MXPA99010459A - Improved fluorescence sensing device - Google Patents

Abstract

A fluorescence sensing device for determining the presence or concentration of an analyte in a liquid or gaseous medium is constructed of a light-emitting diode having a hole (29) generally perpendicular to the P-N junction, such that light is emitted from said junction into said hole (29). The hole is filled with a fluorescent matrix (32) which is permeable to analyte and which contains fluorescent indicator molecules whose fluorescence is attenuated or enhanced by the presence of analyte. A photodetector (23) is positioned at one end of the hole, such that fluorescent light received from the fluorescent indicator molecules is converted to an electrical signal that may be correlated to the presence or concentration of analyte in a gaseous or liquid medium in contact with the fluorescent matrix.

Description

IMPROVED FLUORESCENCE DETECTION DEVICE BACKGROUND OF THE INVENTION 1. FIELD OF THE INVENTION This invention relates to an electro-optical detection device for detecting the presence or concentration of an analyte in a liquid or gaseous medium. In particular, the invention relates to a fluorescence detection device characterized by an extraordinarily compact size, fast response times and high signal-to-noise ratios. 2. TECHNICAL BACKGROUND U.S. Patent No. 5,517,313, the disclosure of which is incorporated herein by reference, discloses a fluorescence detection device comprising a layer arrangement of a material containing fluorescence indicator molecules, a high pass filter and a photodetector. In this device a light source, preferably a light emitting diode ("LED"), is located, at least partially within the indicator material, such that incident light from the light source causes the indicator molecules to fluoresce . The high-pass filter allows the emitted light to reach the photodetector, while filtering the diffuse incident light from the light source. The fluorescence of the indicator molecules employed in the device described in US Pat. No. 5,517,313 is modulated, i.e. attenuated or increased by the local presence of an analyte. For example, the orange-red fluorescence of the complex, tris (4,7-diphenyl-1, 10-phenanthroline) ruthenium (II) perchlorate is extinguished by the local presence of oxygen. Therefore, this complex can be usefully used as the indicator molecule of an oxygen sensor. Similarly, other reporter molecules are known whose fluorescence is affected by specific analytes. In the sensor described in U.S. Patent No. 5,517,313, the material containing the reporter molecule is permeable to the analyte. In this way, the analyte can be dispersed within the material of the surrounding test medium, thus affecting the fluorescence emitted by the indicator molecules. The light source, the material containing indicator molecules, the high-pass filter and the photodetector are configured in such a way that the fluorescence emitted by the indicator molecules impacts the photodetector, generating an electrical signal indicating a concentration of the analyte in the medium surrounding. Although the detection device described in U.S. Patent No. 5,517,313 represents a significant improvement over the prior art devices, there is still a need for even more compact, less expensive sensors and having detection characteristics superior to those described. in that invention. Thus, it is an object of the present invention to provide an improvement to the detection device described in the aforementioned patent.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be illustrated with reference to the accompanying drawings in which: Figure 1 is a perspective view of a conventional light emitting diode. Figure 2 is a perspective view illustrating a detection device in accordance with the present invention. Figure 3 is a cross-sectional view of the detection device of Figure 2, taken along lines 3-3 of Figure 2; Figure 4 is a cross-sectional view of an alternative embodiment of a device; detection in accordance with the present invention. Figure 4a is a perspective view with the separate parts of the detection device of Figure 4. Figure 5 is a cross-sectional view of another alternative embodiment of a detection device in accordance with the present invention.

Figure 6 is a cross-sectional view of another alternative embodiment of a detection device in accordance with the present invention. Figure 7 is a top plan view of the device of detection of Figure 6. Figures 8 and 9 illustrate a photodetector for the detection device in accordance with an alternative embodiment of the present invention. Figure 10 is a cross-sectional view of another alternative embodiment of a detection device in accordance with the present invention. Figure 11 is a cross-sectional view of another alternative embodiment of a detection device in accordance with the present invention. Figure 12 illustrates a multiple detection modality for simultaneously determining the presence or concentration of a plurality of analytes in a gaseous or liquid surrounding medium.

BRIEF DESCRIPTION OF THE INVENTION In accordance with the present invention, a fluorescence detection device for determining the presence or concentration of an analyte in a liquid or gaseous medium comprises: a) A light emitting PN junction (hereinafter referred to as a light emitting diode) ("LED")), said LED has a hole in a direction generally perpendicular to the plane of the PN junction, said hole configured in such a way that, when applying an electrical potential along the junction, it emits light from said junction towards said hole. b) An analyte-permeable fluorescent matrix contained within at least a portion of said orifice, said fluorescent matrix contains fluorescence indicator molecules whose fluorescence is attenuated or increased by the presence of an analyte in said fluorescent matrix; said LED and fluorescent indicator molecules have been chosen in such a way that the wavelength emitted by the LED excites the fluorescence in the indicator molecules. c) A photodetector at one end of said orifice generates an electrical signal response to the fluorescent light emitted by said fluorescent indicator molecules.

DETAILED DESCRIPTION OF THE INVENTION In Figure 1 a conventional LED is illustrated. The LED 10 consists of a layer of a semiconductor type N and a layer of semiconductor type P, which at the interface P-N 12 form a light emitting junction. When an electric potential is applied along the junction P-N 12, for example via electrical terminals 14 and 16, light rays 11 are emitted from the junction in approximately the same plane as the junction. As illustrated in U.S. Patent No. 5,517,313, this edge emission characteristic of LED has been usefully utilized to direct light transversely through a layer of the fluorescent matrix in an electro-optical sensor. LEDs are conventionally made by first preparing a two-layer semiconductor crystalline wafer using known infusion doping techniques and subsequently cutting or cutting the resulting wafer into fragments of suitable size. LEDs are typically small, with measurements of the order of 200 to 300 microns at one edge. In accordance with the present invention, it has been surprisingly discovered that a hole or cavity can be cut into a LED fragment without destroying or substantially damaging the functionality of the light emitting P-N junction. In this way, to the application of an electrical potential along the junction light is emitted from the junction within the hole or cavity, in Fig. 2 a partially cut away perspective view of a device of the present invention is shown. The sensor 20 includes an LED 22 having input terminals 24 and 26 for applying an electrical potential along the PN junction 28. The LED has a hole 29 cut through it in an orientation generally perpendicular to the PN 28 connection. As illustrated in Figure 3, a polymer matrix 32 containing fluorescent indicator molecules whose fluorescence is attenuated or increased by the presence of an analyte is placed in the orifice 29. The fluorescent matrix is permeable to the analyte, such that the analyte present in a gaseous or liquid medium exposed to the open end of the orifice 29 may be dispersed in and out of the fluorescent matrix 32. The orifice 29 may be filled to such a level that the light rays are emitted within the fluorescent matrix 32. For example, the hole 29 can be completely filled (i.e., in parallel with the upper surface of the LED). The hole 29 can also be filled to a level sufficient to cover the PN junction 28. In one embodiment of the present invention, a photodetector 23 can be placed at one end of the LED 22 with a photosensitive area 25 adjacent the hole 29. The photodetector can be a photoelectric device in conventional solid state as a result of the interface of two semiconductors. In a preferred embodiment, the light sensitive area 25 corresponds to the area adjacent to the orifice 29. This light sensitive area can be produced by conventional photocoating techniques already known in the art. The electrical signal generated by the photodetector 23 is transmitted through electrical terminals 33 and 31 to suitable measurement and amplification circuitry (not shown). The operation of the sensor 20 is illustrated in the cross-sectional view of FIG. 3. Upon the application of an electrical potential along the PN junction 28 via terminals 24 and 26, the light rays 34 are emitted into the fluorescent matrix 32 which is contained within the orifice 29. When the light rays impact a fluorescent indicator molecule 36, the molecule emits fluorescence with an intensity that depends on the concentration of the analyte in the fluorescent matrix 32. A portion of the fluorescent light is it directs downwards towards the photodetector 23 and impacts the light sensitive area 25. The photodetector 23 and the light sensitive area 25 generate an electrical signal that is transmitted through the terminals 33 and 31. As illustrated in figure 3 , the light emitted from the PN junction 28 in the fluorescent matrix 32 is effectively trapped within the device through an internal reflectance, thereby improving the overall efficiency of the device. For example, the light beam 37 that is not absorbed in a first passage through the fluorescent matrix 32 can be reflected from the wall of the orifice 29 towards the fluorescent matrix where it has another opportunity to impact a fluorescent indicator molecule. The efficiency of the device can also be improved by covering the walls of the LED 22 with a non-conductive, reflector material 39. For example, a latex material can be used to cover the walls of the LED 22. In this way, the light that would otherwise be Transmitted outside the device is reflected back through the walls of the LED 22 towards the fluorescence matrix 32. The orifice 29 may be formed inside the LED 22 by any convenient technique. It has been found that the hole can be machined into the LED 22 by an excimer laser, preferably one that emits light at a wavelength of about 248 nanometers. The excimer laser can also use a wavelength of approximately 193 nanometers, with a lower efficiency. The X, Y coordinates of the laser beam are controlled by an aperture, and the depth of the hole 29 is controlled by the number of pulses. The dimensions of the hole 29 may vary depending on the applications to which the sensor 20 will be placed. The hole 29 can pass completely through the LED 22. Alternatively, a wall or layer of semiconductor material can remain at the end of the hole adjacent to the photodetector, since it is sufficiently transparent to the light emitted by the fluorescent indicator molecules 36. A shallow hole may be adequate as long as the hole passes through the PN junction. The hole 29 can have any shape that is desired, and conveniently has a cylindrical shape. The diameter of the orifice 29 usefully ranges from about 10 to 300 microns, preferably from about 20 to about 200 microns, and most preferably from about 100 to about 150 microns. The analyte-permeable fluorescent matrix 32 is preferably a polymer matrix having fluorescent indicator molecules dispersed therein. Advantageously, the polymer is one that can be cast into orifice 29, deposited there by evaporation or polymerized from monomers or oligomers in situ. The polymer used in the matrix must be optically transmissible at the wavelength of excitation and emission of the indicator molecules. A variety of polymers can be used for the preparation of the fluorescent matrix 32. The polymer system found useful for preparing an oxygen sensor employs silicone polymer RTV118, available from General Electric Company, Pittsfield, MA, USA. This polymer can be dissolved in a petroleum ether / chloroform mixture of 1: 1 to 1: 6, the ruthenium indicator fluorescence indicator referred to above can be mixed in the polymer solution and at a concentration of about 0.1 to about 1 mM , and the resulting mixture can be placed in the orifice 29. Evaporation of the solvents results in the deposition of a fluorescent matrix 32 within the orifice 29. In one embodiment of the present invention, the electrical terminal can be attached to the upper part. of the semiconductor material that forms the LED 22 and the electrical terminal 24 is connected to the bottom of the LED 22, as illustrated in Figures 2-4. As clearly illustrated in Figure 4a, the electrical terminals 31 and 33 are preferably attached to the upper and lower part of the photodetector 23 respectively. In a preferred embodiment the electrical terminals 24 and 31 can be embedded in an epoxy material that connect the LED 22 to the photodetector 23. In an alternative embodiment, the lower surface of the LED 22 is in electrical contact with the upper surface of the photodetector 23 so that a common electrical contact (not shown) can be used. To facilitate this electrical contact, an electrically conductive adhesive may be used to join the photodetector 23 and the LED 22. Due to the physical configuration of the sensor 20, very little incident light emitted from the junction 28 reaches the photodetector 23. However, small amounts of said light can reach the photodetector through internal reflectance. In addition, the ambient light passing through the hole 29 can reach the photodetector 23. As illustrated in FIGS. 4, an optical cut filter 41 can be interposed between the fluorescent matrix 32 and the photodetector 23. The filter 41 is designed to transmitting fluorescent light emitted from the fluorescent indicator molecules 36 while filtering the incident light emitted by the LED 22, as well as the important portions of ambient light that could otherwise reach the photodetector 23. The photodetector 23, filter 41 and LED 22 can be physically joined by an adhesive. Figure 4a illustrates a perspective view with the separate parts of the sensor of Figure 4. In one embodiment, the optical filter 41 is applied as a reverberation on the photodetector 23. The suitable optical filter coating can be obtained from Optical Coating Laboratory , Inc., Santa Rosa, California, USA, and applied by conventional methods. See U.S. Patent No. 5,200,855. In another embodiment, the optical filter can be a colored epoxy material that can be used to embed the electrical terminal 24 that is connected to the LED 22. For example, a colored epoxy material can be obtained from CVl Laser, Corp., Albuquerque, New Mexico. In yet another embodiment, an optical filter 42 can be placed in the hole 29 between the photodetector 23 and the fluorescent matrix 32, as illustrated in FIG. 5. A suitable optical filter can be an epoxy type filter such as is available from CVl Laser, Corp., Albuquerque, New Mexico.

Figures 6 and 7 illustrate an electro-optical detection device 60 in accordance with another embodiment of the present invention. The sensor 60 includes an LED 62 which is supported by a substrate 64. The LED 62 is preferably formed by depositing a first semiconductor layer 66 (such as a GaN type n material) on the top of the substrate 64 and subsequently depositing a second semiconductor layer 65. (as a GaN type p material) in the upper part of the first semiconductor layer. The PN interface of the semiconductor layers 66 and 65 forms a light emitting junction 68. The LED semiconductor layers 62 vary in thickness from about 2 to 30 microns, preferably from about 5 microns to 20 microns, and most preferably up close from 8 to 12 microns. The LED 62 has input terminals 24 and 26 for applying an electrical potential along the PN junction 68. As shown in FIG. 6, the input terminal 26 is connected to the surface of the anode 65 and the input terminal 24 is connected to the cathode surface 66. In a preferred embodiment, the input terminal 24 is connected to the cathode surface 66 of the LED 62 to a low portion of the cathode surface 66, as illustrated in Figure 6. The input terminal 24 can also be connected to the cathode surface 66 as described above in connection with FIG. 2. Also in accordance with a preferred embodiment, the input terminal 26 is joined to the anode surface 65 of the LED 62 by a connection pad 63 made of a material with high electrical conductivity. The connecting pad 63 is preferably made of gold but can be made of other materials with high electrical conductivity known to those skilled in the art. The input terminal 26 can be attached to the connection pad 63 by any suitable method including, for example, a ball joint or a wedge joint. A hole 69 is formed in the LED 62 in an orientation generally perpendicular to a plane containing the joint P-N 68. As described above in connection with Figures 2-4, the polymer matrix 32 is placed in the orifice 69 which contains the fluorescent indicator molecules whose fluorescence is attenuated or increased by the presence of an analyte. Because LED 62 has an extremely small thickness, as described above, hole 69 is preferably created by covering a portion of LED 62 and etching an orifice 69 using techniques known to those skilled in the art. The coverage and etching technique is preferably used to create a hole 69 in accordance with this embodiment and represents a substantial advantage over the laser ablation technique as described above. The substrate 64 can be made of any suitable material that is optically transmissible substantially at the emission wavelength of the indicator molecules. Preferably, the substrate 64 can be a material that allows the deposition or fabrication of the LED material on its surface. In a preferred embodiment, the substrate 64 is made of a non-conductive material, SIC material. The LED 62 and the substrate 64 can be physically joined by any convenient technique, such as fabrication or deposition. Also in accordance with this embodiment, a photodetector 72 is located on a lower portion of substrate 64 with a photosensitive area below the hole 69, as illustrated in Figure 6. The photodetector 72 may be a photoelectric device in the solid state. resulting from the Interface of two semiconductors. In one embodiment, an N-type 73 semiconductor region and a P-type semiconductor region 74 are formed in the substrate 64, as illustrated in Figure 6. The semiconductor regions 73 and 74 may be formed by techniques known to the skilled artisan. in the technique. For example, the semiconductor region 73 can be created by covering a portion of the substrate 64 and by infusing an uncovered region of the substrate 64, as illustrated in Figure 8. The semiconductor region 74 can be created by covering portions of the substrate 64 and the region. semiconductor 73, and doping by infusion a region not covered by the semiconductor material 73, as illustrated in Figure 9. The electrical signal generated by the photodetector 72 is transmitted via electrical terminals 70 and 71 to the appropriate application and measurement circuits (not shown). An optical cut filter can be interposed between the fluorescent matrix 32 and the photodetector 72. In a preferred embodiment, a filter 75 can be placed in the hole 69 between the fluorescent matrix 32 and the substrate 64, as illustrated in FIG. 10. The filter 75, like the filter 41, is designed to transmit fluorescent light emitted by the fluorescent indicator molecules 36 while filtering the incident light emitted by the LED 62, as well as important portions of ambient light that would otherwise arrive at the photodetector 23. A filter that is preferred is a thin film, deposited electron beam filter of Si? 2 / TiO 2 dichroic as those available from Optical Coating laboratories, Inc., Santa Rosa, California US and are described, for example, in U.S. Patent No. 5,200,855, incorporated herein by reference. Of course, suitable filters having other typical formulations can also be used. As described above together with Figures 6 and 10, the sensor 60 may preferably be of a single monolithic structure having a region of LED and a detection region. In a preferred embodiment the sensor 60 may also have a filter region. According to yet another embodiment of the present invention, a conventional photodetector (such as the photodetector 23 described above) can be placed at one end of the substrate 64 with a photosensitive area 25 below the hole 69, as illustrated in FIG. 11. photodetector 23 can be connected to substrate 64 by a suitable optically transmissible adhesive. The electrical signal generated by the photodetector 23 is transmitted through electrical terminals 31 and 33 to the appropriate amplification and measurement circuits (not shown), as described above. In accordance with this embodiment, the sensor 60 may also have an optical cut filter as described above. As illustrated in Figure 11, the optical cut filter 75 may be interposed in the hole 69 between the fluorescent matrix 32 and the substrate 64. In an alternative embodiment, an optical cut filter may be interposed between the substrate 64 and the photodetector 23, as described above together with Figures 2 through 4. Although both electrical terminals are shown in Figure 11. 31 and 33 extending from the upper and lower regions of the photodetector 23, may extend from the bottom of the photodetector 23. A suitable lower fixation photodetector or "reversible eyelet" as described herein may be obtained, for example, from Advanced Photonics, Camarillo, California. The lower fixing photodetector can also be used with the sensor described above together with Figures 2-5. The sensors for the present invention are characterized in that they have an extremely small size. For example, the general dimensions of the sensor are of the order of 200 to 300 microns in an edge. These sensors can also have overall dimensions as large as approximately 500 microns and as small as 50 microns at the edge. In this way, the sensors can be used in micro applications. For example, the sensors are small enough to be placed under the skin or inside a blood vessel. Although the sensors have been illustrated together with the detection of oxygen concentrations, the analyte-detecting molecules such as glucose, certain hormones, enzymes and the like can be chosen.

The small volume of the fluorescent matrix material and the small photosensitive area of photodetectors 72 and 23 produce devices that have a very low dark current. In this way, the signal-to-noise ratio in the devices of this invention is very good. In view of the extremely small size of the sensors according to the present invention, multiple sensors can be used to simultaneously determine the presence or concentration of a plurality of analytes in a gaseous or liquid surrounding medium. In one embodiment, as illustrated in FIG. 12, a sensor 80 comprises a region of LED that can include a plurality of LEDs 62 and a detection region that can include a plurality of photodetectors 53. LEDs 62 can be formed on substrates 50. by any suitable conventional technique such as, for example, fabrication or deposition. The substrate 50 can preferably be made of a non-conductive material, SiC; however, other suitable substrate materials can also be used as are known to those skilled in the art. Each of the LEDs 62 illustrated in Figure 12 can have essentially the same structure as described above together with Figures 6, 7 or 10. Preferably, each LED contains a fluorescent matrix 32 which includes a fluorescent indicator molecule 36 whose Fluorescence is attenuated or increased by a different analyte. According to this embodiment, the photodetectors 53 can be formed on one side of the substrate 50, as illustrated in Figure 12. The photodetectors 53 preferably include a separate photosensitive area for each LED located on the substrate 50. Each photosensitive area is located such that it receives the fluorescent light emitted by the fluorescent indicator molecules 36 in the orifices 69. In one embodiment, the photodetectors can be formed by coating and doping the substrate 50 by creating separate P-type and N-type semiconductor regions, as described previously together with FIGS. 8 and 9. The electrical signals generated by the photodetectors 53 are transmitted via electrical terminals 70 and 71 to the appropriate amplification and measurement circuits (not shown). As described above in conjunction with Figure 12, the sensor 80 may preferably be a single monolithic structure having a region of LED and a detection region. In a preferred embodiment, the sensor 80 may also have a filter region. The fluorescent sensors of the present invention have been described along with certain preferred embodiments. Those skilled in the art will recognize that modifications and improvements may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (21)

NOVELTY OF THE INVENTION CLAIMS

1. - A fluorescence detection device for determining the presence or concentration of an analyte in a liquid or gaseous medium comprising: a) a light emitting diode ("LED") having a semiconductor junction, said LED having a generally oriented orifice perpendicular to the semiconductor junction configured in such a way that upon application of an electrical potential along the junction, light is emitted from said junction towards said hole; b) a fluorescent matrix permeable to the analyte contained within said orifice, said fluorescent matrix contains fluorescent indicator molecules whose fluorescence is attenuated or increased by the presence of an analyte in said fluorescent matrix, said LED and fluorescent indicator molecule chosen in such a way that the wavelength that emits the LED excites the fluorescence in the indicator molecules; and c) a photodetector at one end of said orifice that generates an electrical signal in response to the fluorescent light emitted by said fluorescent indicator molecules.

2. The fluorescence detection device according to claim 1, further characterized in that the fluorescent matrix comprises fluorescent indicator molecules dispersed in a polymer that transmits light at wavelengths of excitation and emission of the fluorescent indicator molecules.

3. The fluorescence detection device according to claim 1, further characterized in that the longest edge of the LED is less than about 500 microns.

4. The fluorescence detection device according to claim 3, further characterized in that the hole in the LED has a diameter of about 10 to about 500 microns.

5. The fluorescence detection device according to claim 1, further characterized in that it comprises a reflector coating on the external walls of the LED.

6. The fluorescence detection device according to claim 1 or 5, further characterized in that it comprises an optical cut filter interposed between the fluorescence matrix and the photodetector, said optical filter being capable of transmitting light at the wavelength They emit the fluorescent indicator molecules and absorb or block light at a wavelength that emits the LED.

7. The fluorescence detection device according to claim 6, further characterized in that said optical cut filter is covered on said photodetector.

8. The fluorescence detection device according to claim 6, further characterized in that said optical cut filter is placed in said orifice between said fluorescent matrix and said photodetector.

9. The fluorescence detection device according to claim 1 or 5, further characterized in that the indicator molecule is the complex, tris (4,7-diphenyl-1, 10-phenanthroline) ruthenium (II) perchlorate, the Fluorescence detection device is an oxygen detection device.

10. The fluorescence detection device according to claim 1, further characterized in that said LED has a thickness of approximately 10 to 20 microns.

11. The fluorescence detection device according to claim 1, further characterized in that said sensor comprises a plurality of LEDs where each has an orifice containing said fluorescent matrix permeable to the analyte.

12. The fluorescence detection device according to claim 11, further characterized in that said analyte-permeable fluorescent matrix contained in each of said LEDs contains fluorescent indicator molecules whose fluorescence is attenuated or increased by the presence of a different analyte.

13. The fluorescence detection device according to claim 1, further characterized in that said fluorescence detection device is a single monolithic structure.

14. - The fluorescence detection device according to claim 13, further characterized in that said single monolithic structure comprises a region of LED and a detection region.

15. The fluorescence detection device according to claim 14, further characterized in that said single monolithic structure further comprises a filter region.

16. The fluorescence detection device according to claim 14, further characterized in that said LED region has a thickness of approximately 10 to 20 microns.

17. The fluorescence detection device according to claim 1, further characterized in that the longest edge of the LED is less than about 300 microns.

18. A method for determining the presence or concentration of an analyte in a liquid or gaseous medium comprising: a) forming a light emitting diode ("LED") having a semiconductor junction on a substrate; b) forming a hole in said LED oriented generally perpendicular to the junction of the semiconductor configured in such a way that upon application of an electrical potential along the junction, light is emitted from said junction within said hole; c) placing an analyte-permeable fluorescent matrix within said orifice, said fluorescent matrix contains fluorescent indicator molecules whose fluorescence is attenuated or increased by the presence of an analyte in said fluorescent matrix, said LED and fluorescent indicator molecule being chosen in such a way that the wavelength that emits the LED excites the fluorescence in the indicator molecules; and d) forming a photodetector at one end of said substrate that generates an electrical signal in response to the fluorescent light emitted by said fluorescent indicator molecules.

19. The method for determining the presence or concentration of an analyte in a liquid or gaseous medium according to claim 18, further characterized in that it comprises the manufacture of said fluorescence detection device as a single monolithic structure.

20. The method for determining the presence or concentration of an analyte in a liquid or gaseous medium according to claim 18, further characterized in that the step of forming the hole in said LED comprises recording a hole in said LED.

21. The method for determining the presence or concentration of an analyte in a liquid or gaseous medium according to claim 18, further characterized in that the step of forming an LED in the substrate comprises the formation of an LED on a substrate made of SiC.