method according to any of claims 10-12, wherein the protein that is immobilized on the measurement chip (5) is bilirubin oxidase or glucose oxidase.