REUSABLE, PORTABLE, WIRELESS BIOSENSOR

Abstract

The present invention relates to a biosensor device for the determination of analytes, which is non-invasive by means of displaying a colour change, and starting from a sample of liquid. The present invention falls within the field of devices for health, control when playing sports, occupational safety and the food industry.

Claims

1. A wireless and portable biosensor device for displaying a colour change from a first colour to a second colour, wherein said second colour indicates the presence of an analyte in a liquid sample, and the first colour indicates the absence of the analyte or a non-reading state, the device comprising a stratified structure comprising: a transparent substrate comprising a face a and a face b, optionally the substrate comprises at least two holes connecting the two faces, a and b, of the substrate; a conductive structure configured as the main structure of an antenna and at least one conductive track of the working and auxiliary electrodes, comprising conductive silver paste; at least two transparent conductive structures configured to function at least as two transducers, one for the working electrode and the other for the auxiliary electrode; at least one electrochromic structure, configured to function at least as a working electrode; a transparent structure configured to deposit the sample on it and comprising: chitosan; and an oxidase enzyme. a main dielectric structure which is configured to insulate the conductive tracks from the conductive structure; optionally a second dielectric structure located on the conductive structure and/or on the electrochromic one, leaving at least one area of said electrochromic layer exposed, and wherein said dielectric structure layer comprises at least two holes; at least one conductive track configured to connect with the conductive structure in order to close the circuit; and a diode; wherein the device functions in direct current; wherein the antenna is configured so that, with a radio frequency between 10.56 MHz and 16.56 MHz, the measurement of the device is restarted until the non-reading state or state of absence of analyte in the sample; and wherein the device is configured to be reset to the non-reading state or state of absence of analyte thereof when receiving a signal of a certain radio frequency between 10.56 MHz and 16.56 MHz.

2. The device according to claim 1, wherein the conductive structure, comprising conductive silver paste, is located directly on the face a of the substrate.

3. The device according to claim 2, wherein the transparent conductive structure is located on the conductive structure located on the face a of the substrate.

4. The device according to claim 3, the device comprising a stratified structure comprising: a transparent substrate comprising a face a and a face b, and wherein the substrate comprises at least two holes connecting the two faces, a and b, of the substrate; a first layer of conductive structure configured as the main structure of an antenna and conductive tracks of the working and auxiliary electrodes, comprising conductive silver paste, and located on at least one portion of the face a of the substrate; at least one second layer of two transparent conductive structures, located on the first layer of conductive structure, and configured to function at least as two transducers, one for the working electrode and the other for the auxiliary electrode; at least one third layer of electrochromic structure, located on at least one area of the second layer of conductive structure, and configured to function at least as a working electrode; a fourth layer of transparent structure adhered on the third layer of electrochromic structure, and configured to deposit the sample to be determined on it and comprising: chitosan; and an oxidase enzyme. a fifth layer of main dielectric structure located on the previous layers leaving at least one area exposed corresponding to the deposit area of the sample of the fourth layer, and which is configured to insulate the conductive tracks of the main structure of the first layer; a sixth layer of a conductive track comprising conductive silver paste, and located on the face b of the substrate and configured to connect, through the holes in the substrate, with the conductive structure of the first layer and to close the circuit; and a diode on the sixth layer.

5. The device according to claim 1, wherein the transparent conductive structures, configured to function as two transducers, one for the working electrode and the other for the auxiliary electrode, are located directly on the face a of the substrate.

6. The device according to claim 5, wherein the conductive structure, comprising conductive silver paste, is located on the transparent conductive structure.

7. The device according to claim 6, the device comprising a stratified structure comprising: a transparent substrate comprising a face a and a face b, and wherein the substrate comprises at least two holes connecting the two faces, a and b, of the substrate; at least one first layer of transparent conductive structures located on the face a of the substrate and configured to function as two transducers, one for the working electrode and the other for the auxiliary electrode; a second layer of conductive structure, configured as the main structure of an antenna and conductive tracks of the working and auxiliary electrodes, and comprising conductive silver paste, and in contact with at least one portion of the first layer of transparent conductive structure; at least one third layer of electrochromic structure, located on at least one surface of the second layer of conductive structure, configured to function at least as a working electrode; a fourth layer of transparent structure adhered on the third layer of electrochromic structure configured to deposit the sample on it and comprising chitosan; and an oxidase enzyme. a fifth layer of main dielectric structure located on the previous layers leaving at least one area or surface exposed or uncovered corresponding to the deposit area of the sample described in the fourth layer, and which is configured to insulate the conductive tracks of the second layer; a sixth layer of a conductive track comprising conductive silver paste, and located on the face b of the substrate and configured to connect, through the holes in the substrate, with the conductive structure of the first layer and to close the circuit; and; a diode on the sixth layer.

8. The device according to claim 6, the device comprising a stratified structure comprising: a transparent substrate comprising a face a and a face b; at least one first layer of transparent conductive structures located on one of the faces of the substrate and configured to function as two transducers, one for the working electrode and the other for the auxiliary electrode; a second layer of conductive structure, configured as the main structure of an antenna and conductive tracks of the working and auxiliary electrodes, and comprising conductive silver paste, and contact with at least one portion of the first layer of transparent conductive structures; a third layer of dielectric structure configured to insulate the conductive tracks of the second layer; at least one fourth layer of electrochromic structure, located on at least one area of the second layer of conductive structure, configured to function at least as a working electrode; a fifth layer of a second dielectric structure located on the fourth layer of electrochromic structure leaving at least one area exposed or uncovered, corresponding to the deposit area of the sample, and wherein said dielectric structure comprises at least two holes; a sixth layer of transparent structure located, in the area exposed or uncovered by the fifth layer, on the fourth layer of electrochromic structure, configured to deposit the sample on it and comprising: chitosan; and an oxidase enzyme. a seventh layer of a conductive track, configured to connect with the second layer of conductive structure through the holes in the fifth layer of dielectric structure and to close the circuit; and a diode on the seventh layer of conductive structure.

9. The device according to claim 1, wherein the diode is a Schottky diode and has dimensions between 0.8 mm and 1.6 mm.

10. The device according to claim 1, wherein the oxidase enzyme is selected from glucose oxidase, lactose oxidase, maltose oxidase, urate oxidase and ethanol oxidase.

11. The device according to claim 1, wherein the oxidase enzyme is glucose oxidase.

12. The device according to claim 11, wherein it additionally comprises mutarotase enzyme.

13. The device according to claim 1, wherein the device comprises an additional structure on the transducer of the auxiliary electrode configured to function as an auxiliary electrode.

14. The device according to claim 13, wherein the additional structure is selected from a transparent conductive structure and an electrochromic structure.

15. The device according to claim 14, wherein the additional structure is an electrochromic structure.

16. The device according to claim 1, wherein each electrochromic structure comprises: a. microparticles, selected from i. mixed indium and tin oxide; ii. of the core-shell type wherein the core is SiO.sub.2 and wherein the shell is a mixed antimony and tin oxide; and iii. any combination of the above; b. an ink comprising i. pigment, preferably Prussian Blue; ii. a binding resin, preferably a Viton solution; and wherein the microparticle:ink ratio is 2.5:1; and wherein the microparticles and the paint form a homogeneous particulate mixture with a particle size of said mixture between 3 μm and 12 μm.

17. A method for obtaining the device of claim 4, comprising the following steps; a. making holes in a transparent substrate comprising a face a and a face b using a technique selected from CO.sub.2 laser, mechanical punching and a combination of the above, such that they connect the two faces of the substrate; b. printing a conductive structure on the face a of the substrate using conductive silver paste in order to form the spiral structure of the antenna and the conductive tracks of the working and auxiliary electrodes, making at least one printed area coincide with the holes in the step (a); c. printing at least two transparent conductive structures on the printing performed in step (b) by using a transparent conductor, to function as transducers, one for the working electrode and the other for the auxiliary electrode; d. printing an electrochromic structure on a portion of the conductive structure obtained in step (c); e. printing a conductive track on the face b of the substrate, such that the ends of the tracks coincide with the areas of the holes and make contact with the ends of the antenna and with the transducer of the auxiliary electrode; f. depositing a dielectric structure on the face a of the material obtained after step (d) leaving at least one area of the electrochromic structure exposed corresponding to the deposit area of the sample; g. depositing a diode on the conductive track of the face b of the substrate obtained in step (c), between one of the ends of the antenna and one of the electrodes making up the sensor, using a material selected from silver paste and an anisotropic conductive adhesive; and h. adhering a transparent structure comprising chitosan and an oxidase enzyme in the deposit area of the sample which remained exposed after depositing the dielectric structure in step (f).

18. The method for obtaining the device according to claim 17, the method comprising additionally a step (c′) after step (c) of printing a layer on the transducer of the auxiliary electrode configured to function as an auxiliary electrode and selected from a transparent conductive structure and an electrochromic structure.

19. The method for obtaining the device of claim 7, comprising the following steps: a. making holes in a transparent substrate comprising a face a and a face b using a technique selected from CO.sub.2 laser, mechanical punching and a combination of the above, such that they connect the two faces of the substrate; b. printing on the face a of the substrate at least two transparent conductive structures by using a transparent conductor in order to function as transducers, one for the working electrode and the other for the auxiliary electrode, c. printing a second conductive structure on the printing performed in step (b) by using conductive silver paste in order to form the conductive tracks constituting the spiral structure of the antenna and the conductive tracks of the working electrodes; d. printing an electrochromic structure on the second conductive structure obtained in step (c); e. printing a conductive track on the face b of the substrate, such that the ends of the tracks coincide with the areas of the holes and make contact with the ends of the antenna; f. depositing a dielectric structure on the face a of the material obtained after step € leaving at least one area exposed corresponding to the deposit area of the sample of the electrochromic structure and g. assembling a diode on the conductive tracks of the face b of the substrate obtained in step (c), between one of the ends of the antenna and one of the electrodes making up the sensor, using a material selected from silver paste and an anisotropic conductive adhesive; and h. adhering a transparent structure comprising chitosan and an oxidase enzyme in the deposit area of the sample of the structure free from dielectric structure of step (f).

20. The method for obtaining the device according to claim 19, the method further comprising additionally a step (d′) after step (d) of printing a layer on the transducer of the auxiliary electrode selected from a transparent conductive structure and an electrochromic structure.

21. The method for obtaining the device of claim 8, comprising the following steps: a. printing on the face a of the transparent substrate a conductive structure, using conductive silver paste to form the conductive tracks constituting the structure of the antenna and the conductive tracks of the working and auxiliary electrodes; b. printing on the conductive structure obtained in step (a), at least two second transparent conductive structures by using a transparent conductor to function as transducers, one for the working electrode and the other for the auxiliary electrode; c. printing an electrochromic structure on the transparent structure obtained in step (b); d. depositing a dielectric structure on the face a of the material obtained after step (c) leaving at least one area exposed corresponding to the deposit area of the sample; e. printing at least one conductive track on the dielectric structure of step (d), such that the ends of the tracks coincide with the inner ends of the antenna and with the contact of the auxiliary electrode; f. depositing another dielectric structure, leaving the working areas of the electrodes, the contacts for the diode, and the area of the sample to be analysed; g. assembling a diode on the conductive tracks using a material selected from silver paste and an anisotropic conductive adhesive; and h. adhering a transparent structure comprising chitosan and an oxidase enzyme in the deposit area of the sample of the structure free from dielectric structure of step (f).

22. The method for obtaining the device according to claim 21, the method further comprising additionally a step (c′) after step (c) of printing a layer on the transducer of the auxiliary electrode selected from a transparent conductive structure and an electrochromic structure.

23. A patch or label comprising the device according to claim 1.

24. (canceled)

25. (canceled)

26. (canceled)

27. (canceled)

28. A system comprising: a. the portable and wireless biosensor device described in claim 1; and b. an emitter of a signal in a radio frequency between 10.56 MHz and 16.56 MHz configured to restart the measurement of the device until the non-reading state or state of absence of analyte.

29. The system according to claim 28, wherein the emitter of the radio frequency signal are selected from mobile devices with Near-Field Communication capability, Near-Field Communication readers, smart phone apparatuses, smart watches.

30. A method for the qualitative and/or quantitative detection of an analyte in a liquid sample comprising: (i) putting the liquid sample and the transparent structure of the device described in claim 1 in contact; and (ii) detection of the analyte by the colour change.

31. The method according to claim 30, comprising a step after step (ii) of restarting the device by sending a signal at a certain radio frequency between 10.56 MHz and 16.56 MHz, for the detection of another sample according to steps (i) and (ii).

Description

BRIEF DESCRIPTION OF THE FIGURES

[0184] FIG. 1 shows a diagram of the assembly of one of the embodiments and of the portions of the device of the present invention.

[0185] FIG. 2 shows a diagram of the assembly of another of the embodiments and of the portions of the device of the present invention.

[0186] FIG. 3 shows a diagram of the assembly of another of the embodiments and of the portions of the device of the present invention.

[0187] FIG. 4 shows a diagram of the biosensor device of the present invention.

[0188] FIG. 5 shows cyclic voltammograms obtained in supporting electrolyte for the different types of electrodes.

[0189] FIG. 6 shows cyclic voltammetry in supporting electrolyte and derivative voltabsorptogram for SiO.sub.2-ATO electrodes.

[0190] FIG. 7 shows cyclic voltammetry in supporting electrolyte and derivative voltabsorptogram for ITO electrodes.

[0191] FIG. 8 shows the evolution of the absorbance when alternating the potential from −0.1 to +0.4 V vs Ag/Ag.sup.+ in 60 s intervals in a 0.25 mM H.sub.2O.sub.2 solution in supporting electrolyte.

[0192] FIG. 9 shows the temporal evolution of the absorbance spectra for an ITO/PB electrode in a 0.25 mM H.sub.2O.sub.2 solution in supporting electrolyte.

[0193] FIG. 10 shows the temporal evolution of the absorbance measured at 700 nm for different concentrations of hydrogen peroxide in an ITO electrode.

[0194] FIG. 11 shows a representation of the absorbance measured at 700 nm at a fixed time of 100s for an ITO/PB electrode (triangles) and another SiO.sub.2-ATO/PB electrode (circles).

[0195] FIG. 12 shows amperometric calibration curves of the glucose biosensor obtained in an ITO/PB electrode (triangles) and another SiO.sub.2-ATO/PB electrode (circles).

[0196] FIG. 13 shows spectroscopic calibration curves of the glucose biosensor obtained in an ITO/PB electrode (triangles) and another SiO.sub.2-ATO/PB electrode (circles).

EXAMPLES

[0197] The invention, is illustrated below by means of assays carried out by the inventors which reveal the effectiveness of the product of the invention.

Example 1

[0198] Obtaining the Glucose Biosensor Device Comprising PEDOT:PSS as a Transparent Structure Located on the Substrate.

[0199] As seen in FIG. 1:

[0200] (a) First, in a transparent PET substrate with a thickness (1) between 30 μm-100 μm, a series of through holes (2) are made in certain areas, corresponding to the pathways which will serve to facilitate the electrical contact between the two faces of the substrate. The pathways are made by means of a CO.sub.2 laser.

[0201] On said substrate, the main structure of the antenna and the conductive tracks (3) for the working and auxiliary electrodes are printed by means of screen printing. In our case, conductive silver paste is used and the printing is by means of screen printing.

[0202] (b) Next, the transducers for the working (sensor) and auxiliary (4) electrodes are printed. The material of these transducers will be a transparent conductor, preferably PEDOT:PSS.

[0203] (c) Next, the electrochromic material (5) comprising SiO.sub.2-ATO or ITO is printed, at least on the central transducer and, additionally, also on the auxiliary electrode.

[0204] (d) The next step consists of printing a series of conductive tracks on the opposite face of the substrate, such that the ends of the tracks (6) coincide with the areas of pathways and make contact with the ends of the antenna and with the contact of the auxiliary electrode, which is located on the front face;

[0205] (e) having done this, the substrate is turned again;

[0206] (f) and the antenna and the conductive tracks are protected with a structure made of dielectric material (7), Loctite © EDAG-PF 455BC. This structure, in turn, defines the working areas of the electrodes and the area wherein the sample to be analysed will be confined. The reason to print the dielectric after having completed the printing of the conductive material on both faces is to prevent blocking the areas of pathways in case the first printing (a) was not perfect. In this case, a photocurable resin is used.

[0207] (g) The diode (8) is then assembled on the front face. In order to fasten it we will use a silver paste or an anisotropic conductive adhesive. In our case, we preferably use Loctite 3880 conductive silver paste for the assembly of discrete components.

[0208] In particular, a Schottky diode from the Panasonic brand, reference DB2S20500L. The dimensions thereof are 0.8 mm×1.6 mm×0.6 mm (x, y, z).

[0209] (h) Finally, a transparent structure (9) comprising chitosan and an oxidase enzyme is adhered to the deposit area of the sample and which is free of dielectric structure and defined by it. Finally, the biosensor device (10) is obtained.

[0210] Although the sample is placed on the face of the sensor, the fact of having transparent electrodes on a transparent substrate makes the colour change also be able to be read through the rear face of the device.

Example 2

[0211] Obtaining the Glucose Biosensor Device Comprising ITO as a Transparent Structure Located on the Face a of the Substrate.

[0212] As seen in FIG. 2:

[0213] (a) First, in a transparent ITO-PET substrate with a total thickness (1) between 30 μm-100 μm, a series of through holes (2) are made in certain areas, corresponding to the pathways which will serve to facilitate the electrical contact between the two faces of the substrate. The pathways are made by means of a CO.sub.2 laser.

[0214] (b) Next, an etching must be done to define the areas of the working and auxiliary electrodes, in the ITO. This etching is performed as follows: first, the ITO-PET sheet is pre-cut and a series of recording marks are made to facilitate the alignment of the subsequent printing steps. Also, some marks are made in order to align a vinyl mask, which defines the areas which must be etched. Once the areas of the electrodes are protected, the substrate is treated in a bath of diluted aqua regia (HCl—HNO.sub.3) which removes the ITO from areas where it is not needed. Subsequently, the substrate is rinsed with deionised water, and dried. The process continues.

[0215] If the ITO-coated PET sheet is protected by means of plastic, the process can be simplified as follows: By means of a cutting tool, the areas of the electrodes are profiled and recording marks are made on the substrate. Next, the protector is removed from the areas from which the ITO is to be removed, which is etched in a diluted aqua regia bath as described in the previous paragraph. After removing the ITO, the substrate is rinsed with deionised water, and dried. The remaining protector is removed, leaving the transducers of the ITO electrodes uncovered, and the process continues.

[0216] (c) On said substrate, the main structure of the antenna and the conductive tracks for the working and auxiliary electrodes (3) are printed by means of screen printing. In our case, conductive silver paste is used and the printing is by means of screen printing.

[0217] The process continues with steps (d)-(h) of example 1, until the biosensor device 10′ is obtained.

Example 3

[0218] Obtaining the Glucose Biosensor Device Comprising PEDOT:PSS as a Transparent Conductive Layer Located on the Face a of the Substrate.

[0219] As seen in FIG. 3:

[0220] (a) First, on a transparent PET substrate with a thickness (1) between 30 μm-100 μm, the main structure of the antenna and the conductive tracks for the working and auxiliary electrodes (2) are printed by means of screen printing. In our case, conductive silver paste (Loctite EDAG PM-406V1) is used and the printing is by means of screen printing.

[0221] (b) Next, the transducers for the working (sensor) and auxiliary (3) electrodes are printed. The material of these transducers will be a transparent conductor, in this case it is PEDOT:PSS.

[0222] (c) Next, the electrochromic structure comprising SiO.sub.2-ATO (5) is printed on the transducers.

[0223] (d) Subsequently, a structure made of dielectric material is deposited such that it partially protects the antenna, leaving at least the inner end thereof (11) exposed.

[0224] (e) The next step consists of printing a series of conductive tracks on the structure made of dielectric material deposited in the previous step, such that the ends of the tracks (6) coincide with the inner ends of the antenna and with the contact of the auxiliary electrode deposited in step (a);

[0225] (f) Next, a new structure made of dielectric material (12) is deposited, in our case, Loctite© EDAG-PF 455BC). This structure, in turn, defines the working areas of the electrodes, the contacts for the diode and other possible electronic or measurement components, and the area wherein the sample to be analysed will be confined. The aim of this structure is to protect the conductive tracks from degradation by the environment, as well as to prevent possible measurement errors caused by spillage of the sample beyond the area of the electrochromic and auxiliary electrodes.

[0226] (g) Finally, a diode (8) is assembled, in our case it is the reference DB2S20500L, from Panasonic.

[0227] (h) Finally, a transparent structure (9) comprising chitosan and an oxidase enzyme is adhered to the deposit area of the sample and which is free of dielectric structure and defined by it. The finished biosensor device (10″) is shown.

[0228] The sensor device of the present invention according to examples 1 to 3 is shown in a schematic view in FIG. 4, and they show a colour change in the presence of the analyte to be determined. The concentration of said analyte is calculated starting from the speed of the colour change and the intensity thereof.

Example 4

[0229] Electrochemical performance of the electrodes the electrochromic structure of which comprises SiO.sub.2-ATO or ITO and the comparison thereof with the electrodes already present in the state of the art comprising in the electrochromic structure thereof a commercial graphite paste (DropSens electrodes with reference DRP-710), or with commercial modified screen-printed carbon electrodes (screen-printed electrodes using Gwent graphite paste with reference C2070424P2).

[0230] FIG. 5 shows the cyclic voltammograms recorded in the supporting electrolyte for all the electrodes (SiO.sub.2-ATO, ITO, commercial graphite paste, commercial modified screen-printed carbon electrodes). The most striking difference in the voltammetry of the different pastes modified with Prussian Blue is the much higher current observed for the blue pastes presented herein, in comparison to those obtained from commercial materials.

[0231] The blue electrodes show superior electrochemical behaviour based on the peak-to-peak separation thereof compared to commercial graphite-based materials (Table 1)

TABLE-US-00001 TABLE 1 Data obtained from the voltammetry experiments Jp, c/ Charge density/ ΔEp/mV μA .Math. cm.sup.−2 mC .Math. cm.sup.−2 SiO.sub.2-ATO/ 60 ± 2 369 ± 1 8.9 ± 0.1 Prussian Blue ITO/Prussian Blue 31 ± 2 243 ± 1 11.0 ± 0.1  DS/Prussian Blue 82 ± 2  42 ± 1 0.6 ± 0.1 Gwent/Prussian Blue 87 ± 2  73 ± 1 1.4 ± 0.3

[0232] This is due to a better contact between Prussian Blue and the conductive material of the particles. Furthermore, the nanoparticles modified with ITO show the smallest peak-to-peak separation, ca. 30 mV to 5 mVs.sup.−1, probably due to the better conductivity offered by ITO nanoparticles compared to the SiO.sub.2-ATO microparticles. However, the much higher load of Prussian Blue than the state of the art, which arises from the available massive surface area of the nanoparticles, combined with the thickness of the layer printed on the screen, produces much wider peaks in comparison with the other materials.

Example 5

[0233] Spectroelectrochemical performance of the electrodes the electrochromic structure of which comprises SiO.sub.2-ATO or ITO and the comparison thereof with the electrodes already present in the state of the art comprising in the electrochromic structure thereof a commercial graphite paste, or with commercial modified screen-printed carbon electrodes, in the detection of H.sub.2O.sub.2, simulating the conditions of a biosensor based on the reaction of an oxidase wherein said hydrogen peroxide is produced and the optical quantification thereof.

[0234] The black colour of the graphite in the commercial electrodes modified with Prussian Blue prevents the observation of any spectroelectrochemical change.

[0235] In contrast, the cyclic voltammograms, with a sweep speed of 5 mVs.sup.−1, of the electrodes the electrochromic structure of which comprises SiO.sub.2-ATO (FIG. 6) or ITO (FIG. 7) presented herein, the colour change associated with the redox process of Prussian Blue is clearly observed. Furthermore, it is observed together with the derivative of the respective voltabsorptograms thereof with an evolution of the absorbance when alternating the potential −0.1 to +0.4 V vs Ag/Ag+ in intervals of 60 s in a 0.25 mM H.sub.2O.sub.2 solution in the supporting electrolyte. FIG. 8 shows the change in the absorbance as reducing and oxidising potentials are applied. The data shows that the electrodes the electrochromic structure of which comprises ITO show a greater contrast between the oxidised and reduced states than the electrodes the electrochromic structure of which comprises SiO.sub.2-ATO/Prussian Blue.

[0236] For the optical quantification of the hydrogen peroxide the measurement was performed as follows. First, Prussian Blue was reduced to Prussian White by means of applying a potential of −0.1 V vs. Ag/Ag+. Once a stable background colourimetric signal was observed, after approximately 60 seconds of polarisation of the electrode, the potentiostat was turned off, leaving the electrochemical cell in an open circuit. Then, the hydrogen peroxide present in the solution chemically oxidised the Prussian White back to Prussian Blue, and the corresponding colour change was monitored by means of UV-Vis reflectance (FIG. 9). The maximum colour contrast depends on the colour of the underlying conductive particles, which were white (SiO.sub.2-ATO) or pale yellow (ITO), respectively. It was found that these were 675 nm for SiO.sub.2-ATO/Prussian Blue electrodes and 700 nm for ITO/Prussian Blue. FIG. 10 shows diagrams of absorbance as a function of the time at these wavelengths, as a means of monitoring the reduction of hydrogen peroxide in the PW/Prussian Blue surface. As the data shows, the higher the concentration of H.sub.2O.sub.2, the faster the electrode fully recovers the blue colour thereof. FIG. 11 shows the relationship between the peroxide concentration and the reflectance measured at 700 nm, 100 seconds after the depolarisation of the electrode. Similar to the coulometric sensors, the sensitivity and detection limit of which can be adjusted by choosing a suitable integration time, here the performance of the sensor can be adjusted to the concentration of the sample simply by adjusting the integration time of the spectrophotometer and/or the sampling time after the depolarisation of the electrode (in our case 300 ms and 100 s, respectively). This provides control over the sensitivity, the linear range, and the detection limit of the method (see Table 2).

TABLE-US-00002 TABLE 2 Spectroscopic determination of H.sub.2O.sub.2 at different sampling times. SiO.sub.2-ATO ITO Sensitivity/ Dynamic range/ Sensitivity/ Dynamic Range/ AU .Math. mM.sup.−1 mM LD/μM AU .Math. mM.sup.−1 mM LD/μM t = 30 s 0.101 0.05-5 12 ± 10 0.206 0.025-5 8 ± 7 t = 100 s 0.318 .sup. 0.05-2.5 4 ± 3 0.591 .sup. 0.025-2.5 3 ± 2 t = 200 s 0.729 0.05-1 2 ± 1 1.084 0.025-1 2 ± 1 t = 300 s 0.958  0.025-0.5 1 ± 1 1.335 0.025-1 1 ± 1

[0237] As the table shows, the analytical parameters of the sensor can be controlled through the sampling time of the optical measurements. Increasing the sampling time enables the sensitivity of the measurement to be improved, and enables lower analyte concentrations to be detected. Moreover, the dynamic range of the sensor is narrowed, since the photodetector is saturated at lower concentrations as a direct consequence of the longer sampling time.

Example 6

[0238] The devices of examples 1 and 2 are used for the determination of glucose in sweat. The enzymatic oxidation of the glucose produces hydrogen peroxide which, in the electrodes modified with SiO.sub.2-ATO and Prussian Blue, enables the quantification of the glucose electrochemically and spectroscopically.

[0239] FIG. 12 shows the Michaelis-Menten amperometric plots for the glucose by using the two electrochromic devices manufactured as indicated in examples 1 and 2. The dynamic range of both biosensors is comprised between 0.1 mM and 1 mM of glucose, and reaches a saturation greater than 2.5 mM. The analytical performance of our blue biosensors in terms of detection limit and sensitivity, ca. 60 μM and 8.Math.10.sup.−3 A.Math.cm.sup.−2.Math.M.sup.−1 (see Table 3) is comparable to that of other reported amperometric biosensors.

TABLE-US-00003 TABLE 3 Amperometric and spectroscopic determination of the glucose for the two types of electrode Amperometry Spectroscopy Sensitivity/ Sensitivity/ LD/μM A .Math. cm.sup.−2 .Math. M.sup.−1 LD/μM AU .Math. mM.sup.−1 SiO.sub.2-ATO/ 16 ± 4 8.8 .Math. 10.sup.−3 9 ± 1 0.21 Prussian Blue ITO/Prussian Blue 34 ± 6 7.4 .Math. 10.sup.−3 6 ± 1 0.19

[0240] FIG. 13 shows the spectroscopic calibration plots of the glucose biosensor for the two different devices. As seen, no substantial differences are observed when the amperometric or spectroscopic detection method is used, since a similar dynamic range is reached in all cases, reaching a plateau in both cases at concentrations above 2.5 mM. A slight gain in the detection limit is achieved since it decreases to approximately 15 μM probably due to the effect of the high background currents present in amperometric detection which disappear when a spectroscopic detection method is used, but, ultimately, the yields of both types of electrochromic electrodes are comparable, regardless of the device used.