MXPA06005806A - Fiber optic device for sensing analytes - Google Patents

Abstract

A device for sensing analyte concentration, and in particular glucose concentration, in vivo or in vitro is disclosed. An optical conduit, preferably an optical fiber has an optical system at the proximal end of the optical conduit. A sensing element is attached to the distal end of the optical conduit, and comprises at least one binding protein adapted to bind with at least one target analyte. The sensing element further comprises at least one reporter group that undergoes a luminescence change with changing analyte concentrations. Optionally, the sensing element includes reference groups with luminescence properties that are substantially unchanged by variations in the analyte concentrations.

Description

FIBER OPTIC DEVICE TO DETECT ANALYTICS FIELD OF THE INVENTION The present invention relates to a device that can be used to monitor concentrations of compounds found in the physiological medium.

BACKGROUND OF THE INVENTION The monitoring of in vivo concentrations of compounds found in the physiological environment to improve the diagnosis and treatment of some diseases and alterations is a desirable goal and would improve the lives of many people. The advances in this area are promising because they facilitate adequate metabolic control in diabetics. Currently many diabetics use the "finger prick" method to monitor the concentration of blood sugar, patient compliance is problematic because of the pain caused by frequent punctures to the finger. As a result, efforts have been made to find non-invasive or minimally invasive methods. in vivo and more efficient in vi tro for frequent or continuous monitoring of blood glucose or other biological fluids.

Approaches for frequent or continuous monitoring in vivo tend to fall into two general categories: "non-invasive" and "minimally invasive." Non-invasive monitoring determines analyte concentrations by directly tracking spectroscopic changes in skin and tissue. Examples of this technique are infrared radiation spectroscopy and radio wave impedance. Progress in these approaches has been slow due to the requirement of frequent calibration, reproduced illumination of samples and variations in spectroscopic backgrounds between people. A "minimally invasive" approach avoids the direct extraction of blood from the body and depends on the monitoring of signal changes in biological fluids using an intermediate sensor element. Biosensors of this type are apparatuses that can provide quantitative or semi-quantitative, specific analytical information, using a biological recognition element that is combined with a transducer element (detector).

The most common systems for frequent or continuous monitoring of analytes consist of amperometric biosensors that use enzymes such as glucose oxidase (GOx) to oxidize glucose to glucuronic acid and hydrogen peroxide, obtaining an electrochemical signal. These sensors are subject to inaccurate measurement due to oxygen deficiency and the accumulation of by-products of oxidation. An accurate measurement of glucose concentrations requires an excess of oxygen, which is not normally present in human blood or interstitial fluid. Also, the electrochemical reaction itself generates an accumulation of oxidation byproducts that can inhibit and degrade the enzyme and its protective layer.

Bionsensors based on optical rather than electrochemical signals have also been developed and can offer significant improvement in stability and calibration. For example, if a reference is made of an optical signal that depends on the analyte against a second signal independent of the analyte, the sources of noise and instability in the sensor can be corrected. However, the optical detection potential for the detection of analytes in vivo has not yet materialized. One reason for this is that many of the optical detection methods depend on enzymatic chemistry such as glucose oxidase. In a common method an oxygen sensitive fluorescent dye is used to monitor the oxygen consumption by the GOx enzyme reaction. Although this is an optical biosensor, in which the level of the fluorescence signal varies with changing oxygen concentrations, a sensor like this presents the same problems as amperometric devices based on this same chemistry: oxygen deficiency and degradation of enzymes .

To overcome the challenges associated with the detection of enzymes (such as GOx), whether electrochemical or optical, optical or fluorescent detection not based on enzymatic protein is currently being investigated. Concanavalin A and dextran labeled have been used to create a competitive FRET assay; however, this system requires the capture of both components, and the dynamic range of the trial is limited. See Ballerstadt, R., Schultz, J. S., "Competitive-binding assay based on fluorescence quenching of ligands held in cióse proximity by a multivalent receptor". Anal. Chem. Acta 345 (1-3): 203-212 (1997). See also Russell, RJ, Pishko MV, Gefrides C. C, McShane, MJ, Cote, GL, "A fluorescence-based glucose biosensor using cancavalin A and Dextran encapsulated in a poly (ethylene glycol) hydrogel" Anal Chem. 71 (15 ): 3126-3132 (1999)..

Another protein-based detection chemistry uses the periplasmic receptor of Escherichia Coli (E. coli), the glucose-galactose binding protein (GGBP) to generate a fluorescence signal in response to the binding of glucose. See, for example, Tolosa, L., I., Gryczynski, L. R. Eichhorn, J. D. Dattelbaum, F. N. Castellano, G. Rao, and J. R. Lakowicz; "Glucosse sensor for lo -cost lifetime-based sensing using a genetically engineered protein" Anal. Biochem. 267: 114-120 (1999); Hellinga, H., and J. S. Marvin; "Protein engineering and the development of generic biosensors, Trends Biotechnol" 16: 183-189 (1998); Salins, L. L., R. A. are, C. M. Ensor, and S. Daunert, "A novel reagentless sensing system for measuring glucose based on the galactose / glucose-binding protein" Anal Biochem 294: 19-26 (2001); and de Lorimier, R. M., J.J. Smith, M.A. Da yer, L.L. Looger K. M. Salí, C.D. Paavola, S. S. Rizk, S. Sadigov, D. Conrad, L. Loew, and H.W. Hellingá. "Construction of a fluorescent biosensor family" Protein Sci. 11: 2655-2675 (2002). GGBP undergoes a considerable conformational change after ligand binding, capturing the ligand between its two globular domains. See, for example, Shilton, B.M. M. Flocco, M. Nilsson, and S.L. Mowbray; "Conformational changes of three periplasmic receptors of bacterial chemotaxis and transport: the maltose-, glucose / galactose and rbose-binding proteins" J. Mol. Biol. 264: 350-363 (1996). When the protein is specifically labeled in place with a fluorophore sensitive to the environment, it is possible to take advantage of this attribute to obtain a fluorescent signal. See, for example Salins, L.L., R. A. are, C.M. Ensor, and S. Daunert, "A novel reagentless sensing system for measuring glucose based on the galactose / glucose-binding protein" Anal Biochem 294: 19-26 (2001). Since GGBP does not consume glucose or generate reaction products, it is possible to use it as a non-reactive sensor. This fact can provide greater accuracy and reliability than the amperometric biosensors.

While some groups have developed GGBP mutations capable of responding to glucose in the physiological environment, no reports have been found of a functional biosensor device based on binding protein technology that is suitable for monitoring analytes in vivo. A frequent and / or continuous functional biosensor should couple the sensor element to the optical sensor elements while maintaining the integrity and functionality of the sensor, as well as comfort for the patient. For example, the biological recognition element and the accompanying transducer element should preferably be incorporated within a biocompatible material that protects the sensor element against the immune system, allowing the diffusion of the analyte inwards and outwards and avoid leaching of the element sensor in the patient's blood or other biological fluid (for example interstitial fluid). Since the binding proteins require control in the orientation and freedom of conformation to be able to use them effectively, -Multiple strategies of physical absorption, relative random or covalent surface binding or immobilization as taught in the literature are generally suboptimal or unsatisfactory. In addition, a means should be devised to interrogate the sample with light in a reproducible and / or controlled manner.

A generally known approach is to attach the sensor element to one end of an optical fiber and to couple the optical elements such as the excitation sources or detectors to the other end. However, the coupling of the binding proteins to one end of an optical fiber is subject to the aforementioned challenge of preserving the mobility of conformation and / or orientation of the protein. In addition, fiber optic cabling is often impractical from the point of view of patient use, since patients may need to remove or replace the sensor periodically. The replacement of all fiber can be expensive and inconvenient. By last, the optical system, consisting of, for example, excitation sources, detectors and other optical elements must be sufficiently robust to tolerate or correct changes in optical alignment due, for example, to patient movement or to deviation of the electronics in the optical reader. The optical system must also be sensitive enough to detect signals of indicator dyes without depending on a high energy consumption and / or large elements that would make the system non-portable and therefore not usable.

Accordingly, a biosensor incorporating a binding protein with conformational mobility and / or orientation coupled to optical sensing elements that provide a useful and robust device would be convenient in its detector element.

SUMMARY OF THE INVENTION An object of the present invention is to provide a device for detecting the concentration of a target analyte in a sample. The sample may be blood, saliva, tears, sweat, urine, brain spinal fluid, lymphatic fluid, interstitial fluid, plasma, serum, animal tissue and media. The device consists of: (i) an optical conduit having a proximal end and a distal end; (ii) an optical system at the proximal end of the optical conduit containing at least one emitter of electromagnetic energy and at least one electromagnetic energy detector; and (iii) a sensor element in optical proximity with the distal end of the optical conduit containing at least one binding protein that is adapted to bind with at least one target analyte; the sensor element also contains at least one indicator group, and as an option, one or more reference groups.

The optical conduit, which can vary in length from about 0.1 cm to 1 meter, couples light in and out of the optical system and in and out of the sensing element. For example, the optical conduit may be a lens, a reflecting channel, a needle or an optical fiber. The optical fiber can be a single strand of optical fiber (uni or multimodal) or a beam of more than one fiber. In one embodiment, the fiber bundle is bifurcated. The fiber may be unsharpened or sharpened to penetrate a patient's skin.

The optical system consists of a combination of one or more excitation sources and one or more detectors. It can also consist of filters, dichroic elements, a power source and electronics for signal detection and modulation. The optical system, as an option, may include a microprocessor.

The optical system interrogates the sample in a continuous or discontinuous manner by coupling one or more wavelengths of interrogating light in the optical conduit. One or more questionable wavelengths then pass through the optical conduit and illuminate the sensing element. A change in analyte concentration results in a change in wavelength, intensity, lifetime, efficiency of energy transfer and / or polarization of the luminescence of the indicator group, which is a part of the sensing element. The resulting luminescence signal with change returns through the optical conduit to the optical system where it is detected, interpreted and stored and / or presented. In some embodiments, the optical system contains multiple sources of excitation. One or more of these sources can be modulated to allow dynamic processing of the detected signal, thereby improving the signal-to-noise ratio and the sensitivity of the detection. It is also possible to use modulation to reduce the energy consumption of the device or to increase the lifetime of the sensor element by minimizing the unwanted phenomenon as photobleaching. The optical system may also include one or more electromagnetic energy detectors that can be used to detect the indicator luminescent signal and the optional reference groups, as well as internal referencing and / or calibration. The total energy consumption of the optical system is kept small to allow the device to operate using battery current.

The sensing element contains one or more binding proteins that are adapted to bind with at least one target analyte, and at least one indicator group. A suitable binding protein can be any that is adapted to be used as a biosensor. For example, the appropriate binding protein can be any of those described in the copending US Patent Application Serial No. 10 / 039,833 assigned herein, filed on January 4, 2002; US Patent Application Serial No. 10 / 040,077 filed on January 4, 2002; US Patent Application Serial No. 10 / 039,799 filed on January 4, 2002 and the US Patent Application for "Compositions and Methods for Measuring Analyte Concentrations" for Ferry Amiss, et al., (attorney's file No. P -6011) filed on the same date as the present one, and the contents of which are incorporated herein by reference in their entirety. Suitable binding proteins can also be any of those described in US Patent No. 6,277,627, US Patent No. 6,197,534 or WO-03/060464 A2, the total content of which is incorporated herein by reference in its entirety. The reporter group, which is associated with the binding protein, is adapted to undergo a luminescence change upon binding of the target analyte binding protein. When used herein, the term "associated with" means that the reporter group is covalently or non-covalently associated with the binding protein so that upon binding of a target analyte to the binding protein, there is a change in the luminescence properties of the indicator group as wavelength, intensity, half life, efficiency of energy transfer and / or polarization. Examples of indicator groups include, but are not limited to, organic dyes, pairs of organic dyes, fluorescent or bioluminescent fusion proteins, pairs of fluorescent or bioluminescent fusion proteins or any combination of the above. The indicator group may consist of a donor and acceptor that undergo fluorescent resonance energy transfer. Other luminescent labeling portions can be the lanthanides such as europium (Eu3 +) and terbium (Tb3 +), as well as metal-ligand complexes that include those of ruthenium [Ru (II)], rhenium [Re (I)] or osmium [OS (II )], usually in complexes with diimine ligands such as phenanthrins.

The sensor element is in optical proximity to the optical conduit, "optical proximity" means that the components of the device are fairly close together so that an optical signal can be transmitted to or received from one object to another. The sensor element can be placed in optical proximity with the optical conduit in various forms, for example: attached directly to the optical conduit; attached to a conductor that is attached to the optical conduit, attached to a polymeric chain or a polymeric matrix that is attached to the optical conduit; or attached to a polymeric chain or a polymeric matrix that is attached to a connector that is attached to the optical conduit. The sensor element can be permanently fixed to the optical conduit or it can be connected so that it can be replaced so that the sensor element can be replaced conveniently and economically.

In another embodiment, the sensor element may also contain one or more reference groups. Unlike the indicator group, the reference group has a luminescence signal that practically does not change after the binding of the target analyte with the binding protein, "practically does not change" means that the change of luminescence of the reference group is much smaller than the luminescence change suffered by the indicator group. The reference group, which may consist of luminescent dyes and / or proteins, is used for internal reference and calibration. The reference group may be linked to any number of components of the device such as the sensing element, a binding protein that does not contain the indicator group, the polymer matrix, the polymer chain, a biomolecule that is not a binding protein, the optical conduit or a tip.

The sensor element (usually this refers to the binding protein with the associated indicator group and the optional reference group) can be directly attached to the distant end of the optical conduit using for example covalent, ionic or van der aals interactions, coating by immersion, coating by centrifugation, plasma coating or vacuum deposition. The sensor element may also be attached to a connector that allows the sensor element to be easily separated so that it can be replaced.

In another embodiment, the sensor element is attached to or immobilized in a polymeric matrix. The polymeric matrix can be any matrix that allows the free diffusion of the analyte of interest in and out of the matrix, while excluding the interfering immune proteins and proteases and allows the binding protein to retain some degree of mobility. conformation and / or orientation. The matrix can contain multiple layers, an inner layer that serves to retain the binding protein, and one or more outer layers to control permeability and / or achieve biocompatibility. For example, the polymeric matrix may be any of those described in the copending US application, assigned herein, series No. 10 / 428,295, filed May 2, 2003, the total content of which is incorporated herein by reference. reference. Immobilization can be achieved by covalently linking the sensor element to the polymer matrix or by physically capturing the sensor element within the matrix. In the case where the polymer matrix physically traps the sensing element, the pores of the matrix will have the size to retain the sensor element. In an embodiment where the sensor element is attached to the polymer matrix, the sensor element is attached to the matrix using, for example, covalent or ionic bonding. The polymeric matrix may be attached to the distal end of the optical conduit using adhesives, dip coating or centrifugation, plasma coating, covalent, ionic or van der Waals interactions, mechanical connector or combinations thereof.In another embodiment, the sensor element is attached to a polymer chain, the method of joining the sensor element to the polymer chain can be, but is not limited to, covalent, ionic and van der Waals interactions and combinations thereof, the chain polymer is attached to the distal end of the optical conduit "using, for example, dip coating or centrifugation, plasma coating, vacuum deposition, covalent, ionic or van der Waals interactions or combinations thereof.

In another embodiment, the device further contains a tip (sharp or non-sharp) designed to pierce the skin to allow the sensing element to make contact with bodily fluids. Preferably, the tip is disposable. The tip can be made of plastic, steel, polymer glass or any combination of these materials or similar materials. The tip can be attached directly to the optical conduit (fiber) using adhesives or a mechanical adjustment. The tip can also be used to house the optical conduit containing the sensor element, so as to contain the optical conduit and the sensor element. In one embodiment, the sensing element may be contained within the tip.

The device may also contain a connector that can be used to join the components of the device together. The connector can be, for example, any mechanical device, such as normal fiber optic connectors, luer adapters, plastic, metal or glass sleeves or spring loaded housings. For example, it is possible to use the connector to connect the sensor element to the optical conduit, or to connect the optical conduit with the optical system. The fundamental purpose of the connector is to provide a component that allows the other components to be easily separated so that the component can be replaced.

In light of the above comments, it will be noted that the main objective of the present invention is to provide an improved device for detecting the concentration of a target analyte that is useful and easily handled. Preferably, the device can be used for the continuous monitoring of analytes, but those skilled in the art can devise continuous and intermittent monitoring of samples in vivo and / or in vi tro with this device.

Another object of the present invention is to provide a device of compact and portable design.

Another objective of the present invention is to provide a robust device for detecting a target analyte.

Another objective of the present invention is to provide a device that can be used easily and with highly reliable results.

Another objective of the present invention is to provide a device that is accurate and provides readings in a short time interval.

Another objective of the present invention is to provide a device having a tip portion of size that produces little or no pain or sensation when inserted into a patient.

These and other objects and advantages of the present invention will become apparent after considering the following detailed specification together with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood "by referring to the modalities of this which are shown in the figures of the attached drawings, in which: Figure 1 is a general scheme of a biosensor according to an embodiment of the invention; Figure 2 shows two embodiments of the optical configuration in the optical part of the sensor according to an embodiment of the present invention; Figure 3 shows different modalities of the biosensor tip according to one embodiment of the invention.

Figure 4 shows an embodiment of the invention that is an optical biosensor that can be used in vivo; Figure 5 is a graph showing the performance of a fiber optic biosensor according to an embodiment of the invention tracking the changing concentrations of glucose in an anesthetized pig; Figure 6 is a graph showing the performance of a fiber optic biosensor according to one embodiment of the invention using an individual 400 micron core fiber optic sensor and the optical configuration shown in Figure 2A; Figure 7 shows the performance of a fiber optic biosensor according to an embodiment of the invention using an individual 400 micron core fiber optic sensor and the optical configuration shown in Figure 2A; and Figure 8 shows an embodiment of the present invention with multiple electromagnetic energy detectors and an internal reference; In the drawings it should be understood that equal numbers refer to similar characteristics and structures.

DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The preferred embodiments of the invention will now be described with reference to the accompanying drawings. The following detailed description of the invention is not intended to exemplify all modalities. In describing the preferred embodiments of the present invention, specific terminology is employed for the purpose of clarity. However, the invention is not intended to be limited to the specific terminology that was chosen. It should be understood that each specific element includes all technical equivalents that function in the same way to carry out a similar purpose.

The present invention is a manipulated binding protein for binding to an analyte of interest within a desired clinical or analyte environment. In addition, one or more luminescent reporter groups are associated with the binding protein. These luminescent indicator groups can be, but are not limited to, for example, "organic-aromatic coloring molecules" covalently coupled to the cysteine residues in the protein or, for example, luminescent biomolecules as proteins fused to the manipulated binding protein. The cysteine or other amino acid groups can be manipulated in the binding protein to provide binding sites for the luminescent indicator molecule The binding of the analyte to the binding protein results in a change in the luminescent properties of one or more groups Indicators The affected luminescent property may be the wavelength of absorption or emission, the intensity of absorption or emission, the average life of emission, the polarization of the emission and / or the efficiency of the energy transfer. analyte is also reversible, where the resulting separation again produces a change in lum properties iniscentes of the indicator molecule.

The one or more binding proteins together with their associated reporter groups comprise the sensing element. As an option the sensor element may also contain one or more reference groups. Unlike the indicator group, the reference group has a luminescence signal that practically does not change after the binding of the target analyte to the binding protein. The luminescence signal of the reference group provides an optical internal standard that can be used to correct optical artifacts due for example to the electronic derivation in the optical system or to the movement of the sample or optical conduit. The reference group can also be used for calibration. The reference group can be attached to any of the components of the device such as the sensing element, the binding protein that does not contain the indicator group, the polymer matrix, the polymer chain, a biomolecule that is not a binding protein, the duct optical or tip. In one embodiment, the reference group binds to a binding protein that has been manipulated so as not to show an important response to the analyte at physiologically relevant concentrations.

The sensor element, composed of one or more binding proteins, one or more indicator groups and optional reference groups, can be immobilized at the end of the optical conduit or inside a disposable tip that forms the interface with the optical conduit. The immobilization of the sensor element in the optical conduit or inside the disposable tip can be achieved by depositing a thin layer of sensor element., for example, by dip coating or centrifugation, covalent bonding, plasma treatment and the like, directly in the optical conduit or tip. Otherwise, the sensor element can first be immobilized in a polymeric matrix and the polymeric matrix can then be attached to the optical conduit, or the tip by means of adhesives, injection molding, dip coating or centrifugation, plasma coating, vacuum deposition, inkjet technology, covalent, ionic or van der Waals interactions, by mechanical bonding or any combination of these.

The optical system can interrogate the luminescent response of the indicator and reference groups by transmitting light from an electromagnetic excitation source to the optical conduit to the remote end containing the sensing element. The optical system also monitors and interprets the return signals generated by the luminescence response of the indicator group and the reference group. The luminescent properties of the indicator group, the wavelength, intensity, half-life, energy transfer efficiency or polarization change in response to analyte binding or separation of the binding protein.

Now with reference to Figure 1 a specific exemplary embodiment of the present invention will be described. The optical system 2 consists of a combination of elements that can be, but are not limited to, electromagnetic energy emitters, electromagnetic energy detectors, different mirrors, filters, electronic circuits, holographic optics, dichroic elements and the optical patterns needed to send The questioning radiation from the emitter of electromagnetic energy through the optical conduit to the sensor element and then solve and interpret the luminescent return signal. The luminescent return signal of the indicator group changes in response to the changing concentrations of the analyte that would be detected. The optical system 2 may also contain a computer or microprocessor 3 that handles signal processing, the mathematical manipulation of one or more signals and the storage and handling of the data. The computer or microprocessor 3 may be in physical contact with the other components of the optical system or, in a preferred embodiment, be physically separated up to several meters from other components of the optical system. In this embodiment, the information from the electromagnetic energy detectors and the electronic processing elements of the optical system are communicated in wire form to the computer or microprocessor 3. The computer or microprocessor 3 may also store specific calibration information for the detector element. The light of one or more wavelengths produced in the optical system is channeled through an optical conductor 4 to the sensor element 6. The optical conduit 4 can be an optical fiber or a short light guide transmitting light with minimal loss, the sensor element 6 consists of one or more binding proteins with one or more associated luminescent reporter groups immobilized in a polymeric matrix, attached to a polymeric matrix, incorporated in a disposable tip, attached directly to the distal end of the optical conduit or attached to a connector . The detector element 6 may also consist of additional luminescent reference groups which optionally bind to biomolecules, polymers or organic molecules in order to provide a reference or calibration signal. The sensing elements 6 may be attached to the distal end of the optical conduit 4 directly or by a polymeric matrix or, in the preferred embodiment, may be attached to a disposable tip 5, which is attached to the distal end of the optical conduit 4. In this case , the disposable tip 5 is placed against the optical conduit 4 mechanically, by adhesive or by any convenient means known to those skilled in the art. Figure 2 is an enlargement of the optical system 2 in two common modes. In Figure 2A, a dichroic mirror or a shredder of aces 11 is used to direct the light from an electromagnetic energy source 7 to the optical duct 4. The sources of excitation may be, but are not limited to, for example, arc lamps. , laser diodes or LED. In this embodiment, the optical conduit 4 is an optical fiber cable and the same fiber is used to transmit the excitation light from the electromagnetic energy source 7 to the sensor element 6 and also to transmit the luminescence signals from the groups. reference indicator and back to the optical system 2. A dichroic element 11 preferably separates the return signal from the excitation light and directs the signal to the electromagnetic energy detectors 8. The detectors may consist of, but are not limited to, for example, 'photodiodes, CCD chips, or photomultiplier tubes. In case the multiple luminescent signals are returned from the sensor element, it is possible to use other dichroic elements to direct part of the return signals to the multiple detectors. Preferably, a luminescent reference group that is insensitive to the analyte is included together with an analyte-dependent reporter molecule to provide a reference signal. This reference signal can be used, for example, to correct the optical or electronic derivation.

Figure 2B shows a second embodiment in which the bifurcated optical beam or array of fused optical fibers is used to transmit light to and from the sensor element. In this case, the light from the excitation source 7 is transmitted by an arm of the bundle of bifurcated fibers. The luminescent return signals from the sensor element are detected using the second arm of bifurcated fibers, so that in this case the fiber bundle contributes to separate the excitation of the return luminescence. The dichroic optics, the fragmentadotes of aces or polarizers can also be used to further divide the return luminescence, based for example on the wavelength or polarization. As an option, it is possible to use bandpass filters to select the luminescent wavelength to be detected. The power source 9 feeds the current of the optical system 2.

Figure 3 shows the representative methods of attaching the sensor element to the end of an optical conductor, when the optical conductor is an optical fiber. In Figure 3A, the sensor element directly attached to the far end of the optical fiber using, for example, covalent, ionic or van der Waals interactions, dip coating, spin coating, plasma coating, vacuum deposition, ink jet or combinations of these. Otherwise, the sensing element, which contains binding protein, associated indicator groups and optional reference groups, can be attached to a long chain polymer, and the long chain polymer can be attached directly to the distant end of the fiber. optics using, for example, dip or spin coating, plasma coating, vacuum deposition, covalent, ionic or van der Waals interactions, ink jet technology or combinations of these.

In Figure 3B the sensing element is immobilized in a polymer matrix and the polymer matrix is attached to the dietary end of the optical fiber using, for example, adhesives, dip coating or spin coating, plasma coating, injection molding, injection technology of ink, covalent, ionic or van der Waals interactions, a mechanical connector or combinations of these. In a preferred embodiment, the reactive groups of the polymer matrix and / or the protein they use to covalently attach the sensor element directly to the optical fiber, such as for example by introducing amino groups on the surface of a glass or silicon fiber . In Figure 3C, a plastic or polymer sleeve fits over the distal end of the optical fiber and serves to house and protect the sensor element. The sensor element is trapped or attached to a polymer matrix. The polymer matrix containing the sensor element can be introduced into the sleeve by immersion, pouring or immersion and then can be crosslinked or polymerized inside the sleeve, otherwise the sensor element can be polymerized inside the sleeve before the insertion of the optical fiber. Figure '3D shows the optical fiber held inside the needle. The needle may have a modified bevel to control the depth of perforation and / or a lateral portal to allow access of the analyte to the sensor element contained in the needle. The sensor element within the needle may be directly bonded to the optical fiber using any of the methods described in the description of Figures 3A, 3B or 3C or, in another version, may have only mechanical contact with the fiber. The binding schemes shown in Figure 3 are preferred embodiments and can be used individually or in combination.

Figure 4 shows a preferred embodiment of a useful optical biosensor. The tip 21 is a steel needle of approximately 1-10 mm in length which contains inside the sensor element 6, immobilized on an optical fiber 22. The assembly of the fiber, the tip and needle are centered in a plastic assembly 24. The tip, which contains the optical fiber and the sensing element, is inserted perpendicularly into a patient's skin so that the chemistry at the end of the fiber enters the intradermal or subcutaneous space. The adhesive ring 25 then holds the plastic assembly plus the needle assembly in place. The optical system 2, then clamps on the plastic assembly, with the connector 26 serving as an interface of the optical fiber 22 with the optical system. The optical system can be designed, for example, according to the optical mode 2A or 2B. The sources of excitation may consist of, but are not limited to, for example arc lamps, laser diodes or LEDs. The detectors may consist of, but are not limited to, for example photodiodes, CCD chips or photomultiplier tubes.The following examples show some preferred embodiments of the present invention, and are only suggested to exemplify the embodiments. The binding proteins mutated and labeled with fluororphic indicator probes are used herein according to the procedure established by Cass and Col., Anal. Chem. 1994, 66, 3840-3847, or as otherwise described.

EXAMPLE 1 In accordance with one embodiment of the present invention, the glucose galactose binding protein (GGBP) was used with a triple mutation that includes a cysteine substituted by a glutamic acid at position 149, an arginine substituted by an alanine at the position 213 and a serine substituted for leucine at position 238 (E149C / A213R / L238S). The protein was labeled at position 149 with N, N '-dimethyl-N- (iodoacetyl) -N' - (7-nitrobenz-2-oxa-l, 3-diazol-4-yl) ethylenediamine (IANBD amide) oxy . This mutated GGBP "(E149C / A213R / L238S) is specific for glucose, and the reporter group undergoes a change in fluorescence intensity in response to glucose binding.

A multi-coated or multilayer matrix was prepared as follows. A core matrix was formed by mixing a part of the labeled protein with dye binding (15 uM in PBS buffer, pH 7.4, prepared as described in PCT US 03/00203) with 2 to 4 parts of 3% alginate (v / v) in vials for scintillation and shaking with vortex at low speed. 3 mL of the resulting protein-algin mixture was placed in a syringe and infused at a rate of 10 mL / h in 200 mL of 1M CaCl 2 in a mixer, thereby forming beads of approximately 0.4 to 1.5 mm in diameter. The beads were mixed in CaCl 2 solution in the mixer for 15-60 minutes. A containment layer was then formed by placing the beads from above in a solution of poly-L-lysine 0.01% w / v in water, approximately 10 mL, for 1 hour, then drying the beads coated with poly-L-lysine on a absorbent towel for 15 to 30 minutes. At this time the sensor is ready to use it.

The fiber used in this modality was a bifurcated optical fiber. It contained 6 fibers of 40 or arranged around a central fiber of 400 um. The 6 fibers were used as the excitation conduit and the central fiber as the detection conduit. The total diameter of the fibers was 1.4 mm. Once the fiber was polished, Loctite 4011 medical grade adhesive was used to adhere the sensor element to the far end of the optical fiber. The proximal end of the fiber was bifurcated, with one arm going to a source of excitation and the other going to a detector. A 470 nm LED was used as the excitation source, and a commercial fluorescence spectrometer was used as the electromagnetic energy detector. The intensity of the 540 nm emission was then measured.

In one study the far end and the sensor element of a biosensor formed in this way was inserted through a 13 gauge needle to the side of an anesthetized pig, approximately 1-2 nm under the skin. Alternating solutions of lactose-free ringer with and without 10% dextrose were infused through the vein of the pig's ear to increase and decrease the concentrations of glucose in the pig in a controllable manner. At intervals blood samples were taken from the pig's cavity through a throat catheter, and blood sugar readings were tested on a manual blood glucose meter. The fluorescence intensity of the biosensor was observed to track the changing concentrations of glucose in the anesthetized pig, as shown in Figure 5.

EXAMPLE 2 In another embodiment, the binding protein was the glucose-galactose binding protein (GGBP), with a cysteine substituted for a glutamic acid at position 149, an arginine substituted for an alanine at position 213 and a serine substituted for Leucine at position 238 (E149C / A213R / L238S). The protein was labeled at position 149 with N, N '-dimethyl-N- (iodoacetyl) -N' - (7-nitrobenz-2-oxa-l, 3-diazol-4-yl) ethylenediamine (IANBD amide). The biosensor was prepared by inserting the tip of a fiber with a core diameter of 400 microns into a short piece of a catheter tube, and leaving the catheter tube to hang the tip of the fiber 0.1-1 mm. The fiber consisted of a silica core, silica sheath and polyimide buffer. The diameter of the fiber was 400/440/470 microns, where the inclined lines indicate the diameters measured from the outside of the core / sheath / buffer.

The matrix for immobilization was a crosslinked, alginine-based hydrogel prepared by LVG Pronova ™ UP algin with covalent crosslinking through the carboxyls with adipic acid dihydrazide (ADA) through the carbodiimide chemistry. The Pronova ™ UP LVG compound was selected in this modality due to its low viscosity and high glucuronic to mannuronic ratio. A 2% alginate solution was prepared by dissolving one gram of alginate in 50 mL of 0.1 M MES buffer (pH 6.5) and then adding 110 mg of ADA and 79 mg of hydroxybenzitriazole (HOBt). The solution was stored at 4 ° C until use. To the alginate solution were added 145 mg of l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) per 10 mL of solution, using a double syringe mixing technique. The mixture of alginate AAD, HOBt, EDC was aspirated in a 1 mL syringe to which a blunt tip 30 gauge needle was attached. The needle was primed, then the tip was inserted into the catheter tube mold on the optical fiber. The catheter tube in the fiber was filled, ensuring good contact with the tip and the optical fiber and the alginate matrix. The matrix was allowed to crosslink for 15 minutes and then the tip of the fiber and the matrix assembly were transferred to a solution of 0.1 M MES, pH 6.5, where they were stored for 2 hours. At the end of two hours, the sensor tips were placed in excess phosphate buffer solution (PBS, 0.0027 M potassium chloride, 0.137 sodium chloride, pH 7.4) where they were stored a minimum of 30 minutes to quench the reaction.

To bind the binding protein, the tips were incubated in a solution of GGBP labeled in PBS buffer [NBD-E149C / A213R / L238S GGBP] (53 uM, 50 mL) for approximately 8 hours. The sensors were protected from ambient light during incubation. After 8-24 hours of incubation, then 50 uL of EDC / NHS was added (200 mM / 50 mM) to the incubation tube. After 40 minutes. The sensor tips were removed and placed in 50 uL of 1M ethanolamine, pH 8.5 to quench the reaction. After 20 minutes in the ethanolamine solution, the sensor tips were transferred to PBS solution where they were allowed to stand for at least 24 hours while the unreacted protein diffused. The sensors were then transferred to fresh PBS and stored in the dark until they were ready for use.

The fiber of this example was a single multimodal fiber with a core of 400 uM (silica core, silica sheath, polyimide buffer). Since in this embodiment the same fiber transmits excitation and luminescence signals, dichroic optics were used to separate the luminescence from the excitation, as shown in Figure 2A. The excitation was with a 470 nm LED. A commercial dichroic filter was used to reflect the excitation 470 nm towards the end of the fiber input and transmit the fluorescence, centered at 550 nm, to the detector. The spherical glass lenses were used to collimate the has and to focus the light onto the fibers and on the detectors. The dispersed excitation was further removed from the detector using a 550 nm band pass filter. SMA connectors allowed fast connection and disconnection of fiber optic sensors. The electromagnetic energy detector of this modality was a single photon counter photomultiplier tube. The acquisition of the data was carried out on a portable computer that communicated with the detector through an RS-232 connection.

Figure 6: In one study, the remote end and the sensing element of a biosensor formed in this way was inserted into porcine serum solutions containing different concentrations of glucose. Porcine serum solutions were filtered through a 200 micron filter, and glucose concentrations in the solutions were measured by a clinical analyzer. Figure 6 shows the in vitro biosensor performance. The initial concentration of glucose in the serum was measured at 56 mg / dL. The serum samples at 150 and 300 mg / dL were prepared by adding 1 M concentrated glucose in PBS in serum aliquots.

EXAMPLE 3 In another embodiment, of the present invention a biosensor was formed by covalently attaching a thin film to the surface of an optical fiber. The binding protein was the glucose-galactose binding protein (GGBP), with a cysteine substituted for a glutamic acid at position 149, an arginine substituted for an alanine at position 213 and a serine substituted for leucine at position 238 ( E149C / A213R / L238S). The protein was labeled at position 149 with N, N'-dimethyl-N- (iodoacetyl) -N '- (7-nitrobenz-2-oxa-l, 3-diazol-4-yl) ethylenediamine (IANBD amide). The biosensor was prepared by covalently binding an alginate matrix to the amine-functionalized surface of the silica fiber. The composite fiber of the silica core, silica sheath and polyimide buffer. The diameter of the fiber of 400/440/470 microns, where the inclined lines indicate diameters measured from the outside of the core / sheath / buffer.

The polyimide buffer was removed from the fiber optic tip by exposing the last millimeters of the fiber to a torch for approximately 1-2 seconds. Then the residual polyimide was cleaned. The tip with the removed buffer was then placed in 1M sulfuric acid for one hour. Then the tips were rinsed with distilled water, placed in ethanol for 15 minutes and then immersed in anhydrous toluene for 15 minutes, the cleaned rates were then placed in hot anhydrous toluene (60 ° C) with a 1% content of 3%. -aminopropyl triethoxysilane (APTES) and allowed to react for 5 minutes. Then the tips were removed from the APTES solution and washed with ethanol.l for 15 minutes. At the end of this process, the presence of amino groups on the surface of the fiber was verified using photoelectronic spectroscopy.

An alginate matrix was then plicated to the surface of the amine-functionalized fiber as follows. The matrix for immobilization was a cross-linked alginate-based hydrogel prepared by covalent crosslinking of the Pronova ™ UP LVG alginate selected for its low viscosity and high glucuronic to mannuronic ratio, by the carboxyls with adipic acid dihydrazide (ADA) through of the carbodiimide chemistry. A 2% alginate solution was prepared by dissolving one gram of alginate in 50 mL of 0.1 M MES buffer (pH 6.5) and then adding 110 mg of ADA and 79 mg of hydroxybenzitriazole (HOBt). An aliquot of 0.5 mL of this solution was then mixed with 10 mg of EDC in 50 uL of MES buffer using a double syringe mixing technique. The total volume of the solution was approximately 0.55 mL. The mixture of alginate, AAD, HOBt, EDC was then transferred to microcentrifuge vials and fiber tips functionalized with APTES were immersed in the alginate solution for 3-4 minutes until the matrix began to be molded. Then the tips of the alginate solution were removed, the reaction was allowed to continue in air for approximately 1-10 minutes and then transferred to 0.1 M MES buffer, pH 6.5. The tips were left to stand in MES buffer for 2 hours and then the reaction was quenched with a phosphate buffer solution (PBS, 0.0027 M potassium chloride, 0.137 sodium chloride, pH 7.4) for a minimum of 30 minutes.

To bind the binding protein, the tips were incubated in a GGBP solution labeled in a buffer of PBS [NBD-E149C / A213R / L238S GGBP] (20-60 'uM, 50 uL) for several hours. The sensors were protected from ambient light during incubation. After approximately 2-8 hours of incubation it. added 50 uL of EDC / NHS (200 mM / 50 mM) to the incubation tube after 5-40 minutes, the sensor tips were removed and placed in 50 uL of 1 M ethanolamine, pH 8.5 to quench the reaction. After 20 minutes in the ethanolamine solution, the sensor tips were transferred to the PBS solution, where they were left at rest for at least 8 hours while the protein that did not react was diffused. The sensors were then transferred to fresh PBS and stored in the dark until they were ready for use. ' In a study of the modality described above, the optical reader was the same as that described in the previous example, with the exception that excitation at 470 nm was modulated using a solenoid-driven obturator. In addition to being the interface with and controlling the shutters and detectors, the software allowed the timely acquisition of the fluorescence reading, the graphical presentation of the results and the analysis of the data and the calibration algorithms.

The distal end and sensing element of a biosensor thus formed were then inserted into the anesthetized pig side. The insertion was done by intradermal or subcutaneous insertion of the fiber through a hole in the skin formed by an 18-24 gauge needle. Alternative solutions of lactose-free ringer solution with and without 10% dextrose were infused through the vein of the pig's ear to increase and decrease the glucose concentrations of the pig in a controlled manner. At intervals, blood samples were drawn from the vena cava of the pig through a throat catheter and the blood sugar readings were analyzed in a manual blood glucose meter. The fluorescence intensity of the biosensor was observed to track the changing concentrations of blood glucose in the anesthetized pig, as shown in Figure 7.

EXAMPLE 4 In another embodiment, double wavelength detection with an internal, optical reference group was made of the present invention. The binding protein was the glucose-galactose binding protein (GGBP), with a cysteine substituted by a glutamic acid in the position 149, an arginine substituted for an alanine at position 213 and a serine substituted for leucine at position 238 (E149C / A213R / L238S). The protein was labeled at position 149 with the indicator group N, N '-dimethyl-N- (iodoacetyl) -N' - (7-nitrobenz-2-oxa-l, 3-diazol-4-yl) ethylenediamine (IANBD amide). The reference group was Texas Red® C2 maleimide bound to GGBP with a cysteine substituted by a glutamic acid at position 149 (TR-E149C CGBP) In the range of physiological concentrations of glucose, the luminescence of GGBP TR-E149C is practically unchanged, and thus, the GGBP TR-E149C serves as an internal reference for the signal of the analyte-dependent binding protein and the GGBP indicator group [NBD-E149C / A213R / L238S].

The biosensor was prepared by inserting the tip of a fiber of 400 micron core diameter into a short piece of catheter tube, leaving the catheter tube to hang the tip of the fiber 0.1-0.05 mm. The fiber composed of a silica core, silica sheath and polyimide buffer. The diameter of the fiber was 400/440/470 microns, where the inclined lines indicate diameters measured from the outside of the core / sheath / buffer.

The immobilization matrix was a cross-linked alginate hydrogel prepared by covalent crosslinking of the Pronova ™ UP LVG alginate, selected for its low viscosity and the high glucuronic to mannuronic ratio, through the carboxyls with adipic acid hydrazide (ADA ) by carbodiimide chemistry. A 2% solution of alginate was prepared by dissolving 1 gram of alginate to 50 mL of 0.1 M MES buffer (pH 6.5) and then adding 110 mg of ADA and 79 mg of hydroxybenzotriazole (HOBt). The solution was stored at 4 ° C until use. With a double syringe mixing technique, then an aliquot of 0.5 mL of the alginate solution was mixed with 50 uL of an MES solution containing 10 mg of l-ethyl-3- (3-dimethylamino-propyl) carbodiimide (EDC) and 90 .L of the GGBP TR-E149 C 60 .M. the mixture of alginate, AAD, HBOt, EDC, TR-E149C was aspirated in a 1 mL syringe and a blunt-tip 30 gauge needle attached to the syringe. The needle was primed, and then the tip was inserted into the mold of the catheter tube on the optical fiber. The catheter tube on the fiber was filled, ensuring good contact between the tip and the optical fiber and the alginate matrix. The matrix was reticulated for 15 minutes and then the fiber tip of the matrix assembly was transferred to an MES solution, pH 6.5, 0.1 M, where it was stored for 2 hours. At the end of two hours, the sensor tips were placed in excess phosphate buffer solution (PBS, 0.0027 M potassium chloride, 0.37 sodium chloride, pH 7.4) where a minimum of 30 minutes were stored to quench the reaction.

To bind the binding prot the tips were incubated in a solution containing GGBP labeled with IGBD of GGBP NBD-E149C / A213R / L238S in PBS buffer. During the incubation, the sensors were protected from ambient light. After approximately 2-8 hours of incubation, 50 uL of EDC / NHS (200 mM / 50 mM) were added to the incubation tube. After 5-40 minutes, the sensor tips were removed and placed in 50 uL of 1 mM ethanolamine, pH 8.5 to quench the reaction. After 20 minutes in the ethanolamine solution, the sensor pads were transferred to a PBS solution, where they were allowed to stand for at least 8 hours while the unreacted protdiffused. The sensors were then transferred to fresh PBS and stored in the dark until they were ready for use.

In a test of the above-described embodiment, the fluorescence signal was measured using an optical system that followed the configuration shown in Figure 2A. An LED of 470 nm (LS-450) was used for the excitation and, two photomultiplier tubes counters of individual photons as detectors of the electromagnetic energy. A commercial dichroic beamsplitter was used to reflect the 470 nm light from the electromagnetic energy emitter to the fiber and to transmit the luminescence signals from the reference indicator groups to the detectors. A second dichroic beam shredder was used to separate the luminescence signals from the indicator and reference groups, detecting the emission from NBD-E149C / A213R / L238S to a detector, and the emission from the GGBPTR-E149C to the other detector. The 550 nm bandpass filter in front of a detector and a 610 nm bandpass filter in front of the other detector were used to obtain more spectral resolution for the GGBP NBD-E149C / A213R / L238S and TRE-E149C, respectively.

In one test, the remote end and the sensing element of a biosensor formed in this way was inserted into the PBS buffer solutions containing different concentrations of glucose. The concentrations of glucose in the solutions were measured in a clinical analyzer. Figure 8 shows the response of the sensor to the changing concentrations of glucose. The signal at 550 nm of the IANBD indicator group tracks the changing concentrations of glucose. The emission at 610 nm of the Texas Red® indicator group is virtually unchanged when the glucose concentrations vary. However, in this modality, a part of the emission of the indicator group also occurs at 610 nm. The detector in the optical system that tracks the luminescence signals at 610 nm detects the emission of the reference group and also the part of the emission of the indicator group (IANBD) is presented in this wavelength region. Since the contribution of the indicator group to the signal at 610 nm is a constant fraction of the signal at 550 nm, this contribution can be mathematically subtracted from the signal at 610 nm to obtain only the signal from the reference group. When this mathematical manipulation is done, the signal at 610 nm is practically unchanging with the glucose concentration as shown in Figure 8.

Although the invention has been described herusing specific embodiments and embodiments thereof, it is possible to make numerous modifications and variations without departing from the scope of the invention as set forth in the claims.

Claims (48)

1. A device for determining a target analyte in a sample, which consists of: an optical conductor with a proximal end and a distant end; an optical system at the proximal end of the optical conduit containing at least one electromagnetic energy emitter and at least one electromagnetic energy detector; and a sensor element in optical proximity with the distal end of the optical conduit containing at least one binding protein adapted to bind with at least one target analyte and at least one reporter group associated with the binding protein, wherein the indicator group is adapted to undergo a luminescence change upon binding of the target analyte binding protein, and as an option, at least one reference group.

2. The device of claim 1 further contains a tip.

3. The device of claim 2, characterized in that the sensor element is contained within the tip.

4. The device of claim 3, characterized in that the sensor element is attached to the internal surface of the tip and the tip is directly attached to the distal end of the optical conduit.

5. The device of claim 1, characterized in that the sensor element is directly attached to the distal end of the optical conduit.

6. The device of claim 1 further contains one or more connectors.

7. The device of claim 6, characterized in that the sensor element is connected to the distal end of the optical conduit through a connector.

8. The device of claim 1, characterized in that the sensor element is trapped in or attached to a polymeric matrix.

9. The device of claim 8, characterized in that the polymer matrix is directly attached to the distal end of the optical conduit.

10. The device of claim 8, characterized in that the polymer matrix is attached to the inner surface of a tip, wherein the tip is attached directly to the distal end of the optical conduit.

11. The device of claim 8, characterized in that the polymer matrix is attached to the inner surface of a tip, wherein the tip is directly attached to a connector that is directly attached to the distal end of the optical conduit.

12. The device of claim 1, characterized in that the sensor element is connected to a polymer chain.

13. The device of claim 12, characterized in that the polymer chain is attached to the distal end of the optical conduit.

14. The device of claim 12, characterized in that the polymer chain is attached to the inner surface of a tip, wherein the tip is directly attached to the distal end of the optical conduit.

15. The device of claim 22, characterized in that the polymer chain is attached to the inner surface of a tip, wherein the tip is directly attached to a connector that is directly attached to the distal end of the optical conduit.

16. The device of claim 6, characterized in that the optical system is attached to the proximal end of the optical conduit by the connector.

17. The device of claim 6, characterized in that the sensor element is attached to the distal end of the optical conduit by a connector, and the optical system is attached to the proximal end of the optical conduit by a connector.

18. The device of claim 16 and 17, further consists of a tip, wherein the tip is attached to the distal end of the optical conduit through a connector.

19. The device of claim 1, characterized in that the optical conduit contains at least one optical fiber.

20. The device of claim 1, characterized in that the electromagnetic energy emitter is selected from the group consisting of an arc lamp, light emitting diode and laser diode.

21. The device of claim 1, characterized in that the electromagnetic energy detector is a photodiode, photomultiplier tube or charge coupling device.

22. The device of claim 1, characterized in that the optical system also contains optical elements adapted to distinguish multiple wavelengths.

23. The device of claim 22, characterized in that the optical elements also contain optical filters, dichroic components, holographic components or combinations thereof.

24. The device of claim 21, wherein the electromagnetic energy detector is adapted to detect energy emitted by the indicator group in a virtually continuous manner.

25. The device of claim 21, characterized in that the electromagnetic energy detector is adapted to detect the energy emitted by the indicator group periodically.

26. The device of claim 1, characterized in that the optical system consists of electrical or optoelectronic elements for modulating the signal from the electromagnetic energy emitter.

27. The device of claim 1, characterized in that the optical system further contains electrical or optoelectronic elements for modulating the luminescence signal received by the electromagnetic energy detector.

28. The device of claim 1, characterized in that the optical system is adapted to measure the intensity of the luminescence signal.

29. The device of claim 1, characterized in that the optical system is adapted to measure the wavelength of the luminescence signal.

30. The device of claim 1, characterized in that the optical system is adapted to measure the lifetime of the luminescence signal.

31. The device of claim 1, characterized in that the optical system is adapted to measure the polarization of the luminescence signal.

32. The device of claim 1, characterized in that the optical system is adapted to measure the energy transfer efficiency of the indicator group.

33. The device of claim 2, wherein the tip further comprises a metal frame.

34. The device of claim 1, characterized in that at least one reference group is associated with a protein.

35. The device of claim 1, characterized in that the sensor element is further adapted to be inserted in or through the skin of a patient.

36. The device of claim 1, characterized in that at least one indicating group and at least one reference group are excited at the same wavelengths.

37. The device of claim 1, characterized in that at least one indicating group and at least one reference group are excited at different wavelengths.

38. The device of claim 1, characterized in that the luminescence of at least one indicating group and at least one reference group is detected at different wavelengths.

39. The device of claim 1, characterized in that the luminescence of at least one indicating group and at least one reference group is detected at the same wavelengths.

40 The device of claim 1, characterized in that the indicator group consists of a pair of organic dyes chosen so that the energy transfer efficiency between the pair changes with the binding of the analyte.

41. The device of claim 1, characterized in that the reporter group consists of a pair of fusion proteins chosen so that the energy transfer efficiency between the pair changes upon binding of the analyte.

42. The device of claim 1, characterized in that the reporter group consists of an organic dye and a fusion protein, chosen so that the efficiency of the energy transfer between the organic dye and the fusion protein changes after the binding of the analyte.

43 The use of the device of any of claims 1-42 to detect the concentration of a target analyte in a sample.

44. The use of the device of any of claims 1-42 to detect the concentration of a target analyte in a sample, wherein the target analyte is glucose and the sample is a blood sample.

45. A method for detecting glucose concentration, which consists in inserting the device of any of claims 1-42 into a solution containing serum, and optically analyzing the glucose concentration in the solution containing serum.

46. One method for detecting glucose concentration is to penetrate the skin of a patient with the device in any of claims 1-42 and optically analyze the glucose concentration in the blood of the patient.

47. The method of claim 45 or 46, characterized in that the glucose concentration is periodically detected.

48. The method of claim 45 or 46, characterized in that it is detected continuously.