DOUBLE FUNCTIONAL ASSAY DEVICE This application claims the benefit of US Provisional Application No. 60 / 127,442, filed on April 1, 1999 and entitled "Glucose Assay.• 5 Method and Device, "the entirety of which is incorporated herein by reference. BACKGROUND OF THE INVENTION Field of the Invention This invention relates to an apparatus and method 10 for detecting substances, including glucose, in a fluid that is collected from tissue Art Discussion Glucose is an important substance for biological activities, for example, in individuals who can15 being affected by diabetes, there is a need to detect or measure the presence and amount of glucose as part of a daily routine. However, currently available measurement techniques frequently involve invasive testing. A glucose test method includes the20 test test based on blood. The "finger prick" blood test technique is currently a widely accepted methodology for glucose test results in the United States. Of course, this invasive procedure requires the extraction of blood to25 perform the test. This is very uncomfortable for patients,*Fespecially for young patients. In addition, this procedure demands a lot of time. Accordingly, it is desirable to provide non-invasive or minimally invasive techniques for measuring substances, such as glucose concentration, of fluids, such as blood and interstitial fluid. BRIEF DESCRIPTION OF THE INVENTION Briefly, the present invention is directed to a system and method for detecting substances, such as glucose,10 in a fluid that is collected from a tissue. In one aspect, the system according to the present invention has a testing device and an optical apparatus. The assay device is suitable for tissue fixation, wherein the assay device is a dual function device that15 includes a reactive region that is sensitive to at least one substance in the fluid that is collected from the tissue, when the fluid is in contact with the reactive region and such a reactive region is also sensitive to a• first type of optical energy suitable for heating andTransferring heat by conduction to the tissue to remove tissue and form at least one opening in the tissue, from which the fluid is collected. The optical device has an activation head to which the test device is attached and a first source of optical energy that supplies25 the first type of optical energy. A detection device?TO? Optical apparatus is included in the optical apparatus to measure a characteristic of at least one substance from the response of the reactive region, when the reactive region is in contact with at least one substance in the fluid. In another aspect, the present invention provides a method for detecting a substance, such as glucose, in a fluid from a tissue. The method includes the stages of placing a test on an activation head of an optical instrument, where the test is sensitive to at least one substance, placing the activation head in position to the tissue surface in such a way that the assay is in contact with the surface of the tissue, forming at least one opening beneath the assay through the surface of the tissue, thereby allowing the fluid from the tissue to flow through at least one opening and contact the assay, and detect the assay response to the fluid to measure the presence of at least one substance in the fluid. The method can be practiced by using the system according to a preferred embodiment of the present invention. Still in accordance with another aspect of the present invention, there is provided a testing device that includes a base having a first side and a second side and a reactive region disposed or deposited on the first side of the base. The reactive region comprises a material of- .. A, .itoifefa¿tt photosensitization that is placed in contact with the surface of the tissue and is sensitive to a suitable electromagnetic energy emitted thereon, to heat and conductively transfer heat to the surface 5 of the tissue to form at least an opening, to thereby allow the fluid from the tissue, through at least one opening, to contact the assay. On the other hand, the photosensitization material is also sensitive to at least one substance in the fluid,10 from which a characteristic of at least one is detected• a substance, based on the electromagnetic energy dispersed and / or reflected from it. The advantages and additional features of the invention will be set forth in part in the description that follows, and in part will be obvious from the description or can be learned by practicing the invention. The advantages of the invention will be realized and obtained by means of the elements and combinations particularly• indicated in the appended claims. It is going to understand20 that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. These and other features and advantages of the preferred forms of the present invention are described in25 present with reference to the drawing figures.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram of a system for detecting at least one substance in a fluid that is collected from a tissue, according to the present invention. FIG. 2 shows a first side of a testing device in connection with the system shown in Fig. 1 according to the present invention. FIG. 3 shows a second side of a testing device in connection with the system shown in Fig. 1 according to the present invention. FIG. 4 shows a cross-sectional side view of a testing device in connection with the system shown in Fig. 1 according to the present invention. FIG. 5 is a flowchart that generally represents the total process employing the method according to the present invention. FIG. 6 shows a partial view, in cross section, of the testing device and the activation head of the optical apparatus shown in Fig. 1 in operation. FIG. 7 shows a cross-sectional background view of a first embodiment of an activation head of the optical apparatus shown in Fig. 1 according to the present invention.
FIG. 8 shows a cross-sectional background view of a second embodiment of an activation head of the optical apparatus shown in Fig. 1 according to the present invention. DETAILED DESCRIPTION OF THE DRAWINGS Definitions As used herein, the term "biological fluid" or "fluid" at least includes "interstitial fluid" (ISF), which is the clear fluid that occupies the space between the cells in the body, or blood. As used herein, the term "tissue." it means an aggregate of cells of a particular kind,• together with its intercellular substance, which forms a structural material of an animal or plant. At least one surface of the fabric must be accessible to electromagnetic radiation, in such a way that the invention can be carried out. The preferred tissue is the skin. Other tissues suitable for use with this invention include mucosal tissue and soft organs. As used herein, the term "glucose" means a monosaccharide also known as D-glucose, D-glucopyranose, grape sugar, corn sugar, dextrose and cerelosa. Glucose occurs in the fluids of the animal body, for example, in the blood, lymph or interstitial fluid. Glucose enters the bloodstreamby absorption from the small intestine. This is carried out via the portal vein to the liver, where part is stored as glycogen, the rest that re-enters the circulatory system. Another glycogen storage site is muscle tissue. As used herein, "analyte", "substance" or any similar term, means a component that is being detected or measured in an analysis. In particular, the analyte can be any material or chemical or biological compound suitable for the stage through a biological membrane technology known in the art, from which an individual may wish to know the concentration or activity within the body. Glucose is a specific example of an analyte, because it is a sugar suitable for the stage through the skin. Individuals, for example those who have diabetes, may wish to know their blood glucose levels. Other examples of analytes include, but are not limited to, such compounds as sodium, potassium, bilirubin, urea, ammonia, calcium, lead, iron, lithium, salicylates, pharmaceutical compounds, and the like. As used herein, "poration", "microporation" or any similar term means the formation of a small hole or pore or opening at a desired depth in or through the biological membrane,";;; * * -0W-. I,? AM * tiL? Ét ** M? * K. S $ such as skin or membrane, or the outer layer of an organism that decreases barrier properties from this biological membrane to the passage of biological fluids, such as analytes, from below the surface for analysis .. Preferably, the hole or micropore will not be larger than about 1 mm (1000 μm) in diameter and will extend to a depth selected, as hereinafter described, as used herein, "micropore" orÍC "pore" means an opening formed by the microporation method. As used herein, the term "reactive", "active component" or any other similar term means any chemical material or compound15 suitable for use by methods previously known in the art and / or by the methods taught in the present invention, which induces a desired effect, such as a biological or optical effect, or other observable effect, which may include, but is not limited to, (1) producing a detectable change in this measurable response of the compound or formulation to the application of energy to this area which may be electromagnetic, mechanical, thermal, optical or acoustic when in contact with at least one substance in a fluid that is collected from a tissue, (2) producing a response when it is in contact with at least one substance in a fluid that is collected from a tissue, to allow a characteristic of at least one substance to be measured or detected of the response and / or (3) be sensitive to a type of electromagnetic energy emitted on it when it is in contact with a tissue, to heat and transfer heat mediant and conduction to the tissue to remove the tissue and form at least one opening in the tissue from which a fluid can be collected. As used herein, the term "material of10 photosensitization "means a material that contains• less an active reagent or component, which is at least sensitive to at least one substance in a fluid and to a type of electromagnetic energy emitted thereon when in contact with a tissue, to heat and15 transfer heat by conduction to the tissue to remove the tissue. The present invention is directed to a system and method for detecting at least one substance in a fluid that is collected from a tissue. For example, the system andThe method is described in connection with an application for detecting glucose in interstitial fluid or blood collected from a human being. Obviously, the system and method according to the present invention can be used to detect another substance (s) in any fluid25 biological.
Specifically, Fig. 1 shows a system 100 using a disposable testing device 20 in combination with an optical apparatus 50 for detecting a substance, such as glucose, in a fluid that is collected from a tissue 40. The optical apparatus 50 includes a box 52 that is about the size of a human hand. A first power source 54, a second power source 56 and a detection instrument 58 are placed inside the box 52. The first power source 54, the second10 power source 56 and the detection instrument 58 are• coupled to an activation head 70 via the optical fiber (s) 60. The activation head 70 is received at an open end 74 of a holder 72 of the case 52. The holder 72 can have any depending on, among other things, the shape of the activation head 70 and, consequently, alternatively can be referred to as an activation head receiving element. In a preferred embodiment as shown in Fig. 1, the holder 72 is• conical shape The holder 72 can be a piece20 or part of the box 52. It is preferable that the holder 72 be able to receive the activation head 70, to allow a glucose measurement to be made by using a disposable testing device 20, but then to allow the device to test disposable 20 sea25 easily removed after a measurement is made and. ^ s ± ^ m mm m & Then allow a new test device 20 to be fixed to the trigger head 70 again, so that the system 100 is ready to perform a new measurement. The optical apparatus 50 shown in Fig. 1 is deduced from an apparatus described in commonly assigned U.S. Patent No. 5,792,049, which is incorporated herein by reference. In a preferred embodiment, the first energy source 54 transmits a first type of energy in the form of electromagnetic radiation 39 with sufficient intensity. Preferably, the first energy source 54 is an optical energy source, such as a laser, that provides stimulated emission of radiation and operates in the infrared, visible or ultraviolet region and is suitable for practicing the present invention. Alternatively, the first energy source 54 may be a laser diode, a radio signal generator, a microwave signal generator, a sound signal generator, a visible signal generator, an ultraviolet signal generator, a generator of X-ray, a? ray generator, a-ray generator, a ß-ray generator or any other type of electromagnetic signal generator. The second energy source 56 provides a second type of energy as supplied to an object, that is, the testing device 20. Preferably, the secondmk &? a * s < ~ * J ** »* -? A ** Mik ¿a * ¿? £ * energy source 56 is an optical energy source such as an electric bulb, a halogen tungsten bulb, a tungsten bulb filled with noble gas , one or several LEDs or other similar optical devices that cover the desired regions of an objective optical spectrum. The second power source 56 transmits the second type of energy to the activation head 70 through the optical fiber (s) 60. The activation head 70 projects the second type of energy onto the activation device 70. Test 20. Alternatively, the second energy source 56 may be placed in a location within the box 52 and near the holder portion 72 to supply the second type of energy to the testing device 20 directly. For embodiments where the second energy source 56 provides optical energy, the optical energy is supplied to the testing device 20 through the activation head 70 to illuminate the testing device 20, which is in contact with the tissue fluid. The scattered and / or reflected optical energy of the testing device 20 can be collected and transmitted to the detection instrument 58 through the activation head 70 to detect and / or measure the presence of at least one substance in the fluid of the tissue 40, such as glucose. It should be noted that although in the embodiment shown in Fig. 1, the first and second energy sources 54, 56 are separate elements, it is also contemplated that a single energy source can provide both the first and the second type of energy . An example of such a power source is a laser with an adjustable intensity and bandwidth. The optical apparatus 50 may include a control unit (not shown) for controlling the application of the first type of energy from the first energy source 54, the second type of energy from the second energy source 56 and the energy processing received by the detection instrument 58. 10 Even with reference to FIG. 1, the instrument of"Detection 58 is an optical detection device, such as a spectrometer. The spectrometer may include, for example, a microspectrometer offered by American Laubscher Corporation of Farmingdale, New York, called the15 microspectrometer VIS / NIP. The detection instrument 58 may be of another class of electromagnetic signal detectors such as a specific band detector (s). The detection instrument 58 is coupled to the activation head 70 through one of the optical fibers20 60 to detect and / or measure a characteristic of at least one substance, such as glucose, in a fluid collected from the tissue 40, based on the energy spectrum corresponding to an interaction between the test device 20 and the glucose in a fluid collected from the tissue25 40. The energy spectrum includes electromagnetic energy^^ m-áit-iil dispersed and / or reflected from the test device 20 which is irradiated by at least one of the first energy source 54 and the second optical energy source 56. For the w mode where the second source of 56 energy is used5 to illuminate the testing device 20, the energy spectrum includes light within a waveband indicative of the substance, such as glucose, in the fluid, scattered and / or reflected from the testing device 20 and the desirable optical interaction can Include appearance and / or change10 color (visible or invisible) in a region of the device• of assay 20. Alternatively, the presence of a substance can be measured if the energy spectrum detected by the detection instrument 58 does not have a component with a specific waveband, otherwise indicative of the15 substance. In addition, depending on the types of the first and second energy sources 54, 56 and / or the type of photosensitizing material used in the testing device 20, as discussed in more detail later herein, the presence of a substance in a fluid, such as glucose,20 can be measured using Fluorescence intensity, Fluorescence life duration, surface plasmon resonance, Fluorescence polarization, dichroic or circular, Raman scattering and other known technologies or a combination of at least two of these technologies in25 conjunction with the embodiments of the present invention. HekAvJ? tbtíA? assay device 20 has a reactive region that is sensitive to glucose and in contact with the fluid, as discussed in more detail later herein. The detection instrument Wm 58 preferably has a sensor (not shown)5 sensitive to the energy reflected and / or scattered by the testing device 20 and a processor (not shown) coupled to the sensor to receive and process a supplied energy from the sensor to determine the presence of at least one of the substances. In addition a screen (LCD u10 other type) disposed on the outside of the optical apparatus 50 can be coupled to the detection instrument to visualize a measurement. The optical fiber (s) 60 can be a single flexible transparent fiber device containing a15 bundle of optical fibers or a bundle or set of flexible transparent fiber devices. Preferably, the optical fiber (s) 60 are light guides having fiber properties and requirements for image transfer, in which the information is continuously transmitted over distances20 relatively short. The optical fiber (s) 60 may be any one or a combination of multirate, stepped refractive index pre fibers, multimodal fiber of graduated index and a unimodal stepped index fiber. However, preferably the optical fiber (s) 60 is25 a single or combination of multimodal, stepped refractive index pre fibers. For example, the optical fibers manufactured by 3M Corporation, which have a diameter range of 1-1000 microns, can be used to carry out the present invention. In one embodiment of the present invention as shown in Fig. 7, the optical fiber (s) 60 includes the optical fibers 60a, 60b and 60c for transmission of electromagnetic energy for the first energy source 54 , the second energy source 56 and the detection instrument 58, respectively. In another embodiment of the present invention as shown in Fig. 8, the optical fiber (s) 60 includes a bundle or set of several flexible transparent fiber devices 60a, 60b and 60c. For example, the optical fiber 60a couples the first energy source 54-to the activation head 70, the optical fibers 60b couple the second energy source 56 to the activation head 70 and the optical fibers 60c couple the detection instrument 58 at the activation head 70. It should be noted that as shown in Fig. 8, there are several optical fibers 60b and 60c to increase the capacity of the activation head 70 to supply the second type of energy to, and collect the energy dispersed and / or reflected from the testing device 20. Referring again to Fig. 1, the curved portion 66 of the box 52 allows a user's hand...... ^^^. ^^ .... K ...?, S * «AA.Lt. ^ LBaaJ ^ -a ^ comfortably hold and position the system 100, which includes the optical device 50 with the test device 20 fixed, for pressing the test device 20 firmly against the tissue 40 to carry out a measurement. A person can initiate a measurement such as may be the case, by pressing a push button 61 with his thumb. The actuating head 70 has a portion "concavely curve 71, as shown in Fig 6. Note that FIG 6 shows for purposes of explanation the actuating head 70 in position spaced from the tissue 40,.. In operation In fact, the testing device 20 is fixed to the activation head 70 and is in contact with the tissue 40. A concavely formed activation head 70 allows the testing device 20 to come into close contact with the tissue 40, when the device of test 20 is pressed against the tissue 40 by the activation head 70. In addition, the activation head 70 is preferably made of material suitable for absorbing heat from the tissue generated by the reactive region 24 during the operation. Activation 70 serves as a heat absorber to reduce sensation to the subject, such as a patient, by removing heat from incidentally created tissue during the process The material of the activation head 70 isaluminum or other metals or suitable alloys that have good heat absorption characteristics. With reference now to Figs. 2-4 in conjunction B | with FIG. 6, according to a preferred embodiment of the 5 present invention, the assay device 20 includes a base or supporting member 21 having a first side 22 and a second side 32. The base 21 may be an element of a small disk shape made of fiber or other suitable material (s), transparent to the first and second type of10 energy supplied by the first and second sources ofTWF energy 54, 56. Alternatively, the base 21 may be oval, square, triangular or any other geometric shape. In the same way, the base 21 can be made of plastics, polymers, thin metal film, cartons or15 other types of materials. As shown in Figs. 2, 4 and 6, the first side 22 of the testing device 20 has a reactive region 24 or a microdot arranged or deposited on the first side 22. Preferably, the reactive region• 24 is located substantially in the central area of the first20 side 22. In a preferred embodiment of the present invention, the reactive region 24 includes a layer of photosensitizing material, which is sensitive to the electromagnetic energy supplied by the first energy source 54 to heat and conductively transfer heat to25 the surface of the fabric 40 to form at least one opening- ^ A ^ to ^ A ^ ^, -.. ^^ '. - trr or micropore 41 as shown in Fig 6, to thereby allow the tissue fluid 40, through at least an opening or micropore 41, is contacted with Wm the first side 22 of the testing device 20. This technique5 of microporation is described in commonly assigned U.S. Patent No. 5,885,211, which is incorporated herein by reference. In addition, the reactive region 24 or the layer of photosensitization material is sensitive to a substance of interest in the fluid, to alter in a10 detectable electromagnetic energy dispersed by, and / or# Reflected reactive region 24 in response to the second type of optical energy to thereby indicate a characteristic of at least one of the substances in the tissue 40. The first side 15 of the device 22 assay 20 optionally has adhesive material 26 disposed or deposited thereon, to leave the reactive region 24 substantially uncovered, as shown in Fig. 2. Adhesive material 26 can be used to fix the20 testing device 20 to the tissue 40, when the activation head 70 presses the test device 20 to the tissue 40. The testing device 20 optionally has adhesive material 36 deposited on the second side 32. The adhesive material 36 can be used for fix the device25 test 20 to the activation head 70 of the optical apparatus 50.
Optionally, the adhesive material 36 is disposed on the base 21 to form a cover around a window 34 opposite the reactive region 24 of the first side 22. The window 34 allows the supply of electromagnetic energy 39a from the first power source 54, such as a laser, to reach and heat the reactive region 24 of the first side 22, which then transfers heat 39c 'to the surface of the fabric 40 to form at least one opening or a micropore 41 as shown in Fig. 6, for thus allowing the tissue fluid, through at least one opening 41, to contact the reactive region 24 of the first side 22. The window 34 also allows the supply of electromagnetic energy 39b from the second source of energy. energy 56 to reach the reactive region 24 and cause a desirable optical interaction with the reactive region 24 which can then be detected from the scattered and / or reflected energy 39c, as explained or previously in the present. With reference again to Figs. 2 and 3, optionally, the testing device 20 has a separation tab 28. The separation tab 28 can be an integral part of the base 21 or a separate component fixed to the base 21 by glue or rubber or other kind of material adhesive or heat seal, etc. The separation tab 28 can be used to handle or transport the deviceof test 20, before, during or after a measurement. For example, before a measurement is made, the separation tab 28 can be used to fix the testing device 20 to the activation head 70 of the optical apparatus 50. In the same way, once a measurement has been made, the separation tab 28 can be used to detach the testing device 20 away from the activation head 70. A new testing device 20 can then be attached to the activation head 70 and the system 100 is now ready to make another measurement on the fabric 40. The photosensitization material used in the reactive region 24 preferably includes a formulation of active components and / or inactive components. As explained hereinabove, the formulation of the photosensitizing material provides at least two functions: a function to react with one or more substances of interest to allow their detection by electromagnetic means; and a second function for absorbing a certain type of electromagnetic energy focused thereon, for heating and conductively transferring heat to the adjacent tissue and forming at least one opening therein. In one embodiment of the present invention, the inactive components include a number of well-known polymeric binders that can both stabilize and sustain the active components in theirKÜ- 'place. These polymeric binders include, but are not limited to, polyvinylpyrrolidone, polyvinyl alcohol, polyethylene glycol, bovine serum albumin, and collagen. Optionally, a surfactant that will allow a more uniform diffusion and faster re-solubilization of the active components may be added as an inactive component. There are many options for the surfactant suitable for the present invention, such as sodium dodecylisulfate, Triton X-100, cholate, dioctylsulfosuccinate, polyoxyethylene sorbitan such as Tween 20 and Span 20 and polyoxyethylene ethers such as Brij 35, etc. In another preferred embodiment of the present invention, a regulatory solution can be included in the formulation as an inactive component. The regulatory solutions commonly used are citrate, phosphate and a variety of "biological regulatory solutions" such as HEPES, MES, Bis-Tris, BES, ADA, ACES, MOPSO, MOPS, Bis-Tris propane, TES, etc. The addition of a buffer solution to the formulation can improve the stability and performance of the photosensitization material. However, the selection of the regulatory system will depend largely on the selection of an indicator system, as discussed below in the present. The active components of the layer of photosensitizing material include an enzyme system and an indicator of at least one of the substances in the fabric 40 to be measured. In a preferred embodiment of the present invention, the active components include specific enzymes or compounds with a high binding affinity for glucose and may include an auxiliary or mediating enzyme. These components are used in conjunction with one or more indicators such as chromogens or fluorescent probes to produce a change in absorption or absorption and emission spectra, respectively. An enzyme system that is useful in a modalityThe preferred embodiment of the present invention is the oxidase / glucose peroxidase system. This enzyme system can be used in conjunction with a variety of indicators such as either 4-aminoantipyrine (4-AAP) or 3-methyl-2-benzothiazolone hydrazone (MBTH) with a variety of derivatives15 of phenol or aniline. These derivatives include phenol, p-hydroxybenzoic acid, p-hydroxybenzene sulfonate, aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3,5-dimethyl aniline, N-ethyl- N- (2- hydroxy-3-sulfopropyl) -3-methyl aniline, N-ethyl-N- (2-hydroxy-3-sulfopropyl) aniline, N- (2-hydroxy-3-sulfopropyl) 0 aniline, etc. Some indicator systems that can be used with the oxidase / glucose peroxidase system require only one chromogen and can be used without 4-AAP or MBTH. Examples of such indicators are ortho-dianisidin, ortho-toluidine, 3, 3 ', 5, 5' -tetramethylbenzidine, 5 ABTS and others.
Another enzyme system that is useful in a preferred embodiment of the present invention is dehydrogenase < 3p glucose and NAD. This enzyme system can be used either as it is with ultraviolet detection of NADH or coupled with either an electron mediator (or diaphorase) with a chromogen. The electron mediator can come to form the class of compounds such as ferrocyanide, phenazine methosulfate or phenazine ethosulfate. The indicator can be one of the common tetrazolium dyes, such as yodonitrotetrazolium, neo-tetrazolium blue, nitrotetrazolium blue or some of the more recent water-soluble tetrazoliums (WTSs). There is a great classification of dye solutions (dyes and pigments) used for cytological staining, which can be used with either the oxidase / glucose peroxidase system or the glucose dehydrogenase system, which serve in the function of absorbing electromagnetic energy ( optical) of the first type to form openings in the fabric, as described hereinabove. In addition, to detect the presence of glucose, instead of using enzymes, glucose binding proteins can be used in a preferred embodiment of the present invention. Such glucose binding proteins are non-destructive and are based on a signal change in the glucose link. The glucose detection system that uses glucose binding proteins as active components is commonly based on fluorescence. At least two types of glucose binding proteins can be used in the present invention. One is a single molecule system and the other is a bimolecular or multimolecular system. In a single molecule system, according to one embodiment of the present invention, the binding molecule has conjugated to these two fluorophores with the property that the emission spectrum of one of the fluorescent dyes (donor) overlaps with the spectrum of absorption of the other dye (acceptor). In the link there is usually a confor ational change in the protein molecule that changes the relative distance between the two dyes. Typically, the dyes move closer together. The glucose binding proteins that are candidates for this type of work are the Glucose-Galactose Linker Protein (GGBP), hexokinase (in the absence of ATP) and apo-glucose oxidase. Any of a large number of molecules that undergo conformational change in the glucose link can be used to carry out the present invention. In the irradiation with a wavelength that excites the donor dye, the proximity of the two dyes determines what percentage of the excited donor dyes will be transferred non-radiatively to the acceptor dye; the closer the two dyes, the more this quantum energy transfer occurs. This process is called k Fluorescence Resonance Energy Transfer5 (FRET). The amount of FRET measured is directly related to the glucose concentration. This non-radiative transfer can be measured in a number of ways: by measuring the intensities of the light emitted from the donor dye and the acceptor dye, by measuring the duration of the10 fluorescence life of the donor dye and / or by measuring the decrease in fluorescence polarization relative to the incident light. According to another preferred embodiment of the present invention, in a bimolecular system, aA macromolecule that includes a single or multiple glucose molecules is conjugated with a donor or acceptor fluorescent dye. While one glucose binding protein is conjugated to the other dye, that is, if the molecule that• it carries glucose is conjugated with a donor dye, then theThe glucose binding protein is conjugated with the acceptor dye. A common glucose binding protein used in this application is Concanavalin A. Other lectins and GGBP, hexokinase and apo-glucose oxidase can also be used to bind glucose in this system. Again,25 the amount of FRET that is presented in this systembimolecular is proportional to the glucose concentration and is measured in the same ways as in the monomolecular system, described hereinabove. The photosensitization material is disposed or deposited on the base 21 as a thin film, or as a micro-dot, as is known to those skilled in the art, or as an aggregation of powders containing a formulation of inactive components and active components, as described above. The reactive region 24 is formed and defined by the photosensitization material. - FIG. 5 depicts the steps involved in using system 100 to perform a measurement on tissue 40 in accordance with the present invention. In particular, step 502 comprises placing a testing device 20 on the activation head 70 of the optical apparatus 50. As discussed hereinabove, the testing device 20 is sensitive to at least one substance in a tissue fluid. 40. An application of the present invention is where the system 100 is used to measure the presence of glucose in a fluid that is collected from the tissue 40; in this case the test device 20 is sensitive to glucose. The adhesive material 36 fixes the test device 20 to the activation head 70 to maintain an appropriate position during the measurement.
Step 504 comprises positioning the activation head 70 to the tissue surface 40, so that the testing device 20 is in contact with the tissue surface 40. It is preferable to press the activation head 70 firmly, but in a manner soft, against the tissue 40, such that the reactive region 24 is in direct contact with the surface of the tissue 40. The step 506 comprises forming at least one opening or micropore 41 below the test 20 through the tissue surface. 40, to thereby allow tissue fluid 40 to flow through at least one opening 41 and contact test 20 to wet or impregnate reactive region 24. In particular, with reference to Fig. 6, step 506 comprises irradiating the reactive region 24 of the base 21 with energy 39a, whereby the photosensitizing material in the reactive region 24 is sensitive to the energy 39a for heating and conductively tra nferring heat 39c 'to the surface of the fabric 40 to form at least one opening 41. Alternatively, multiple openings or micropores spaced from each other in the fabric can be formed. The micropore is formed through a tissue surface, such as skin, at a predetermined depth range in the tissue. One type of micropore depth control is described in more detail in commonly assigned U.S. Patent No. 6,022,316, which is incorporated herein by reference. After the opening (s) is formed, the activation head 70 can be pressed against the tissue 40 to assist in extracting fluid from the tissue 40 to the testing device 20. The step 508 comprises detecting the response of the test device 20 to the fluid, to measure the presence of at least one substance in tissue 40. With reference to Fig. 6, step 508 comprises irradiating test device 20 with energy, such as optical energy 39b or light from the second energy source. 56, detect the reflected and / or dispersed energy 39c of the reactive region 24 of the testing device 20 and evaluate the reflected and / or scattered energy 39c to determine the presence (and / or measurement) of at least one substance in the tissue 40. The detection can be performed by an optical instrument or detection unit 58. Optionally, after a measurement is made on the tissue 40, the testing device 20 can be removed from the optical apparatus 50 and discarded. The steps 502-508, as discussed hereinabove, can then be repeated to perform a new measurement. Various modifications and alterations of this invention will become apparent to those experienced in the art without departing from the scope andspirit of this invention and it should be understood that this invention will not be unduly limited to the illustrative embodiments set forth herein.•