DETECTION OF A CHEMICAL SPECIES IN THE SWEAT OF A SUBJECT

Abstract

A detection apparatus for placement on an investigation zone of an epidermis of a human or animal subject for detecting at least the nitric oxide dissolved in sweat, said apparatus comprising: a structure (1) defining a microfluidic circuit for guiding a flow of sweat, the structure comprising an entry orifice (4) allowing passage of sweat from the epidermis, the microfluidic circuit comprising at least one microfluidic channel (9) in communication with the entry orifice, at least one electrochemical sensor (10) comprising at least four electrodes disposed successively in a longitudinal direction of the microfluidic channel, the at least four electrodes comprising a reference electrode, at least two working electrodes and a counter-electrode, the electrochemical sensor being configured to produce a signal that is representative of a concentration of nitric oxide and further for implementing a depletion and/or for producing a signal that is representative of a flow rate of the flow of sweat.

Claims

1. A detection apparatus for placement on an investigation zone (97) of an epidermis of a human or animal subject for detecting at least nitric oxide dissolved in sweat, said detection apparatus (100) comprising: a structure defining a microfluidic circuit (8), the structure comprising an entry orifice (4) allowing passage of sweat from the epidermis, the microfluidic circuit (8) comprising at least one microfluidic channel (9) for guiding a flow of sweat (98), the microfluidic channel (9) being in communication with the entry orifice (4), at least one electrochemical sensor (10) configured to produce at least one signal that is representative of a concentration of the nitric oxide dissolved in the flow of sweat (98) in the microfluidic channel (9), wherein the electrochemical sensor (10) comprises at least four electrodes disposed successively in a longitudinal direction of the microfluidic channel (9), the at least four electrodes comprising a reference electrode (21), at least two working electrodes (20, 23) and a counter-electrode (30), and wherein the electrochemical sensor is further configured to perform at least one additional operation from among the following: depleting a chemical species in the flow of sweat in the microfluidic channel (9), said chemical species having an oxidation potential lower than the oxidation potential of nitric oxide, and producing a signal that is representative of a flow rate of the flow of sweat in the microfluidic channel (9).

2. The detection apparatus as claimed in claim 1, in which the structure is a multilayer structure (1) comprising a lower layer (3) and at least one layer atop the lower layer (3), the microfluidic circuit (8) extending parallel to the lower layer (3), and the lower layer (3) comprising said entry orifice (4).

3. The detection apparatus as claimed in claim 2, in which the multilayer structure (1) comprises an upper layer (7) and at least one middle layer (6) situated between the lower layer (3) and the upper layer (7), the microfluidic circuit (8) being formed in a thickness of the at least one middle layer (6).

4. The detection apparatus as claimed in claim 3, in which the at least one middle layer comprises a first middle layer (6) and a second, sealing middle layer (26) situated between the first middle layer (6) and the upper layer (7), the second, sealing middle layer (26) comprising an opening (28) at the electrodes.

5. The detection apparatus as claimed in claim 2, in which the multilayer structure (1) comprises an upper layer (7) and an outlet orifice (13) traversing the upper layer (7), in which the at least one microfluidic channel (9) is in communication with the outlet orifice (13).

6. The detection apparatus as claimed in claim 3, in which the at least four electrodes are disposed on an inner face of the upper layer (7) closing the microfluidic channel (9) at a top and/or on an upper face of the lower layer (3) closing the microfluidic channel (9) at a bottom.

7. The detection apparatus as claimed in claim 1, in which, in the direction of the flow (98), the at least four electrodes comprise in succession the first working electrode (20), in the form of a depletion electrode, the second working electrode (23) for measuring the concentration of nitric oxide, and the counter-electrode, the reference electrode (21) being placed at a position immediately upstream of the first working electrode (20) or immediately downstream of the second working electrode (23).

8. The detection apparatus as claimed in claim 1, in which, in the direction of the flow (98), the at least four electrodes comprise in succession the first working electrode (20) for measuring the concentration of nitric oxide, the second working electrode (23) for measuring the concentration of nitric oxide, and the counter-electrode, the reference electrode (21) being placed at a position immediately upstream of the first working electrode (20) or immediately downstream of the second working electrode (23).

9. The detection apparatus as claimed in claim 7, in which the electrochemical sensor (10) is configured to produce the signal that is representative of the flow rate by measuring a delay (?t) between a variation in current in the first working electrode (20) and a variation in current in the second working electrode (23).

10. The detection apparatus as claimed in claim 1, in which the electrochemical sensor (10) is configured to produce a signal that is representative of instantaneous production of nitric oxide in the investigation zone (97) on the basis of the signal that is representative of the concentration of nitric oxide and of the signal that is representative of the flow rate of the flow of sweat (98).

11. The detection apparatus as claimed in claim 1, in which the electrochemical sensor (10) is configured to produce the signal that is representative of the concentration of nitric oxide by an electrical, especially amperometric, measurement between at least one of said working electrodes (20, 23) and the counter-electrode (30).

12. The detection apparatus as claimed in claim 1, in which the electrochemical sensor (10) is configured to polarize at least one of said working electrodes (20, 23) to an electrical potential for oxidation of nitric oxide.

13. The detection apparatus as claimed in claim 1, in which the electrochemical sensor (10) is configured to produce a signal that is representative of a concentration in the flow of sweat of at least one of the following chemical compounds: nitrite ion, hydrogen peroxide and peroxynitrite, dissolved in sweat.

14. The detection apparatus as claimed in claim 13, in which the electrochemical sensor (10) comprises a third working electrode (25) between the first or second working electrode (20, 23) and the counter-electrode for measuring the chemical compound.

15. The detection apparatus as claimed in claim 1, comprising a colorimetric detection device (18) connected to the channel (9) downstream of the electrochemical sensor (10), the colorimetric detection device (18) comprising a hydrophilic porous body impregnated with a chemical reagent capable of reacting with one of the following chemical compounds: nitrite ion, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide and hypochlorous acid, dissolved in sweat, so as to provide a colored indicator indicating a quantity of said chemical compound in the flow of sweat (98).

16. The detection apparatus as claimed in claim 15, in which the chemical reagent comprises a Griess reagent capable of reacting with the nitrite ion dissolved in the flow of sweat (98).

17. The detection apparatus as claimed in claim 5, comprising a colorimetric detection device (18) connected to the channel (9) downstream of the electrochemical sensor (10), the colorimetric detection device (18) comprising a hydrophilic porous body impregnated with a chemical reagent capable of reacting with one of the following chemical compounds: nitrite ion, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide and hypochlorous acid, dissolved in sweat, so as to provide a colored indicator indicating a quantity of said chemical compound in the flow of sweat (98); and wherein the colorimetric detection device (18) is disposed in the outlet orifice (13).

18. The detection apparatus as claimed in claim 1, in which the electrochemical sensor (10) is configured to polarize at least one said working electrode (20, 23, 25) during a determined time with a periodic recurrence.

19. The detection apparatus as claimed in claim 1, in which the microfluidic circuit (8) comprises a plurality of microfluidic channels (9) each guiding a flow of sweat, which are connected in derivation from one another to the entry orifice (4).

20. The detection apparatus as claimed in claim 19, in which the plurality of microfluidic channels (9) comprises an additional microfluidic channel (9) comprising an electrochemical sensor (10), the electrochemical sensor (10) comprising at least three electrodes disposed successively in a longitudinal direction of the additional microfluidic channel (9), the at least three electrodes comprising a reference electrode (21), a counter-electrode (30) and at least one working electrode (20, 23, 25), the additional electrochemical sensor (10) being configured to polarize the electrodes to an electrical potential for oxidation of a chemical compound selected from nitrite ion, hydrogen peroxide and peroxynitrite and being configured to produce at least one signal that is representative of a concentration of said chemical compound dissolved in a flow of sweat in the additional microfluidic channel (9).

21. The detection apparatus as claimed in claim 19, in which the plurality of microfluidic channels comprises an additional microfluidic channel (109) comprising a colorimetric detection device (18), the colorimetric detection device (18) comprising a hydrophilic porous body impregnated with a chemical reagent capable of reacting with one of the following chemical compounds: nitrite ion, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide and hypochlorous acid, so as to provide a colored indicator indicating a concentration or a quantity of the chemical compound dissolved in a flow of sweat in the additional microfluidic channel (109).

22. The detection apparatus as claimed in claim 21, in which the additional channel (109) comprises a chrono-sampling system connected to the entry orifice (4), the chrono-sampling system including a plurality of chambers configured to fill sequentially with sweat, and in which a plurality of colorimetric detection devices (18) are disposed in said chambers, each colorimetric detection device (18) comprising a chemical reagent capable of reacting with a chemical compound, such that the colorimetric detection devices disposed in said chambers provide a colored indicator indicating a cumulative quantity of said chemical compound in the flow of sweat in the additional microfluidic channel (109).

23. The detection apparatus as claimed in claim 15, comprising an optical sensor configured to produce a measurement signal that is representative of an intensity of a color of the chemical reagent in the visible or ultraviolet spectrum.

24. The detection apparatus as claimed in claim 1, comprising a communication device (17) configured to transmit one or more measurement signals produced by the detection apparatus (100) to a storage or post-processing apparatus.

25. A portable device comprising a detection apparatus (100) as claimed in claim 1, the portable device being implemented in the form of: a watch, a telephone, a fabric, a headband, a garment or an undergarment.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0118] For better understanding of the subject matter of the invention, embodiments thereof as shown in the accompanying drawings will be described below, by way of purely illustrative and nonlimiting example. In these drawings:

[0119] FIG. 1 represents a schematic view of a subject seen from the back on whom a detection apparatus according to one embodiment has been placed,

[0120] FIG. 2 is a perspective view partially representing a multilayer structure for a detection apparatus according to one embodiment,

[0121] FIG. 3 represents a view in section, along the line II-II of the figure, of the multilayer structure,

[0122] FIG. 4 is an expanded view of the multilayer structure according to one embodiment,

[0123] FIG. 5 is an expanded view of the multilayer structure according to another embodiment,

[0124] FIG. 6 is an enlarged perspective view of an electrochemical sensor of the multilayer structure according to one embodiment,

[0125] FIG. 7 is an expanded view of the multilayer structure according to another embodiment,

[0126] FIG. 8 is a functional schematic representation of a microfluidic circuit which can be used in a detection apparatus,

[0127] FIG. 9 is another functional schematic representation of another microfluidic circuit which can be used in a detection apparatus,

[0128] FIG. 10 is another functional schematic representation of another microfluidic circuit which can be used in a detection apparatus,

[0129] FIG. 11 is a schematic perspective representation of an electrochemical sensor which can be used in the microfluidic circuit of FIGS. 2 to 10,

[0130] FIG. 12 is a chronogram illustrating a detection method which can be carried out with the electrochemical sensor of FIG. 11.

[0131] FIG. 13 (A) represents a scheme of an electrochemical sensor which can be used in the microfluidic circuit of FIGS. 2 to 10 of the detection apparatus according to one embodiment, and FIG. 13 (B) represents a method for detecting a sweat flow rate that may be carried out with the electrochemical sensor of FIG. 13 (A),

[0132] FIG. 14 represents a scheme of an electrochemical sensor of the detection apparatus according to one embodiment with the depletion function,

[0133] FIG. 15 represents an explanatory scheme for the depletion function according to one embodiment,

[0134] FIG. 16 is a chronogram illustrating a detection method which may be carried out with the electrochemical sensor of FIG. 14,

[0135] FIG. 17 illustrates schematically an electrochemical sensor according to an embodiment with five electrodes,

[0136] FIG. 18 illustrates schematically an electrochemical sensor according to an embodiment with six electrodes,

[0137] FIG. 19 illustrates schematically an embodiment of the multilayer structure further comprising a colorimetric detection device,

[0138] FIG. 20 is an expanded view of the multilayer structure according to another embodiment with a plurality of channels,

[0139] FIG. 21 is a functional schematic representation of a detection apparatus which can be used in the apparatus of FIG. 1,

[0140] FIG. 22 is a diagram of steps illustrating a method which may be implemented with the apparatus of FIG. 1,

[0141] FIG. 23 is a graph illustrating a result of the measurements which may be obtained with the apparatus of FIG. 1.

DESCRIPTION OF THE EMBODIMENTS

[0142] FIG. 1 represents a detection apparatus 100 disposed on the skin of a human subject 2, for example on the back of the subject, which is intended for performing quantitative measurements of chemical species dissolved in sweat, including nitric oxide dissolved in sweat, and optionally the nitrite ion or hydrogen peroxide. The detection apparatus 100 may be disposed on another part of the body, for example the nape of the neck, the shoulder, the arm or the leg.

[0143] The detection apparatus 100 comprises a microfluidic part and other functional parts which will be described further below, in particular a control device 40 (FIG. 21).

[0144] With reference to FIG. 2, the multilayer structure 1 takes the form, for example, of a low-profile casing which comprises a lower layer 3 made of a flexible and biocompatible material, preferably self-adhesive, which may be positioned directly on the skin of the subject, and a second layer 6 which lies atop the lower layer 3. The multilayer structure 1 is made from watertight materials, for example from polymer.

[0145] The second layer 6 is hollowed out in its thickness to form a microfluidic channel 9 and a sampling dome 99 situated over an opening 4 formed in the lower layer 3.

[0146] With reference to FIG. 3, the lower layer 3 is stuck to the skin 2 by an adhesive layer 96. A central part of the lower layer 3 and of the adhesive layer 96 comprises the circular opening 4 delimiting an investigation zone 97 on the skin 2 of the subject, being for example a few mm to a few cm in diameter. The circular opening 4 may adopt another form, for example, an ellipse, a triangle, a rectangle, a square, a polygon or some other form. The circular opening 4 is an entry orifice allowing a flow of sweat 98 to be conducted to, in particular for bringing the sweat into, the microfluidic channel 9. The flow of sweat 98 passes from the skin 2 of the subject to the microfluidic channel 9, passing across the circular opening 4.

[0147] In the embodiment of FIG. 3, an upper layer 7 covers the second layer 6 to form the microfluidic circuit at the top. The microfluidic circuit may therefore be formed through the whole thickness of the second layer 6, thereby making it easier to manufacture, for example by cutting or engraving.

[0148] A hydrophilic collector element (not shown), for example a fibrous body, such as cotton or a non-woven material, may be disposed in the circular opening 4 and the dome 99. The function of the collector element is to bring the sweat produced in the investigation zone to the microfluidic circuit.

[0149] With reference to FIG. 4, the multilayer structure comprises: [0150] a lower layer 3 comprising an entry orifice 4 allowing passage of sweat, [0151] an upper layer 7 comprising an outlet orifice 13, [0152] a middle layer 6 situated between the lower layer 3 and the upper layer 7, the microfluidic circuit being formed in the thickness of the at least one middle layer 6 and extending parallel to the lower layer 3.

[0153] The microfluidic circuit consists of a microfluidic channel 9 which is in communication with the entry orifice 4 at a first end and in communication with the outlet orifice 13 at a second end. Accordingly, the flow of sweat from the skin 2 of the subject is conducted in the microfluidic channel 9, which conducts the sweat from the entry orifice 4 to the outlet orifice 13 by capillary action.

[0154] An electrochemical sensor 10 comprises four electrodes disposed on the inner face of the upper layer 7 closing the microfluidic channel at the top. The electrodes are therefore situated in the internal space of the microfluidic channel.

[0155] As a dimensional example, the entry orifice has a diameter of between 1 mm and 15 mm, the microfluidic channel has a length of between 0.5 cm and 5 cm and a width of between 25 ?m and 500 ?m, the middle layer has a thickness of between 10 ?m and 200 ?m, and the layers of the multilayer structure have a width of between 1 cm and 5 cm and a length of between 2 cm and 15 cm.

[0156] For example, the entry orifice 4 has a diameter of 5 mm, the microfluidic channel 9 has a length of 1.8 cm and a width of 100 ?m, the middle layer has a thickness of less than 70 ?m, for example 20 ?m, and the layers of the multilayer structure have a width of 3 cm and a length of 9 cm.

[0157] With reference to FIG. 5, the multilayer structure is similar to FIG. 4. However, in this embodiment, the outlet orifice 13 is situated in the middle layer 6, at one end of the middle layer. The detection apparatus 100 comprises an electrochemical sensor 10 comprising respectively four electrodes; each electrode comprises respectively two parts disposed facing one another, a first part disposed on an inner face of the upper layer 7 closing the microfluidic channel 9 at the top, and a second part disposed on an upper face of the lower layer 3 closing the microfluidic channel at the bottom. Each electrode part comprises a connector illustrated by a black rectangle, via which the electrodes may be electrically connected.

[0158] According to one embodiment, not illustrated, the detection apparatus may comprise a single electrochemical sensor 10 comprising four electrodes disposed on an upper face of the lower layer 3 closing the microfluidic channel 9 at the bottom.

[0159] FIG. 6 illustrates four electrodes arranged in a microfluidic channel, which may be the microfluidic channel 9 presented in FIGS. 4 and 5, for example. The four electrodes are applied metals disposed on the inner face of the upper layer 7 closing the microfluidic channel 9 at the top. The four electrodes are disposed successively in a longitudinal direction of the microfluidic channel and comprise: a working electrode 20, implemented here in the form of a depletion electrode which operates in a manner that will be detailed further below, a second working electrode 23, a reference electrode 21, and a counter-electrode 30. The height of the electrodes is between 1 and 50 nanometers (nm), the space between the electrodes is between 10 and 10 000 micrometers (?m), and the width of the electrodes is between 1 and 1000 ?m. The electrodes may be manufactured, for example, of platinum (Pt), of gold (Au), of silver (Ag), or of silver chloride (AgCl).

[0160] At least one of the electrodes may also be covered entirely or partly with polyeugenol. platinum black or polyphenol. The electrodes are configured to perform one or more of these actions: depleting, measuring the concentration of nitric oxide, measuring the concentration of at least one other chemical component, and measuring the flow rate of the flow of sweat flowing in the microfluidic channel 9.

[0161] FIG. 7 illustrates the multilayer structure 1 similar to FIG. 4, in which a second, sealing middle layer 26 is situated between the first middle layer 6 and the upper layer 7, and the second, sealing middle layer 26 comprises an opening 27 at the electrodes, allowing the electrodes to be contacted with the flow of sweat circulating in the microfluidic channel 9. The second, sealing middle layer 26 further comprises an intermediate opening 28 which communicates with the outlet orifice 13 of the upper layer 7 so as to enable discharge of sweat. For example, the opening 27 is rectangular in form and has a length of 5 mm and a width of 200 ?m.

[0162] With reference to FIG. 8, the middle layer 6 comprises a microfluidic circuit 8 fed with a flow of sweat. The sweat is received from the entry orifice of the lower layer toward the microfluidic circuit 8. The microfluidic circuit 8 may comprise one or more microfluidic channels 9, specifically four parallel microfluidic channels 9 in the example represented. However, the microfluidic circuit 8 may adopt different forms; FIG. 9, for example, represents a microfluidic circuit 8 comprising parallel microfluidic channels 9, and FIG. 10 represents four microfluidic channels 9 distributed radially. The microfluidic channels 9 are formed, for example, in the thickness of the middle layer 6 and are separated by partitions 11. Each microfluidic channel 9 is respectively separated from the other microfluidic channels, within which the sweat may circulate independently. The number of microfluidic channels 9 may be higher or lower than in these figures.

[0163] Each fluidic circuit 9 is equipped with a sensor 10A, 10B, 10C or 10D. The arrows 12 illustrate the direction of flow of sweat in the microfluidic channels 9. Via the outlet orifice 13, the microfluidic channels 9 end preferably in a drainage reservoir which retains the analyzed fluids, so as to prevent the reaction products of the electrolysis coming back into contact with the subject's skin.

[0164] The sensors 10A, 10B, 10C and 10D arranged in the microfluidic circuits 9 for analyzing the sweat are preferably electrochemical sensors. The operating principle of an electrochemical sensor is that of wholly or partially electrolyzing the solution present in the fluidic channel 9 between a working electrode and a counter-electrode. An electrochemical sensor of this kind may be implemented in a variety of ways, in particular in miniaturized form with dimensions of the order of a millimeter.

[0165] A description will now be given of a number of embodiment examples of the electrochemical sensors, with reference to FIG. 8.

EXAMPLES

Example 1

[0166] The sensor 10A is intended for detecting hydrogen peroxide. It therefore operates with a potential difference equal to the oxidation potential of hydrogen peroxide, E.sub.H.sub.2.sub.O.sub.2. The sensor 10B is intended for detecting nitric oxide. It therefore operates with a potential difference equal to the oxidation potential of nitric oxide, E.sub.NO. The sensor 10C is intended for detecting the nitrite ion. It therefore operates with a potential difference equal to the oxidation potential of the nitrite ion, E.sub.NO.sub.2?.

[0167] The sensors 10A, 10B, 10C carry out synchronous measurements of the instantaneous intensities, denoted i.sub.oxdn, of the faradaic currents linked to the electrochemical oxidation of the aforesaid chemical species. The sensors 10A, 10B, 10C therefore allow the detection and quantification of the instantaneous concentration of the aforesaid chemical species.

[0168] Each of the three aforesaid chemical species can be detected by amperometric measurements with the aid of microelectrodes. The latter consist, for example, of strips of platinum covered with a thin layer, for example of micrometric dimensions, of platinum black applied by electrochemical reduction, in an aqueous medium, of the anion of a platinum salt, Pt(Cl).sub.6.sup.4?.

[0169] The three chemical species (NO, NO.sub.2.sup.? and H.sub.2O.sub.2) can be distinguished by the fact that their oxidation potentials on these electrodes are clearly separated, occurring in the following order: E.sub.H2O2<E.sub.NO<E.sub.NO2?. However, the faradaic currents are additive. The measured current at the oxidation potential of each chemical species therefore adds to the elementary currents linked to the oxidation of this chemical species and to the oxidation of all of the chemical species which have lower oxidation potentials.

[0170] Thus, only the species H.sub.2O.sub.2 can be oxidized at the oxidation potential EH.sub.2O.sub.2. The species H.sub.2O.sub.2 and NO can be oxidized at the oxidation potential E.sub.NO. The three species can be oxidized at the oxidation potential E.sub.NO2?. The currents measured by the sensors 10A to 10C, respectively denoted i.sub.oxon (EH.sub.2O.sub.2), i.sub.oxon(E.sub.NO) and i.sub.oxon (E.sub.NO2?), therefore satisfy the following equations:

[00001]$i_{\mathrm{oxdn}}\left(E_{H2O2}\right)=a1i_{H2O2}$$i_{\mathrm{oxdn}}\left(E_{\mathrm{NO}}\right)=a2i_{H2O2}+a3i_{\mathrm{NO}}$$i_{\mathrm{oxdn}}\left(E_{\mathrm{NO}{2}^{-}}\right)=a4i_{H2O2}+a5i_{\mathrm{NO}}+a6i_{\mathrm{NO}{2}^{-}}$

in which the coefficients a1 to a6 represent calibration constants for the sensors, which can be measured experimentally.

[0171] Accordingly, via subtractions which are easily implemented on an electronic circuit, the following are obtained:

[00002]$i_{H_2{O}_2}=\left(1/a1\right)i_{\mathrm{oxdn}}\left(E_{H_2{O}_2}\right)$$i_{\mathrm{NO}}=\left(1/a3\right)i_{\mathrm{oxdn}}\left(E_{\mathrm{NO}}\right)-\left(a2/a1\right).Math.\left(1/a3\right)i_{\mathrm{oxdn}}\left(E_{H_2{O}_2}\right)$$i_{{\mathrm{NO}}_{2^{-}}}=\left(1/a6\right)i_{\mathrm{oxdn}}\left(E_{{\mathrm{NO}}_{2^{-}}}\right)-\left(a4/a6\right)i_{H_2{O}_2}-\left(a5/a6\right)i_{\mathrm{NO}}$

[0172] At any time t, the instantaneous intensity of the faradaic oxidation current, i.sub.s(t), for each chemical species S is proportional to its concentration, C.sub.s(f), in the volume of fluid situated above the electrodes which detect it. The proportionality factor depends on a form factor, denoted ?, which is a function of the geometry of the sensor, and on the Faraday constant, denoted n.sub.s, consumed per mole of the chemical species, i.e.:

[00003]$n_{H2O2}=n_{\mathrm{NO}{2}^{-}}=2\mathrm{and}{n}_{\mathrm{NO}}=1$

[0173] It will be recalled that F denotes the faraday, i.e. 96 500 coulombs, the value for the charge of one mole of electrons.

[0174] The form factor ? is a constant factor imposed by the geometry of the electrochemical sensor, which can be evaluated theoretically and measured experimentally by calibration. For the sake of simplicity, the three sensors 10A to 10C are considered below to have identical geometries, such that the form factor ? is the same for all of the sensors.

[0175] The result is that the concentrations of the chemical species can be obtained from the currents measured by the sensors 10A to 10C, with the aid of the following expressions, in which the temporal variable t has been specified:

[00004]$C_{H_2{O}_2}\left(t\right)=i_{\mathrm{oxdn}}\left(E_{H_2{O}_2},t\right)/\left(2F?\right)$$C_{\mathrm{NO}}\left(t\right)=\left[i_{\mathrm{oxdn}}\left(E_{\mathrm{NO}},t\right)-i_{\mathrm{oxdn}}\left(E_{H_2{O}_2},t\right)\right]/\left(F?\right)$$C_{{\mathrm{NO}}_{2^{-}}}\left(t\right)=\left[i_{\mathrm{oxdn}}\left(E_{{\mathrm{NO}}_{2^{-}}},t\right)-i_{\mathrm{oxdn}}\left(E_{\mathrm{NO}},t\right)\right]/\left(2F?\right)$

[0176] In example 1, the three sensors 10A to 10C can therefore operate in parallel, each with a constant oxidation potential, namely E.sub.H2O2, E.sub.NO and E.sub.NO2? respectively.

[0177] In a variant embodiment, only NO and NO.sub.2.sup.? are detected. This embodiment is particularly advantageous when the measurement of H.sub.2O.sub.2 is not significant and does not influence the results of the intended objective. The concentration C.sub.H2O2(t) presented above is then considered to be uniformly zero, i.e. C.sub.H2O2(t)=0. The system of equations is therefore simplified.

Example 2

[0178] In example 2, a single microfluidic channel 9 and a single sensor 10A are used; the others being able to be omitted.

[0179] In this case, the sensor 10A operates sequentially in order to detect the aforesaid chemical species during three successive steps. The oxidation potential is therefore switched between three potential stages, respectively equal to the three oxidation potentials mentioned above, for example periodically in accordance with the sequence E.sub.H2O2.fwdarw.E.sub.NO.fwdarw.E.sub.NO2?.fwdarw.E.sub.H2O2.fwdarw.E.sub.NO.fwdarw.E.sub.NO2?.fwdarw. etc.

[0180] In this case, each oxidation potential is maintained for a duration that is very long compared with the time constant for the working electrode, this time constant being, for example, a few milliseconds for the microelectrodes employed in the microfluidic channels, and measurements of the current are carried out at the end of each constant potential stage.

[0181] The remaining measurement signals can be processed using the same equations as in example 1.

Example 3

[0182] Because nitric oxide is a small molecule that is both hydrophilic and lipophilic, it can easily pass through thin layers of organic polymer, in contrast to the other two species H.sub.2O.sub.2 and NO.sub.2.sup.?. Thus, it can be detected in isolation with the aid of an electrochemical sensor protected by a layer of this type, for example with a working electrode made of platinized platinum coated with a thin layer of polyeugenol (4-allyl-2-methoxyphenol) applied by electropolymerization.

[0183] In example 3, the working electrode of the sensor 10D is therefore coated by the layer which is schematically represented by the numeral 19. The instantaneous concentration of nitric oxide can therefore be measured independently of that of the chemical species H.sub.2O.sub.2 and NO.sub.2.sup.?, in accordance with the expression:

[00005]$C_{\mathrm{NO}}\left(t\right)={\left[i_{\mathrm{oxdn}}\left(E_{\mathrm{NO}},t\right)\right]}_{\mathrm{eugenol}}/\left(F?\right).$

[0184] Here, i.sub.oxdn(E.sub.NO, t)].sub.eugenol denotes the current measured by the sensor 10D.

[0185] The other sensors 10A to 10C and the other microfluidic channels 9 can be omitted. This method can therefore advantageously be used with a single sensor when only the concentration of NO is desired.

Example 4

[0186] In this case, the sensor 10D of example 3 is amalgamated with the sensors 10A to 10C of example 1 or with the sensor 10A of example 2. This configuration can be used to obtain two measurements that are independent of the concentration of dissolved nitric oxide, and thus to check the consistency of the measurements, in particular by verifying that the sensors do not exhibit drift, which is linked for example to partial deactivation of the surface of one of the electrodes.

[0187] In this case, the electrochemical electronic control device 40 (FIG. 21) is preferably configured to compare the two measurements of the concentration of nitric oxide and to emit an alarm when the result of the comparison satisfies a predefined criterion, for example if it exceeds a predefined threshold.

[0188] In examples 1 to 4 above, the measured instantaneous faradaic currents can be used to measure the concentration of the chemical species in the analyzed solution. As a consequence, in a static system, the intensity of the current is sufficient to document the production of the detected species.

[0189] However, when the detection apparatus 100 is applied to an essentially dynamic physiological system, it is desirable also to be able to have quantitative access to the dynamics of production of each chemical species by the cardiovascular system, for example during exertion tests or during medical monitoring. Under dynamic conditions, in order to access the instantaneous quantity of a chemical species, denoted ?Q(t), produced over a short period of time, denoted ?t(t), it is desirable to know the mean concentration, C.sub.s(t), of the chemical species and the volume flow rate of the analyzed fluid simultaneously, namely:

[00006]$d\left(t\right)=\left(?V/?f\right)$ [0190] in which ?V designates the volume scanned during the time interval ?t. Thus, the intensity of the production flow, denoted P.sub.s(t), of a chemical species S at a time t is given by:

[00007]$P_s\left(t\right)=\left[?Q/?t\right]\left(t\right)=C_s\left(t\right).Math.d\left(t\right)$ [0191] in which the mean concentration C.sub.s(t) is obtained from the mean intensities of the electrochemical oxidation currents measured between the times t and t+?t.

[0192] In the context of the dynamic applications envisaged, it is therefore desirable for the detection apparatus 100 to measure, at the same time and at each time t required by the desired accuracy for monitoring the physiological status of the patient over time, for example once per minute, the mean intensities, i.sub.av(f), of the faradaic current linked to the electrochemical oxidation of the one or more chemical species being monitored and the value for the volume flow rate d(t) of sweat at the time t in the corresponding fluidic circuit.

[0193] FIG. 11 illustrates an embodiment of an electrochemical sensor 10 that can meet this dual requirement in an integrated manner. This electrochemical sensor 10 comprises at least one pair of working electrodes 20, 23. A strip microelectrode of this type may be produced from platinized (platinum black) platinum, which may or may not be covered with a layer of electropolymerized eugenol of micrometric dimensions. A strip microelectrode of this type may be implanted by microfabrication, for example by CVD and/or lithography. This strip microelectrode or these strip microelectrodes can be used to electrochemically oxidize the selected chemical species.

[0194] The microfluidic circuit 9 of FIG. 11 is further equipped with a reference electrode 21 that is produced, for example, in the form of an Ag/AgCl microstrip and placed upstream of the pair of working electrodes 20, 23. Finally, the fluidic circuit 9 is equipped with a counter-electrode 30 made of platinized platinum and placed downstream of the pair of working electrodes 20, 23. Notwithstanding the functional schematic representation of FIG. 11, the surface area of the counter-electrode 30 is in fact two to three times larger than that of the other electrodes.

[0195] The assembly of the microfluidic channel 9 with the electrodes 20, 21, 23, 30 is bathed in a lamina of sweat, not shown, and thus constitutes a microfluidic electrochemical cell with four electrodes. Each of the electrodes 20, 21, 23, 30 is connected to an electrochemical electronic control device 40 (FIG. 21) by means of electrical contacts insulated from the sweat.

[0196] This embodiment of an electrochemical sensor 10 may be employed in one or more of the aforementioned microfluidic circuits 9.

[0197] In order to measure the volume flow rate d(t), the electrochemical sensor 10 has to include the pair of working electrodes 20, 23. The solution described here is simple and readily industrializable, because it has no moving parts and it makes no claim to be hydrodynamic. It does not require any intervention aimed at modulating the flow rate of the fluid, while being suitable for any reasonable physiological flow rate.

[0198] The two working electrodes 20, 23, for example two strips of platinized platinum, may act as working microelectrodes, are electrically independent and are spaced apart by a distance L along the path of the fluid analyzed in the microfluidic circuit 9. The two working electrodes 20 and 23 are, for example, installed on the bottom of a linear channel, the section of which has a constant area A.

[0199] The working electrode 23 positioned downstream is used in accordance with the method illustrated in FIG. 12, which comprises two steps. The graph 81 represents the electrical potential applied to the working electrode 20 as a function of time. The graph 82 represents the electrical potential applied to the working electrode 23 as a function of time. The potentials indicated as 0 on graphs 81 and 82 in fact signify disconnection of the corresponding electrode (open circuit). The graph 83 represents the faradaic current measured at the working electrode 20 as a function of time. The graph 84 represents the faradaic current measured at the working electrode 23 as a function of time.

[0200] During a first step carried out over a range of time prior to the time to, the potential E.sub.oxdn applied to the working electrode 20 is sufficient to allow oxidation of the one or more target chemical species, while the downstream working electrode 23 is disconnected. The working electrode 20 positioned upstream can then be used to continuously record the instantaneous electrochemical current, i.sub.oxdn(f), which, following any calculations indicated further above, then indicates the concentration C(f) of the one or more target chemical species in the fluid analyzed.

[0201] During a second step carried out over a range of time from the time to, the working electrode 20 is disconnected and the potential E.sub.oxdn is applied to the downstream working electrode 23.

[0202] At the time to, the flow of sweat passing above the working electrode 23 has already been electrolyzed (completely or partially) during its passage above the working electrode 20 which is located upstream, in a manner such that the concentration of the target chemical species there is zero, or at least much lower than before it entered the electrochemical sensor. The intensity i.sub.oxdn of the current detected by the working electrode 23 (graph 84) is therefore zero (or at least much lower than that of the current i.sub.oxdn detected at the working electrode 20 before the time t.sub.0).

[0203] At the time t.sub.0+?t, the working electrode 23 starts to analyze a non-electrolyzed solution and the current intensity i.sub.oxdn that it detects becomes of the same order as that detected by the working electrode 20 before the time t.sub.0. The growth in the current, schematized by a step in FIG. 12, is detected by an ad-hoc electronic circuit. The duration ?t, which is the delay between this growth and the moment t0 of disconnection of the working electrode 20, represents the time necessary for the flow of sweat to transit between the two working electrodes 20 and 23. The duration ?t is represented by a double-headed arrow at the bottom of FIG. 12. In order to simplify the representation, it has been assumed in FIG. 12 that electrolysis of the target chemical species is complete when the working electrode 20 is connected. The same measurement principles are applicable when this electrolysis is only partial.

[0204] The rate of flow v(t) and the flow rate d(t) may therefore be estimated as follows:

[00008]$v\left(t\right)=L/?t$$d\left(t\right)=A.Math.v\left(t\right)$

[0205] The potential E.sub.oxdn applied to the working electrode 23 is sufficient to enable oxidation of the one or more target chemical species, while the working electrode 20 is disconnected.

[0206] The measurement of concentration may therefore optionally be continued for a certain period with the working electrode 23. The second step ends with the disconnection of the working electrode 23 at the time t.sub.1. The working electrode 20 may then be reconnected and the method can be repeated as many times as necessary in order to evaluate the flow rate d(t) at successive times.

[0207] The distance L between the two working electrodes 20 and 23 is preferably sufficiently small, for example of the order of 1 mm, for the changes in the physiological response of the patient to be negligible over the period ?t.

[0208] A second method of measuring the volume flow rate of the sweat flow is illustrated in FIG. 13. In contrast to the explanation above, the measurement principle here is that of detecting a drop in the measurement current of a chemical compound, for example nitric oxide. FIG. 13A illustrates schematically an assembly of four electrodes disposed in a microfluidic channel 9 according to one embodiment. The electrodes are disposed successively in a longitudinal direction of the microfluidic channel 9. The electrodes are disposed as follows: a reference electrode 21 disposed upstream and receiving the flow of sweat 98 first; a first and a second working electrode 20, 23, spaced apart by a distance g represented by the double-headed arrow; and a counter-electrode 30. The measurement principle is illustrated in FIG. 13 (B) and comprises the connection and the polarization of the two working electrodes 20, 23 simultaneously, then the monitoring over time of the change in the current of the second working electrode 23 situated downstream in the microfluidic channel 9. From the start of the polarization (at t=0), the two working electrodes 20, 23 oxidize the same species. The first, upstream working electrode 20 brings about depletion of these species and, after a duration dt, induces a decrease in the current of the second working electrode 23, situated downstream. dt is the time required for the depletion zone comprising the species oxidized by the first working electrode 20 to reach the second electrode 23, by convection. It is observed, therefore, that after the simultaneous polarization of the first 20 and the second 23 working electrode at t=0, the current of the second working electrode 23, situated downstream, reduces in the duration dt. By way of example, the duration dt in FIG. 13 (B) is estimated at five seconds. Accordingly, the linear rate v of the flow is obtained by the simple relation v=g/dt.

[0209] This principle may be exploited in combination with a depletion function described with reference to FIGS. 14 to 16 below.

[0210] FIG. 14 illustrates the microfluidic circuit comprising an electrochemical sensor comprising four electrodes disposed successively in a longitudinal direction of the microfluidic channel 9. A depletion electrode 20 is placed upstream of the working electrode 23, for the depletion of the interfering species for which downstream detection is unwanted. By polarization of the depletion electrode 20, the interfering chemical species are oxidized selectively. The depletion electrode 20 is wide, so as to optimize depletion and to remove virtually all of the one or more interfering species, for example to remove hydrogen peroxide H.sub.2O.sub.2. The chemical species of interest will be oxidized by the working electrode 20 situated downstream. A configuration of this kind may be used for depletion of one or more interfering chemical species having an oxidation potential lower than the oxidation potential of nitric oxide, and for improving measurement of a concentration of a chemical compound, nitric oxide for example. The counter-electrode 30 and the reference electrode 20 are necessary for controlling the potentials and for circulating currents in the measuring zone of the electrochemical sensor.

[0211] In the microfluidic channel 9 equipped with this depletion electrode 20, a concentration of nitric oxide can be obtained directly, with no need to resolve the system of linear equations presented further above.

[0212] In a variant embodiment in which it is desired to obtain the measurement of the concentration of NO.sub.2.sup.? in the microfluidic channel 9, the depletion electrode 20 may be configured for removing nitric oxide.

[0213] FIG. 15 presents an example of depletion of hydrogen peroxide with the sensor of FIG. 14. The reference electrode and the counter-electrode are not shown. The depletion electrode 20 and the working electrode 23 are polarized independently of one another under constant sweat flow rate conditions. The direction of the flow of sweat is represented via the arrows in the microfluidic channel 9. The depletion electrode 20 is very large relative to the working electrode 23, for example eight times larger, so as to remove all of the interfering species upstream, here hydrogen peroxide (H.sub.2O.sub.2), by oxidation. The chemical species for which measurement of the concentration is desired, here nitric oxide, is not oxidized by the depletion electrode 20. The nitric oxide will be oxidized downstream by the working electrode 23. Accordingly, the depletion electrode 20 and the working electrode 23 are polarized to potentials such that E.sub.DE<E.sub.WE, so as to obtain the selectivity in the detection of nitric oxide.

[0214] As set out above, the flow rate of the flow of sweat may be measured via the working electrode 20, which performs the depletion, and the working electrode 23, which oxidizes the nitric oxide, for example. FIG. 16 illustrates measurement of the flow rate of the flow of sweat 98 according to a method similar to that described in FIG. 13A and B. Accordingly, the depletion electrode 20, which oxidizes the chemical species whose detection is not desired, for example hydrogen peroxide, and the working electrode 23, which oxidizes nitric oxide, are connected and polarized simultaneously. In a first period, therefore, the working electrode 23 will detect nitric oxide NO and also hydrogen peroxide, as the volume of sweat situated between the depletion electrode 20 and the working electrode 23 will not be depleted by the depletion electrode 20. In a second period, the depletion electrode 20 brings about depletion of hydrogen peroxide, thereby, in a manner similar to that described with FIG. 13, after a duration dt, inducing a decrease in the current of the working electrode 23. In this embodiment, the current of the working electrode 23 reduces but does not become zero or virtually zero, because following the depletion, the working electrode 23 selectively detects the nitric oxide.

[0215] The methods for flow rate measurement described above may be employed simultaneously in all of the parallel microfluidic channels. However, if these channels are configured and fed in a similar manner, a single flow rate measurement may be sufficient. In that case, the flow rate measurement method described above may be employed in a single microfluidic channel 9. Furthermore, these flow rate measurement methods can be combined with the sensors of the various examples.

[0216] With reference to FIGS. 18 and 19, electrochemical sensors are described that employ a larger number of electrodes.

[0217] The electrodes presented in FIGS. 17 to 19 may be disposed on the inner face of the upper layer closing the microfluidic channel at the top and/or disposed on an upper face of the lower layer closing the microfluidic channel at the bottom. The schematic representation does not differentiate the different layers.

[0218] FIG. 17 represents an electrode configuration according to an embodiment in which the electrochemical sensor comprises, from left to right in FIG. 17, a reference electrode 21, a depletion electrode 20, a first working electrode 23, a second working electrode 24, and a counter-electrode 30. With this configuration it is possible, in the following order, to carry out: [0219] depletion of an unwanted chemical species such as hydrogen peroxide via polarization of the depletion electrode 20 to the potential for oxidation of hydrogen peroxide; oxidation of nitric oxide via the first working electrode 23, with an oxidation potential greater than the potential for oxidation of hydrogen peroxide; [0220] oxidation of nitrites via the second working electrode 24, with an oxidation potential greater than the potential for oxidation of hydrogen peroxide and than the potential for oxidation of nitric oxide, and measurement of the flow rate of the flow of sweat by a delay between the first working electrode 23 and the second working electrode 24. The flow measurement may also be measured by a delay between the depletion electrode 20 and the first working electrode 23.

[0221] FIG. 18 illustrates an embodiment similar to the preceding figure. The embodiment differs in that the electrochemical sensor comprises a third working electrode 25, situated between the second working electrode 24 and the counter-electrode 30. The flow rate of the flow of sweat 98 is measured between the second working electrode 24 and the third working electrode 25. By spacing the electrodes 24 and 25 apart, in other words by shifting the electrodes 25 and 30 further toward the end of the channel 9, it is possible to improve the resolution of the flow rate measurement for the flow of sweat 98.

[0222] FIG. 19 represents a variant embodiment in which the detection apparatus comprises a microfluidic circuit 8 comprising two parallel microfluidic channels 9 and 109. A first microfluidic channel 9 comprises an electrochemical sensor. The assembly of the electrochemical sensors disclosed here may be integrated in this first microfluidic channel 9. The second microfluidic channel 109 comprises a colorimetric detection device 18. The colorimetric detection device 18 comprises a hydrophilic microporous membrane. The hydrophilic microporous membrane comprises at least one chemical reagent capable of reacting with a chemical species for detection, for example, of: nitrite ion, hydrogen peroxide, peroxynitrite, sulfur dioxide, hydrogen sulfide, nitric oxide, carbon monoxide and hypochlorous acid. In proportion with the accumulation of the chemical species for detection in the porous body impregnated with the reagent, the reagent changes color, and the intensity of its specific color increases. The intensity of color may be detected to provide a quantitative measure of the quantity of the chemical species dissolved in the flow of sweat. For example, the reagent used is the Griess reagent, allowing detection of the nitrite ion by providing a red-colored indicator.

[0223] According to a variant embodiment presented in FIG. 20, the colorimetric detection device 18 is placed at the outlet of the microfluidic channel 9 in series with an electrochemical sensor 10. For this, FIG. 20 shows a multilayer structure 1 identical to FIG. 4. Since the colorimetric detection device 18 is disposed downstream of the electrochemical sensor, it receives a solution which has been electrolyzed by the electrochemical sensor 10. Colorimetric measurement is possible, however, with the proviso that the species for detection has not been substantially adversely affected by the operation of the electrochemical sensor 10.

[0224] The methods for detecting concentration and flow rate described above may be carried out in an automated manner with the aid of an electronic control device 40, which is preferably integrated into the detection apparatus 100.

[0225] With reference to FIG. 21, an embodiment of the electronic control device 40 which can be integrated into the detection apparatus 100, for example in the form of an electronic circuit board, is now described.

[0226] The or each electrochemical sensor 10 is connected to an analog-to-digital converter 14, which in turn supplies a processor 15. The processor 15 is, for example, programmed to execute the methods for detecting concentration and flow rate described above.

[0227] An energy source 16, for example a battery, supplies the electronic control device 40. A communication module 17, which may be wired or wireless, may also be provided in order to communicate the results of the measurements of concentration, flow rate and/or quantitative material flow, for one or each target chemical species, to a storage or post-processing apparatus.

[0228] FIG. 22 represents a method which may be executed by the processor 15 in one embodiment.

[0229] In step 31, the instantaneous concentration Cs(t) of a chemical species S is determined from electrochemical measurements.

[0230] In step 32, the volume flow rate d(t) in the corresponding fluidic circuit is determined.

[0231] In step 33, the quantitative material flow for the chemical species under consideration is determined on the basis of Cs(t) and d(t), for example:

[00009]$P_s\left(t\right)=C_s\left(t\right).Math.d\left(t\right)$

[0232] FIG. 23 is a graph illustrating a measurement signal for the quantitative material flow as a function of time that may be obtained with the detection apparatus 100, for example during an exertion test on a subject for the species NO.

[0233] The electronic control device 40 optionally comprises other functional modules, for example a gyroscopic module and/or an accelerometer module for detecting the orientation and movements of the subject and also the level of activity of the subject, and a temperature sensor in order to measure the temperature of the subject's epidermis. It is useful to know the temperature of the skin for the purposes of correlations between the temperature and the dilation of the vessels.

[0234] Certain elements of the detection apparatus 100, in particular the electronic control device 40, may be realized in different forms, in a unitary or distributed manner, using physical and/or software components. Physical components that may be used are application-specific integrated circuits (ASICs), field programmable gate arrays (FPGAs), or microprocessors. The software components may be written in various programming languages, for example C, C++, Java or VHDL. This list is not exhaustive.

[0235] Although the invention has been described in connection with a number of particular embodiments, it is clear that it is not in any way limited to them and that it encompasses all equivalent techniques for the means described and also combinations thereof if they fall within the scope of the invention. For example, the detection apparatuses described may comprise an additional microfluidic channel or different electrochemical sensors and/or sensors comprising a different number of electrodes.

[0236] The use of the verb comprise, encompass or include and the conjugated forms thereof does not exclude the presence of elements or steps other than those set out in a claim.

[0237] In the claims, none of the reference signs in parentheses should be interpreted as limiting the claim.