Entrapped binding proteins as biosensors

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DESCRIPTION

Binding proteins trapped as biosensors

Background of the invention

1. Field of the invention

The invention belongs to the field of Biotechnology. Specifically, the invention is directed to imprisoned mutated binding proteins, mutated binding proteins containing reporter groups, mutated binding protein compositions containing reporter groups in analyte-permeable matrices, and their use as analyte biosensors both in vitro and in vivo. .

2. Description of the relevant technique

Monitoring glucose concentrations to facilitate adequate metabolic control in diabetics is a desirable goal and would improve the lives of many individuals. Currently, most diabetics use the fingertip prick procedure to control their blood glucose levels, being its acceptance problematic by the patient due to the pain caused by frequent needle sticks (several times a day). Accordingly, efforts have been made to develop more efficient non-invasive or minimally invasive in vivo and in vitro procedures for frequent and / or continuous monitoring of glucose in blood or other glucose-containing biological fluids. Some of these more promising procedures involve the use of a biosensor. Biosensors are devices capable of providing specific quantitative or semi-quantitative analytical information using a biological recognition element combined with a transduction (detection) element.

The biological recognition element of a biosensor determines selectivity so that only the compound to be measured produces a signal. Selection can be based on biochemical recognition of the ligand, in which the chemical structure of the ligand (eg, glucose) remains unchanged, or on biocatalysis in which the biological recognition element catalyzes a biochemical reaction of the analyte.

The transducer translates the recognition of the biological recognition element into a semi-quantitative or quantitative signal. Possible transducer technologies are optical, electrochemical, acoustic / mechanical, or colorimetric. The optical properties that have been exploited include absorbance, fluorescence / phosphorescence, bio / chemiluminescence, reflectance, light scattering, and refractive index. Conventional reporter groups, such as fluorescent compounds, can be used or, alternatively, the opportunity exists to perform direct optical detection without the need for a label.

Biosensors specifically designed for glucose detection that use biological elements for signal transduction commonly use electrochemical or colorimetric detection of glucose oxidase activity. The use of this procedure is associated with difficulties that result from the influence of oxygen levels, the presence of inhibitors in the blood and problems with electrodes, among others. In addition, detection results in consumption of the analyte which can cause difficulties when measuring low glucose concentrations.

A rapidly advancing part of biosensor development is the use of fluorescently labeled periplasmic binding proteins. As published by Cass (Anal Chem. 1994, 66 3840-3847), the utility of a labeled maltose-binding protein (MBP) as a maltose sensor was demonstrated. In this work, MBP, which has no native cysteine ​​residues, was mutated to provide a protein with a single cysteine ​​residue at position 337 (S337C). The position of this mutation was in the maltose-binding cleft and underwent a large environmental change after maltose-binding. Numerous fluorophores were studied, some either blocked ligand binding or interfered with the change of protein configuration. Among those studied, M - ((2-iodoacetoxy) ethyl) -M-methyl) amino-7-nitrobenzoxadiazole (IANBD) produced a substantial increase in fluorescence intensity (160%) after binding to maltose. This result coincides with the change in location of the fluorophore from a hydrophilic or solvent-exposed medium to a more hydrophobic medium, as would have been theoretically predicted by closing the hinge after binding to maltose. In either case, this mutant protein and associated reporter group do not bind diagnostically to important sugars in mammalian body fluids. Cass also revealed the association of this protein with TiÜ2 surfaces. However, the protein bound to the surface underwent reduction in activity with time and required constant hydration (Analytical Chemistry 1998, 70 (23), 5111-5113).

Hellinga, et al., (US 6,277,627) published the engineering of a glucose biosensor by introducing a fluorescent transducer into a galactose / glucose binding protein (GGBP) mutated to contain a cysteine ​​residue, taking advantage of the large configuration changes that take place after binding with glucose. Hellinga et al (US 6,277,627) reveal that the transmission of configuration changes in mutated GGBPs can be exploited to create integrated signal transduction functions that convert a glucose binding to a change in fluorescence via a coupling mechanism. allosteric. Fluorescent transduction functions have been reported to minimally interfere with the intrinsic binding properties of the sugar binding pocket of GGBP.

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To accurately determine the glucose concentration of biological solutions, such as blood, interstitial fluids, eye solutions, perspiration, etc., it may be desirable to adjust the binding constant of the detection molecule of a biosensor to match the operating range. physiological and / or pathological of the biological solution of interest. Without the appropriate binding constant, the signal may be out of range for a certain physiological and / or pathological concentration. In addition, biosensors can be configured using more than one protein, each with a different binding constant to provide accurate measurements over a wide range of glucose concentrations as disclosed by Lakowicz (Us 6,197,534).

Despite the usefulness of mutated GGBPs, few of these proteins have been designed and examined, either with or without reporter groups. Site-specific mutations and / or the binding of certain reporter groups can act to modify a binding constant in an unpredictable way. Furthermore, a biosensor containing reporter groups may have a desirable binding constant, but not produce an easily detectable signal upon binding to the analyte. One of the primary factors that determines the sensitivity of a given reporter probe bound to a given protein for the detection of a specific analyte is the nature of the specific interactions between the selected probe and the amino acid residues of the protein. Currently, it is not possible to predict these protein interactions using computational procedures, nor is it possible to employ a rational design methodology to optimize the choice of indicator probes. Furthermore, it is not possible to predict the effect on either the binding constant or the selectivity based on the position of any indicator group or amino acid substitution in the protein (or vice versa).

To develop reagent-free, self-contained and / or implantable and / or reusable biosensors with the use of proteins, the transduction element must be in communication with a detection device to interrogate the signal going to and from the transduction element. . The most common procedures include placing proteins on or on the surface of optical fibers or planning waveguides using immobilization strategies. Such immobilization strategies include, without limitation, entrapment of the protein in semipermeable membranes, organic polymer matrices, or inorganic polymer matrices. The immobilization strategy used can ultimately determine the performance of a working biosensor. The prior art details numerous problems associated with immobilization of biological molecules. For example, many proteins undergo irreversible configuration changes, denaturation, and loss of biochemical activity. Immobilized proteins can exist in a large selection of possible orientations on any particular surface, for example, with some proteins oriented so that their active sites are exposed and others oriented so that their active sites are not exposed (and thus , are not capable of undergoing selective binding reactions with the analyte). Immobilized proteins are also subject to time-dependent denaturation, denaturation during immobilization, and leakage of the trapped protein after immobilization. This causes problems including, for example, the inability to maintain the calibration of the sensing device and signal drift. In general, binding proteins require orientation control to allow efficient use, therefore, physical absorption and random or bulk covalent binding to surfaces, or immobilization strategies such as those commonly shown in the literature do not give good results.

Several reports have been published on the encapsulation of proteins and other biological systems in simple inorganic silicon matrices formed by low temperature sol-gel process procedures (eg, Brennan, JD "Journal of Fluorescent" 1999, 9 (4 ), 295-312 and Flora, K .; Brennan, JD Analytical Chemistry 1998, 70 (21), 4505-4513). Some sol-gel matrices are optically transparent, making them useful in the development of chemical and biochemical sensors based on optical transduction, for example, absorption or fluorescence spectroscopic procedures. However, to be functional, trapped or immobilized binding proteins must still be capable of at least some analyte-induced configuration change. Changes of configuration of the binding proteins can be substantially restricted in most of the sol-gel matrices shown in the literature. It has been reported that sol-gel entrapped proteins can exhibit dramatically modified binding constants or binding constants that change over relatively short periods of time or under varying environmental conditions. Furthermore, it has been reported that the function of the protein entrapped in the sol-gel matrix is ​​time-dependent, a characteristic that limits the general applicability of sol-gels in biosensors for in vitro as well as in vivo use.

Therefore, there is a need in the art to design more useful mutated proteins and mutated GGBP proteins that generate detectable signals upon binding with an analyte for use as biosensors and, furthermore, there is a need to incorporate these proteins into matrices permeable to analytes to serve as an interface between the signal transmitting and receiving elements.

Summary of the invention

The disclosure provides entrapped or encapsulated mutated binding proteins and mutated binding proteins having reporter groups attached thereto for use as in vivo or in vitro biosensors. Furthermore, the disclosure provides a glucose biosensor that includes (a) a mutated binding protein and at least one reporter group attached thereto, such that said reporter group provides a detectable signal when said mutated binding protein is exposed. to glucose; and (b) an analyte-permeable matrix in which the mutated glucose / galactose binding protein and the reporter group are capable of being encapsulated in the matrix.

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The disclosure also provides compositions comprising a mixture that includes (a) at least one mutated glucose / galactose binding protein and at least one reporter group attached thereto; and (b) a hydrogel, dialysis membrane, sol-gel, or a combination thereof to provide an analyte-permeable matrix, in which the mutated glucose / galactose-binding protein and the reporter group are encapsulated in the matrix. .

The disclosure also provides a device that includes (a) a mutated maltose-binding protein (MBP) and at least one reporter group attached thereto, such that the reporter group provides a detectable signal when the mutated MBP binds to the maltose and wherein MBP includes a cysteine ​​present at position 337; and (b) a maltose permeable matrix, in which the mutated MBP and the reporter group are encapsulated in the matrix.

Furthermore, the disclosure provides a device and compositions thereof suitable for in vivo use that include (a) a mutated glucose / galactose binding protein and at least one reporter group attached thereto, such that the reporter group provides a detectable and reversible signal when the mutated glucose / galactose binding protein is exposed to varying concentrations of glucose; and (b) an analyte permeable matrix, in which the mutated glucose / galactose binding protein and reporter group are encapsulated in the matrix.

Brief description of the figures

FIG 1 illustrates the change in fluorescence response to glucose of GGBP-Ηβ with NBD A213C / L238C amide in solution.

FIG. 2 illustrates the increased signal from entrapped binding proteins in the absence and presence of analyte compared to solution.

FIG. 3 illustrates an entrapped binding protein in the absence and presence of analyte compared to solution.

FIG. 4 illustrates the reversible signal of a trapped binding protein of one embodiment of the present invention upon exposure to glucose solutions at the indicated concentrations.

Detailed description of the invention

The term "biosensor" refers generally to a device that uses specific biochemical reactions mediated by isolated enzymes, immunosystems, tissues, organelles, or whole cells to detect chemical compounds, usually by electrical, thermal, or optical signals. As used herein, a "biosensor" refers to a protein capable of binding to an analyte that can be used to detect the analyte or a change in analyte concentration by a detecting means as described herein. . More specifically, a biosensor of the invention includes at least one mutated glucose / galactose binding protein and at least one reporter group bound thereto and an analyte-permeable matrix, wherein the mutated glucose / galactose binding protein and the indicator group are capable of being encapsulated in the matrix.

The term "binding proteins" refers to proteins that interact with specific analytes in a manner capable of transducing or providing a detectable and / or reversible signal that differs well from a signal in the absence of analyte, from a signal in the presence of concentrations. analyte variables over time or in a concentration-dependent manner, by means of the procedures described. Transduction includes continuous, scheduled, and episodic procedures that include disposable or reusable applications. Reversible signal transduction can be instantaneous or time dependent, provided a correlation is established with the presence or concentration of the analyte. Binding proteins mutated in such a way are preferred to effect transduction.

The term "galactose / glucose binding protein" or "GGBP" or "maltose binding protein" or "MBP", as used herein, refers to a type of protein that occurs naturally in the periplasmic compartment of bacteria. These proteins are naturally involved in chemotaxis and transport of small molecules (eg, sugars, amino acids, and small peptides) in the cytoplasm. For example, GGBP is a single-chain protein consisting of two globular α / β domains that are connected by three chains to form a hinge. The binding site is located in the cleft between the two domains. When glucose enters the binding site, GGBP undergoes a configuration change, centered on the hinge, that joins the two domains and traps glucose at the binding site. X-ray crystallographic structures have been determined for the closed form of E coli GGBP (NK Vyas, MN Vyas, FA Quiocho Science 1988, 242, 1290-1295) and S. Typhimurium (SL Mowbray, RD Smith, L B. Cole Receptor 1990,1, 41-54) and are available in the protein database (http: //www.rcsb.org/.pdb/) as 2GBP and 3GBP, respectively. The DNA and amino acid sequence of GGBP from E. coli wild-type can be found at www.nc-bi.nlm.nih.gov/entrez/ accession number D90885 (genomic clone) and accession number 230520 (amino acid sequence). The preferred GGBP is derived from E. coli.

"Mutated binding protein" (eg, mutated GGBP "), as used herein, refers to bacterial binding proteins that contain one or more amino acids that have been substituted with, removed from, or added to the one (s). amino acids present in natural protein. Preferably, said substitutions, deletions

ES 2 380 922 T3 or insertions involve less than 5 amino acid residues, more preferably one or two residues. The binding protein mutation includes substitution of a non-reactive amino acid with a reactive amino acid to provide covalent bonding of electrochemical or photosensitive reporter groups. "Reactive" amino acid is intended to mean an amino acid that can be modified with a labeling agent analogous to cysteine ​​labeling with a thiol-reactive dye. Non-reactive amino acids include alanine, leucine, phenylalanine, and others, which have side chains that cannot be easily modified once they are incorporated into a protein (see Greg T. Hermanson, "Bioconjugate Techniques," Academic Press, 1996, San Diego , pp. 4-16 for classification of amino acid side chain reactivity).

Exemplary mutations of the GGBP protein include: a cysteine ​​substituted with a lysine at position 11 (K11C); an aspartic acid substituted cysteine ​​at position 14 (D14C); a cysteine ​​substituted with valine at position 19 (V19C); an asparagine substituted cysteine ​​at position 43 (N43C); a cysteine ​​substituted with a glycine at position 74 (G74C); a cysteine ​​substituted with a tyrosine at position 107 (Y107C); a cysteine ​​substituted with threonine at position 110 (T110C); a cysteine ​​substituted with serine at position 112 (S112C); a double mutant including a cysteine ​​substituted with a serine at position 112 and serine substituted with a leucine at position 238 (S112C / L238S); a cysteine ​​substituted with a lysine at position 113 (K113C); a cysteine ​​substituted with a lysine at position 137 (K137C); a cysteine ​​substituted with glutamic acid at position 149 (E149C); a double mutant including a cysteine ​​substituted with a glutamic acid at position 149 and a serine substituted with leucine at position 238 (E149 / L238S); a double mutant comprising a histidine substituted cysteine ​​at position 152 and a methionine substituted cysteine ​​at position 182 (H152C / M182C); a double mutant including a serine substituted with an alanine at position 213 and a cysteine ​​substituted with a histidine at position 152 (H152C / A213S); a cysteine ​​substituted with a methionine at position 182 (M182C); a cysteine ​​substituted with an alanine at position 213 (A213C); a double mutant including a cysteine ​​substituted with an alanine at position 213 and a cysteine ​​substituted with a leucine at position 238 (A213C / L238C), a cysteine ​​substituted with a methionine at position 216 (M216C); an aspartic acid substituted cysteine ​​at position 236 (D236C); a cysteine ​​substituted with a leucine at position 238 (L238C); a cysteine ​​substituted with an aspartic acid at position 287 (D287C); a cysteine ​​substituted with an arginine at position 292 (R292C); a cysteine ​​substituted with a valine at position 296 (V296C); a triple mutant including a cysteine ​​substituted with a glutamic acid at position 149, an alanine substituted with a serine at position 213 and a serine substituted with leucine at position 238 (E149C / A213S / L238S); a triple mutant including a cysteine ​​substituted with a glutamic acid at position 149, an arginine substituted with an alanine at position 213 and a serine substituted with leucine at position 238 (E149C / A213R / L238S); a quadruple mutant that includes a serine at position 1, a cysteine ​​at position 149, an arginine at position 213, and a serine at position 238 (A1S / E149C / A213R / L238S); a quadruple mutant that includes a serine at position 1, a cysteine ​​at position 149, a serine at position 213 and a serine at position 238 (A1S / E149C / A213S / L238S); and a quadruple mutant that includes a cysteine ​​at position 149, a cysteine ​​at position 182, a cysteine ​​at position 213, and a serine at position 238 (E149C / M182C / A213C / L238S). More examples are listed in Table 2 hereinafter. The amino acid residue numbers refer to the published sequence of E. coli having 309 residues, as detailed below, or the corresponding amino acid residue of any substantially homologous sequence from an alternative source (eg, glucose / galactose binding proteins from Citrobacter freundii or Salmonella typhimurium, accession numbers of the sequences P23925 and P23905, respectively).

The imprisoned or encapsulated mutated binding proteins of the present invention can be used in an analysis of analytes in vitro or in vivo that, for example, is capable of following the kinetics of biological reactions in which an analyte participates (e.g. ., glucose), as well as clinical trials and industrial testing of food or beverages. It is preferable that the concentration of the binding protein in the matrix is ​​less than the binding constant (Kd) of the protein with its analyte.

The mutation can have one or more different targets. For example, it is possible to mutate a natural protein to change the long-term stability of the protein; to conjugate the protein with a certain matrix or encapsulating polymer; to provide binding sites for detectable reporter groups; to adjust its binding constant with respect to a certain analyte; or any combination thereof.

In the present invention, the analyte and the mutated protein act as binding partners. The term "associates" or "binds", as used herein, refers to binding partners that have a relative binding constant (Kd) strong enough to allow detection of protein binding by methods. detection. Kd can be calculated as the concentration of free analyte to which half of the protein binds, or vice versa. When the analyte of interest is glucose, the Kd values ​​for the binding partners are preferably between about 0.0001mM and about 30mM.

In the present invention, it has been shown that mutated GGBPs can be used to detect glucose binding by linking them with a reporter group that provides a detectable signal upon glucose binding. That "provides a detectable signal", as used herein, refers to the ability to recognize a change in a property of a reporter group in a manner that allows for the detection of binding between ligand and protein. For example, in one embodiment, the mutated GGBPs comprise a detectable reporter group whose detectable characteristics are altered following a change in protein configuration that occurs upon binding to glucose. In a

In a preferred embodiment, the reporter group is a luminescent marker that produces a mutated GGBP with an affinity for glucose that exhibits a detectable change in luminescence characteristics upon binding to glucose. The change in detectable characteristics may be due to an alteration of the marker environment bound to the mutated GGBP.

The luminescent marker can be a fluorescent marker or a phosphorescent marker. The use of fluorescent labels that can be excited to fluorescence by exposure to light of certain wavelengths is preferred.

In one embodiment, the reporter group is a fluorophore. As used herein, "fluorophore" refers to a molecule that absorbs energy and then emits light. Non-limiting examples of fluorophores useful as indicator groups in the present invention include (tetramethylrhodamine-5-iodoacetamide), Quantum

M - ((2-iodoacetoxy) ethyl) -M-methyl) amino-7-nitrobenzoxadiazole (acrylodan), pyrene, Lucifer Yellow, -Cy5, fluorescein, Red®, (IANBD), Dapoxyl® coumarins, rhodamines, 5-TMRIA

Texas Red®, Cy3,

6-acryloyl-2-dimethylamino-naphthalene (2-bromoacetamidoethyl) sulfonamide, (M- (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a, 4a-diazas-indacene-2- il) iodoacetamide (Bodipy507 / 545 IA),

M- (4,4- difluoro-5,7-diphenyl-4-bora-3a, 4a-diaza-s-indacene-3-propionyl) -M-iodoacetylethylenediamine (BODIPY®

530/550 IA), 5 - ((((2-iodoacetyl) amino) ethyl) amino) naphthalene-1-sulfonic acid (1,5-IAEDANS) and carboxy-X-rhodamine, 5/6-iodoacetamide (XRIA 5 , 6). Preferably, IANBD is used. Many detectable intrinsic properties of a fluorophore reporter group can be monitored to detect glucose binding. Some of the properties that may change upon glucose binding include duration of fluorescence, intensity of fluorescence, anisotropy or polarization of fluorescence, and spectral changes of fluorescence emission. Changes in these properties of fluorophores can be brought about by changes in the medium of the fluorophore, such as those produced by changes in the configuration of the protein. Medium sensitive dyes, such as IANBD, are particularly useful in this regard. Other changes in the properties of fluorophores can be generated by interactions with the analyte itself or interactions with a second reporter group, for example, when a FRET (fluorescence resonance energy transfer) is used to monitor changes in the distance between two fluorophores.

Although the use of fluorescent labels is preferred, the use of other reporter groups is contemplated. For example, electrochemical indicator groups could be used in which an alteration of the indicator medium results in a change in the redox state of the indicator. Such a change can be detected, for example, by using an electrode.

Furthermore, the possibility of using other spectroscopically detectable markers, for example, markers detectable by NMR (nuclear magnetic resonance) is envisaged.

The reporter group can be attached to the mutated protein or GGBPs by any method known in the art. For example, the reporter group can be linked via amines or carboxyl residues to the protein. However, covalent coupling through thiol groups at cysteine ​​residues is especially preferred. In the present invention, for example, for mutated GGBP, cisternae located at position 11, position 14, position 19, position 43, position 74, position 107, position 110, position 112, position 113, position 137, are preferred. position 149, position 152, position 213, position 216, position 238, position 287 and position 292.

Any thiol-reactive group known in the art can be used to attach reporter groups, such as fluorophores, to a cysteine ​​of a genetically engineered or mutated protein. Lodoacetamide, bromoacetamide, or maleimide are widely known thiol-reactive moieties that can be used for this purpose.

Fluorophores that operate at long excitation or emission wavelengths (e.g., excitation or emission wavelengths of about 600 nm or greater) are preferred when the molecular sensor is to be used in vivo, e.g., incorporated into an implantable biosensor device (the skin being opaque below 600 nm). Currently, there are few environmentally sensitive probes available in this region of the spectrum and perhaps none with thiol-reactive functional groups. However, thiol-reactive derivatives of Cy-5 can be prepared, for example, according to the teachings of HJ Gruber, et al, Bioconjugate Chem., (2000), 11, 161-166. Conjugates containing these fluorophores, for example bound to various cysteine ​​groups contained in mutated GGBPs, can be screened to identify which ones produce the greatest change in fluorescence after binding to glucose.

The mutated GGBPs useful in the present invention can be genetically engineered or mutated to have a histidine marker at the N-terminal, C-terminal, or both terminals of the protein. Histidine fusion proteins are widely used in the field of molecular biology as an aid in protein purification. Exemplary labeling systems produce proteins with a label containing approximately six histidines, and preferably such labeling does not compromise the binding activity of the mutated GGBP.

As used herein, "matrix" refers to an essentially three-dimensional medium capable of immobilizing, trapping, or encapsulating at least one binding protein in order to measure a detectable signal from a ligand-protein interaction. The relationship between matrix constituents and binding protein includes, but is not limited to, covalent, ionic, and Van derWals interactions, and combinations thereof. The spatial relationship between the matrix and the binding protein includes the heterogeneous and homogeneous distribution in

ES 2 380 922 T3 and / or over any or all of the matrix volume. The matrix can be composed of organic, inorganic, glass, metal, plastic material, or combinations thereof. The matrix provides the binding protein with a transduction element configuration that can be incorporated, for example, into the distal end of a fiber or other small, minimally invasive probe to be inserted into a patient's tissue to allow a reading. episodic, continuous or programmed of the patient. The patient transduction element information can be provided, for example, by telemetry, a visual, audio or other means known in the art, for example, according to the teachings of US 5,517,313, US 5,910,661, US 5,894,351 and US 5,342,789, as well as Beach, RD, et al., "IEEE Transactions on Instrumentation and Measurement" (1999) 48, 6, p. 1239-1245. The information includes electrical, mechanical and actinic radiation appropriate to obtain the analyte concentration or change in concentration as appropriate.

In one aspect, the biosensor is used to detect the analyte in vivo. In this regard, the biosensor is encapsulated in a matrix that can then be used as an implantable device. The "matrix" can be in any desirable form, including one or more of a disk, cylinder, patch, nanoparticle, microsphere, porous polymer, open cell foam, as long as it is permeable to the analyte. The matrix also prevents leakage of the biosensor. The array allows light from optical sources or any other questioning light to or from the reporter group to pass through the biosensor. When used in an in vivo application, the biosensor will be exposed to a substantially physiological range of analyte, and determination or detection of a change in analyte concentration would be desirable, considering that determination or detection includes the means of detection. continuous, scheduled and episodic. Thus, the envisaged in vivo biosensor of the present invention comprises at least one mutated binding protein in an analyte-permeable encapsulating or entrapment matrix, such that the mutated binding protein provides a detectable and reversible signal when the mutated binding protein is exposed to varying analyte concentrations, and that the detectable and reversible signal can be related to the analyte concentration. Implantable biosensors can be implanted, in some embodiments, in or under the skin of a dermal-epidermal junction of a mammal to interact with interstitial fluid, tissue, or other biological fluids. Information obtained from the implant in the patient can be provided, for example, by telemetry, a visual medium, an audio medium, or other means known in the art, as set forth above.

Preferably, the matrix is ​​prepared with biocompatible materials or incorporates materials capable of minimizing adverse reactions with the body. Adverse reactions from implants include inflammation, protein encrustation, tissue necrosis, immune response, and leakage of toxic materials. Such materials or treatments are widely known and used in the art, for example, according to the teachings of Quinn, CP; Pathak, CP; Heller, A .; Hubbell, IA "Biomaterials" 1995, 16 (5), 3 89-396 and Quinn, CAP; Connor, RE; Heller, A. "Biomaterials" 1997,18 (24), 1665-1670.

The biosensor can be encapsulated in a matrix made substantially from hydrogel. The polymeric portion of the hydrogel may contain functionality that is suitable for hydrogen bonding or covalent coupling (eg, hydroxyl groups, amino groups, ether linkages, carboxylic acids and esters, and the like) with either the protein or with the reporter group.

In the present invention, a number of hydrogels can be used. Hydrogels can be, for example, polysaccharides, such as agarose, dextran, carrageenan, alginic acid, starch, cellulose or derivatives thereof, such as, e.g. g., carboxymethyl derivatives or hydro-expandable organic polymers, such as, eg. eg, polyvinyl alcohol, polyacrylic acid, polyacrylamide, polyethylene glycol, copolymers of styrene and maleic anhydride, copolymers of vinyl ether and maleic anhydride, and their derivatives. Derivatives that provide networks crosslinked by covalent bonds are preferred. The synthesis, and the biomedical and pharmaceutical applications of hydrogels on which they are based, which comprise polypeptides, have been described by numerous investigators. (See, p. eg, Biosensors Fundamentals and Applications, edited by ADF Turner, I. Karube and GS Wilson; published in Oxford University Press, in 1988). An exemplary hydrogel matrix derived from a water soluble UV crosslinkable polymer comprises poly (vinyl alcohol), M-methyl-4 (4'-formyl styryl) pyridinium-methosulfate-acetal (CAS Registry No. [107845-59-0 ]) available from PolyScience Warrington, PA.

In one embodiment of the encapsulation process, one or more hydrogels in water are added to the mutated binding protein in an aqueous buffer solution having a pH in the range of about 4 to about 10 depending on the protein. Subsequent drying of the matrix, for example crosslinking, gives it a physical shape. With the use of this technique and a conventional manufacturing procedure (p. For example, block casting, reverse emulsion polymerization, tracing or contact printing, melt-bed coating, or dip or spin coating), it is possible to obtain matrices of various configurations (e.g. granules , nanoparticles, microparticles, monoliths, and thick and thin films) suitable for in vitro and in vivo use.

In one embodiment, the matrix can be composed of modified sol-gels. Modified sol-gels include at least partial cured (or gelled) preparations composed of permeable metal oxide glass structures containing, in addition to the sol-gel precursor materials, preferably one or more organic components which hydrolytically condense together with the sol-gel precursor, so that the resulting sol-gel matrix confers properties suitable for, for example, implantation. Suitable properties include low volume reduction over time, resistance to cracking and other physical defects,

ES 2 380 922 T3 maintenance of protein function and compatibility with the protein and / or reporter group, and compatibility with the implantable animal or subject. Suitable organic materials include polyols, such as glycerol, ethylene glycol, propylene glycol, polyethylene glycol and the like, for example according to the teachings of Gill and Ballesteros, Journal of the American Chemical Society, 1998, 120 (34), 8587-8598. It is understood that those skilled in the art can appreciate that the attributes described are generally not predictable for a given protein / sol-gel / reporter group combination, thus optimization of the sol precursor materials can be expected. -gel, organic component and protein solution for any given binding-reporter protein pair. Applicants have found that such optimization can provide unexpected signal enhancement, change in binding constants, improved physical performance attributes of the matrix, and combinations thereof versus other matrices or aqueous solutions thereof. Optimization of the performance attributes of the protein-reporter pair and the functional performance attributes of the encapsulating matrix can be achieved, for example, by means of combinatorial or other design-based statistical procedures that are known in the art.

Sol-gel matrices useful for the present invention include material prepared by well-known conventional sol-gel processes, and include inorganic material, organic material, and mixed organic / inorganic material. Materials used to produce sol-gel can include, but are not limited to, aluminates, aluminosilicates, and titanates. These materials can be augmented with organically modified silicates (ormosyl) and functionalized siloxanes to provide the ability to confer and manipulate hydrophilicity and hydrophobicity, ionic charge, protein covalent bonding, and the like. As used herein, the term "hydrolytically condensable siloxane" refers to sol-gel precursors having a total of four substituents, at least one, preferably two, and most preferably three or four substituents, being alkoxy substituents covalently linked to silicone through oxygen and mixtures thereof. In the case of precursors with three, two and one alkoxy substituent, at least one of the remaining substituents is preferably covalently bonded with silicone through carbon, and the rest of the substituents contain an organic functionality selected from alkyl, aryl , amine, amide, thiol, cyano, carboxyl, ester, olefinic, epoxy, silyl, nitro and halogen.

In one embodiment of the encapsulation process, one or more of the hydrolytically condensable siloxanes are hydrolyzed in water, either spontaneously or under acid or base catalysis, forming derivatives with an organic polyol component present in a molar amount relative to the hydrolytically condensable siloxane. up to about 10: 1 to 1:10, preferably up to about 5: 1 to 1: 5; and most preferably, up to about 1: 1. To this mixture, prior to final gelation, mutated binding protein is added in an aqueous buffer solution having a pH in the range of about 4 to about 10 depending on the protein. At least partial condensation reactions give rise to the final matrices.

In another embodiment, the hydrolytically condensable siloxane hydrolyzed in water, either spontaneously or under acid or base catalysis to form derivatives with the organic polyol, is mixed with a water-soluble polymer component. Suitable water-soluble polymers include polyvinyl alcohol (PVA), sodium salt of poly (maleic acid-co-olefin) (PMSA), sodium salt of poly (vinyl sulfonic acid) (PVSA), and polyvinylpyrrolidone (PVP). Poly (maleic acid-co-olefin) includes copolymers of maleic anhydride with styrene, vinyl ester, and (C1-C8) olefins and their salts, for example, of sodium, potassium, ammonium, tetraalkylammonium, and the like. Preferably, the water soluble polymer component is 0 to about 30% by weight of the sol-gel composition.

In another embodiment, the hydrolytically condensable siloxane hydrolyzed in water, either spontaneously or under acid or base catalysis to form derivatives with the organic polyol, is mixed with one or more functionalized silicone additives (FSA) in amounts of proportions from 0 to about 0.6 mol% with respect to the hydrolytically condensable siloxane. Exemplary FSAs include alkyl derivatives, eg, methyltrimethosilane (MTMOS); amine derivatives, eg, 3-aminopropyl-triethoxysilane (ATEOS); and bis-silane derivatives, for example, (bis (3-methyldimethoxysilyl) propyl) polypropylene oxide (BIS).

In another embodiment, both the water-soluble polymer component and the functionalized silicone additive are mixed with the hydrolyzed hydrolytically condensable siloxane, either spontaneously, or under acid or base catalysis to form derivatives with the organic polyol, providing a suitable matrix to entrap or encapsulate the binding protein. With the use of the aforementioned sol-gel technique and a conventional manufacturing procedure (p. g., block casting, reverse emulsion polymerization, tracing or contact printing, melt bed coating, or dip or spin coating), it is possible to obtain airgel or xerogel matrices of various configurations (e.g. g., granules, nanoparticles, microparticles, monoliths, and thick and thin films) suitable for in vitro and in vivo use.

In another embodiment, the matrix can be formed from dialysis membranes. Dialysis membranes can be produced to encapsulate or physically imprison the protein. Covalent bonding with the membrane is considered within the scope of the described embodiment. The choice of the membrane has to be made based on the molecular weight cutoff so that the analytes of interest can easily pass through the membrane while the high molecular weight materials have restricted entry, or in the case of proteins binding partners, leave the membrane matrix. The required molecular weight cutoff would be such that it meets the above requirement and is common in the art. Commonly, membranes having a molecular weight cutoff of

ES 2 380 922 T3 between about 1,000 and about 25,000 daltons are suitable. Using this technique, matrices of various configurations and shapes suitable for use in vitro and in vivo can be prepared.

It is also contemplated that the matrices containing the binding protein and the reporter group are combinations of one or more hydrogels, sol-gels and dialysis membranes. For example, a protein entrapped or encapsulated in a hydrogel or a sol-gel can be placed on a dialysis membrane of a suitable shape and size, as well as allow implantation into a subject or manipulate the properties of mass transport or permeability to the matrix analytes.

Biosensors with binding proteins entrapped or encapsulated in matrices are capable of measuring or detecting analyte concentrations in the micromolar order (10^-6 molar) to molar without reagent consumption. In some embodiments, their sensitivity to the analyte may allow the use of biosensors to measure low concentrations of analyte known to be present in low volume samples of interstitial fluid. Implantable biosensors can be implanted, in some embodiments, in or under the skin of a dermal-epidermal junction of a mammal to interact with interstitial fluid, tissue, or other biological fluids. Protein-binding biosensors provide the means to monitor the analyte on a continuous, episodic, or on-demand basis as deemed appropriate by the user or according to the treatment of a condition.

In other embodiments, the sensitivity of the biosensors to analyte (eg, glucose) is such that they can be used to analyze levels of analyte in blood or to determine the concentration of analyte in a biological solution or other solution. As used herein, a "biological solution" includes, but is not limited to, blood, perspiration, and / or ocular or interstitial fluid, and combinations thereof.

Examples

The following examples illustrate certain preferred embodiments of the present invention, but are not intended to illustrate all embodiments. Mutated maltose binding protein MBP S337C was labeled with the fluorophore reporter probe NBD used herein according to the procedure set forth by Cass, A. et al. (Anal. Chem. 1994, 66, 3840-3847). Fluorescence emission spectra of the mutated tagged protein were measured using a SLM Aminco fluorimeter (Ontario, Canada) with slot settings of 8 and 4 for excitation and settings of 5 and 5 for the MC250 emission monochromator, in order to to compare the ligand binding performance of the fluorophore-labeled proteins entrapped in various matrices with the performance of the same proteins in solution. The initial fluorescence emission intensity is defined as I₀. The relative ratio of the maximum of the emission intensity in the presence of the respective protein ligand (If) to the absence of ligand (Io) is defined as AF.

Binding constants were determined by assessing increasing glucose concentrations in a protein solution with mixing after each glucose addition. The slot configurations were the same as listed above. Kd was determined from the following relationships adapted from Pisarchick and Thompson (1990):

F ^ + Fp-F ^ + x / Kj (1) where F is the fluorescence intensity, F_itlf is the fluorescence at infinity, F0 is the fluorescence at zero glucose and x is the free glucose concentration ([GLC] free) determined by the relationship:

<img file="ES2380922T3_D0001.tif" />

[6 £ d_or [Pradal - Kj + - EQ |} 2 + 4&gt; EL, where [GLC]_toty [Pro]_tot are the total concentrations of glucose and protein, respectively.

Example 1. This example describes the procedure for the expression and purification of mutant proteins without histidine markers. GGBP is encoded by the E. coli Mg1B-1 gene. This protein was modified by introducing the cysteine ​​amino acid at various positions by site-directed mutagenesis of the Mg1B-1 gene. These proteins were then expressed in E. coli and purified.

Mutagenesis of Mg1B-1 cassettes was performed as follows. The wild type Mg1B-1 gene was cloned into a pTZ18R vector (Dr. Anthony Cass, Imperial College, London, England). Mutant plasmids were generated from this parent plasmid using cassette mutagenesis producing randomized amino acid sequences, essentially as described by Kunkel (1991) and cloned into E. coli JM109 (Promega Life Science, Madison, WI). Mutant plasmids were identified by sequencing. The mutant protein was induced in JM109 and purified as described below. An E. coli JM109 colony containing the mutant plasmid was grown overnight at 37 ° C with shaking (220 rpm) in LB broth containing 50 pg / ml ampicillin (LB / Amp). The overnight growth was diluted 1: 100 in 1 L of fresh LB / Amp and incubated at 37 ° C with shaking until the OD600 of the culture was 0.3-0.5. The expression of the mutant was induced by adding a concentration

ES 2 380 922 T3 final 1mM IPTG (Life Technologies, Gaithersburg, MD) with continued incubation and shaking at 37 ° C for 4-6 hours. Cells were harvested by centrifugation (10,000 xg, 10 min, 4 ° C).

The mutant protein was harvested by osmotic shock and purified by column chromatography. The cell pellet was resuspended in a sucrose buffer (30mM Tris-HCl, pH 8.0, 20% sucrose, 1mM EDTA), incubated at room temperature for 10 min and then centrifuged (4000 xg, 15 min , 4 ° C). The supernatant was decanted and kept on ice. The cell pellet was resuspended, and the addition of 10 ml of ice cold sterile deionized H2O was repeated, and the suspension was incubated on ice and centrifuged. The remaining supernatant was mixed with other collected supernatants and recentrifuged once (12,000 xg, 10 min, 4 ° C). The mixed osmotic shock product was filtered through a 0.8 pm filter and then 0.45 pm. Streptomycin sulfate (Sigma Chemical Co., St. Louis. MO), 5% w / v, to the product of the osmotic shock and stirred once for 30 min followed by centrifugation (12,000 xg, 10 min, 4 ° C). The osmotic shock product was then concentrated using Amicon Centriprep 10 (PMCO 10,000) filters (Charlotte, NC) and dialyzed overnight against 5mM Tris-HCl, pH 8.0, 1mM MgCl2. The dialyzed osmotic shock product was centrifuged (12,000 xg, 30 min, 4 ° C). The resulting supernatant was used for a pre-equilibrated DEAE fast-flow sepharose column (Amersham Pharmacia Biotech, Piscataway, NJ) at 0.5 ml / min. The column was washed with 5-10 column volumes. A linear gradient of 0-0.2M NaCl was applied to the column and the fractions were collected. Fractions containing mutant protein were identified by SDS-polyacrylamide gel electrophoresis with Coomassie brilliant blue staining (MW approx. 32 kDa). Fractions were pooled and dialyzed overnight (4 ° C) against phosphate buffered saline (PBS) or 10mM ammonium bicarbonate (pH 7.4), concentrated using Amicon Centriprep 10 filters and stored at 4 ° C or -20 ° C with glycerol. The dialyzed protein was lyophilized with ammonium bicarbonate.

Example 2. The present example describes the expression and purification of mutant GGBPs containing histidine markers. Mutant GGBPs were genetically engineered either by site directed mutagenesis or by cassette mutagenesis. Site directed mutagenesis (QuikChange, Stratagene, La Jolla, CA) was performed to modify the individual amino acids of the pQE70 vector by replacing one amino acid with another, especially the selected amino acid. The cassette mutagenesis procedure (Kunkel 1991) was performed to randomize amino acids from a specified region of the GGBP gene. The mutated cassettes were then subcloned into the expression vector pQE70. The plasmid pGGBP-His contained the GGBP gene cloned in the expression vector pQE70 (Qiagen, Valencia, CA). This construct places six histidine residues at the C terminus of the GGBP gene. The strain of E. coliSG13009 to overexpress the mutant GGBP-His following standard procedures (Qiagen). Following overexpression of a 250 ml culture, cells were harvested by centrifugation (6,000 rpm) and resuspended in 25 ml of Bugbuster (Novagen, Madison, WI). Lysozyme (25 mg) was added to the lysate and the mixture was gently mixed at room temperature (RT) for 30 min. A clear lysate was produced by centrifugation (6,000 rpm) and to this, 0.5 ml of imidazole (1M) and 3 ml of Ni-NTA beads (Qiagen) were added. After 30 minutes of gentle mixing at RT, the mixture was centrifuged (6,000 rpm) and the lysate was removed. The beads were washed with 25 ml of solution (1M NaCl, 10mM Tris, pH 8.0) and centrifuged again. Mutant GGBP-His was eluted from the beads by adding 5 ml of solution (160mM imidazole, 1M NaCl, 10mM Tris, pH 8.0) and mixing for 15 min. The protein solution was immediately filtered through a Centriplus YM-100 filter (Amicon, Charlotte, NC) and then concentrated to 1-3 mg / ml using a Centriplus YM-10 filter. The protein was dialyzed overnight against 2 L of storage solution (1M NaCl, 10mM Tris, 50mM NaPO4, pH 8.0).

Example 3. The present example generically describes the labeling of the reporter probe binding protein. An aliquot of mutant GGBP containing cysteine ​​(4.0 nmol) in PBS was treated with 2mM dithiothreitol (5 µl, 10 nmol) for 30 min. A stock solution of

M, M'-dimethyl-M- (iodoacetyl) -M '- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) ethylenediamine (IANBD amide, 0.5 mg) in DMSO (100 pl, 11.9mM) and 3.36 pl (40 nmol) were added to the protein. The reaction continued at room temperature for 4 h on a Dynal rotary mixer in the dark. The labeled protein was purified by gel filtration on a NAP-5 column (Amersham Pharmacia). Labeling ratios were determined using an estimated extinction coefficient (50mM^-1 cm^-1) for GGBP calculated in GeneWorks 2.45 (IntelliGenetics), 8478 (IANBD amide) = 25mM^-1cm^-1) and an OD measurement for an IANBD amide standard solution at 280 nm and 478 nm. The protein dye concentration was calculated as C dye = A478 / 8478. The absorbance of the protein at 280 nm was calculated as Aprat (280) 280 = Atotal (280) Acolorante (280), where Acolorante (280) = A479 x (A280 / A478⁾coloring pattern. Therefore, the protein concentration was Cprot (₂so) = A_prat(₂₃₀)/8₂to₀. FIG 1 illustrates the change in fluorescence response to glucose concentration of a representative example, GGBP-H6 with NBD A213C / L238C amide in solution. Table 1 summarizes the change in fluorescence of various mutant GGBPs labeled with reporter groups, including reporter groups that have either excitations or an emission maximum of at least 600 nanometers. Table 2 summarizes the change in fluorescence and Kd values ​​determined from mutations of one, two, three, and four amino acid substitutions. These data clearly show that reporter group-tagged GGBP mutations can provide desirable attributes as glucose biosensors. The data show the relationship between the mutation and the reporter group of the samples tested.

ES 2 380 922 T3

<td colspan="7">TABLE 1 Percent Change in Fluorescence for GGBP Mutants¹</td>

<td>Colorant</td><td>Excitation / emission (nm)</td><td>S112C</td><td>M182C</td><td>A123C</td><td>A213C His6</td><td>M216C</td>

<td>IANBD Amida</td><td> 470/550</td><td> 0</td><td> 4</td><td> 3</td><td> 51</td><td> 7</td>

<td>IANBD Ester</td><td> 470/550</td><td></td><td></td><td></td><td></td><td></td>

<td>IAEDANS</td><td> 336/490</td><td> -7</td><td> -8</td><td> 0</td><td></td><td> -9</td>

<td>Bodipy 530/550 IA</td><td> 530/550</td><td> 7</td><td> -10</td><td> 33</td><td></td><td> 4</td>

<td>XRIA 5.6</td><td> 575 / 600</td><td> -21</td><td> -19</td><td> -38</td><td></td><td> -15</td>

<td>Lucifer Yellow IA</td><td> 426/530</td><td></td><td></td><td> -14</td><td></td><td> -3</td>

<td>Bodipy 507/545 IA</td><td> 507 / 545</td><td></td><td></td><td> 25</td><td></td><td> -3</td>

<td>Cy5</td><td> 640 / 660</td><td> 2</td><td> 0</td><td> 11</td><td></td><td> -7</td>

<td>Maleimide from Texas Red®</td><td> 580/610</td><td></td><td></td><td></td><td> -13</td><td></td>

<td>Dapoxil</td><td> 375/580</td><td> 15</td><td> 7</td><td> 12</td><td></td><td> 2</td>

<td colspan="7">0 to 1mM glucose F at [dye] = 0.5uM. Unless otherwise indicated, all mutants lacked histidine markers.</td>

Table 2. Summary of GGBP-H6 NBD mutations

<td></td><td>Solution</td><td></td><td></td><td>Sol-gel</td>

<td>ID</td><td>AF (%)¹</td><td>Kd (mM)²</td><td>Colorant / protein</td><td>AF (%) Kd (mM)</td>

<td>Natural type</td><td>intrinsic</td><td> 0,0002</td><td></td><td></td>

<td>A1C</td><td></td><td></td><td></td><td></td>

<td>A1S</td><td></td><td></td><td></td><td></td>

<td>A1 S, E149C, A213R, L238S</td><td> +21³</td><td></td><td> 0,31</td><td></td>

<td>A1S, E149C, A213R, L238S</td><td> +480</td><td> 0,37</td><td> 0,9</td><td></td>

<td>K11C</td><td> 10</td><td></td><td> 1,8</td><td></td>

<td>D14C</td><td></td><td></td><td> 1,5</td><td> 21</td>

<td>V19C</td><td> -56</td><td> 0,0001</td><td> 0,38</td><td> -0,99</td>

<td>N43C</td><td> 40</td><td> 0,0002</td><td> 0,28</td><td></td>

<td>G74C</td><td> -3</td><td> 0,0009</td><td> 1,43</td><td></td>

<td>Y107C</td><td> -30</td><td> 0,001</td><td> 0,93</td><td></td>

<td>T110C</td><td> -9</td><td></td><td></td><td></td>

<td>S112C</td><td> 220</td><td> 0,05</td><td> 1,15</td><td></td>

<td>S112C, L.238S</td><td> 6</td><td></td><td> 1,5</td><td></td>

<td>K113C</td><td> 15</td><td></td><td> 0,65</td><td></td>

ES 2 380 922 T3

<td>K137C</td><td> -5</td><td> 0,00004</td><td> 1,17</td>

<td>E149C</td><td> 300</td><td> 0,0002</td><td> 0,96</td>

<td>E149C, A213C</td><td> + 110</td><td></td><td> 0,70</td>

<td>E149C, A213R</td><td> 660</td><td></td><td> 1,1</td>

<td>E149C, A213S</td><td> 240⁴</td><td> 0,0023</td><td> 1,1</td>

<td>E149C, A213T</td><td> 350</td><td></td><td> 0,6</td>

<td>E149C, A213L</td><td> 280</td><td> 0,1</td><td> 1,1</td>

<td>E149C, A213Y</td><td> 280</td><td> 0,1</td><td> 1,1</td>

<td>E149C, A213C, L238C</td><td> +39³</td><td></td><td> 1,08</td>

<td>E149C, A213S, K223N</td><td></td><td></td><td></td>

<td>E149C, K223N</td><td> 260</td><td> 0,003</td><td> 0,7</td>

<td>E149C, L238C</td><td> 260</td><td> 5</td><td> 1,6</td>

<td>E149C, L.238S</td><td> 660⁴</td><td> 0,08</td><td> 1,36</td>

<td>E149C, K223N, N256R</td><td></td><td></td><td></td>

<td>E149C, N256S</td><td> 1</td><td></td><td> 0,93</td>

<td>B149C, N256R</td><td> 200</td><td> 742⁶</td><td> 0,9</td>

<td>E149C, M182C, A213C, L238S</td><td> 200</td><td> 216⁶</td><td> 3,2</td>

<td>E149C, A213S, L238S</td><td> 480</td><td> 0,47</td><td> 0,76</td>

<td>E149C, A213R, L238S</td><td> 500</td><td> 12</td><td> 1,1</td>

<td>H152C</td><td> 210</td><td> 0,07</td><td> 1,3</td>

<td>H152C, A213S</td><td> 100</td><td> 0,16</td><td></td>

<td>H152C, A213R</td><td> -3</td><td></td><td> 1,2</td>

<td>m52C, K223N)</td><td> 200</td><td> 0,003</td><td> 1</td>

<td>H152C, M182C</td><td></td><td></td><td></td>

<td>M182C</td><td> 11</td><td></td><td></td>

<td>A213C</td><td> 50</td><td> 0,124</td><td> 0,68</td>

<td>A213C, L238C</td><td> 24, 67³</td><td> 6</td><td> 1,4</td>

<td>A213C, L255C</td><td> -5</td><td></td><td> 0,98</td>

<td>M216C</td><td> 67</td><td> 0,008</td><td> 0,91</td>

<td>D236C</td><td> +2³</td><td></td><td> 0,43</td>

<td>L238C</td><td> -6,+3³</td><td>0.003 (SPR)</td><td> 1,3</td>

<td>D287C</td><td> 4</td><td></td><td> 1,1</td>

<td>R292C</td><td> -34</td><td> 0,0008</td><td> 1,5</td>

317

0,36

ES 2 380 922 T3

<td rowspan="2"></td><td colspan="4">(continuation)</td>

<td colspan="2">Solution</td><td colspan="2">Sol-gel</td>

<td>ID</td><td>AF (%)¹</td><td>Kd (mM)²</td><td>Colorant / protein</td><td>AF (%) Kd (mM)</td>

<td>V296C</td><td> -10</td><td> 0,00001⁵</td><td> 1,08</td><td></td>

'' Glc AF 0 to 1mM at [dye] = 0.5mM ²Kd measured at [dye] = 0.1mM ³Af when measured with Glc from 0 to 100mM ⁴Af when measured with Glc from 0 to 10mM ⁵Estimated; lime. with Sigma graph it was not convergent⁶Estimated; the curve did not reach saturation

Example 4. The present example describes the immobilization of a biosensor of the present invention using glycerol modified silicate condensate (GMSC). Glycerol additions modified the silicate condensate (GMSC). Glycerol additions were made directly after acid hydrolysis of tetraethoxyorthosilicate (TEOS) or tetramethoxyorthosilicate (TMOS). A range of hydrolysis times, pH levels, reagent addition order, and TEOS: glycerol ratios were evaluated to determine the optimal conditions to start the glyceration reaction. Preferred conditions were found using a 10 to 30 minute interval between hydrolysis and glycerol addition, a pH range of 0.5 to 1, and a 1: 1 molar ratio of TEOS to glycerol. The following information describes a modified Gill and Ballesteros procedure for a TEOS-based glycerol modified silicate condensate (GMSC) preparation, using the following reagent ratios: TEOS or TMOS: 1; H2O: 1, Methanol: 4, Glycerol: 1. TEOS or TMOS in methanol was added to a flask and cooled to 0 ° C on ice. Then 0.6M HCl was added dropwise to the solution. After 20 minutes of stirring, glycerol was added dropwise. The reaction was slowly heated over 1-2 hours at 20-25 ° C. Thereafter, the reaction vessel was heated further and was kept at a temperature range of 60-70 ° C under nitrogen for between 36 and 42 hours. The optimal duration was 40 hours. Observation of phase separation indicated incomplete glyceration for reactions stopped before 36 hours. Reactions held longer than 42 hours produced GMSC sol-gel monoliths with greatly reduced physical properties, eg, increased brittleness. After reaction for 40 hours at 60-70 ° C, the volume of the solution was reduced by rotary evaporation until it became viscous and clear, at which point methanol was added to the solution in a 4: 1 weight ratio. The GMSC solution was shown to be stable, providing consistent results for several months when stored at refrigerated temperature. When the GMSC solution was to be used, the methanol was removed by rotary evaporation and distilled water in a 1: 1 weight ratio was added to the GMSC reagent to catalyze final hydrolysis / gelation. Monoliths, thin films and powders were created with this procedure using an appropriate container as a mold. GMSC sol-gel monoliths were not found to be brittle and underwent approximately 8% shrinkage upon drying at 4 ° C at 50% relative humidity for 2 weeks (% shrinkage was the mean of changes in diameter and length measured with a microcalibrator and compared to the original dimensions of the mold). An electron microscopy (SEM) further illustrated the significant improvements in surface fracturing between monoliths created with TEOS hydrolysis and monoliths created by the GMSC procedure described above. This set of experiments demonstrates how sol-gels with better physical characteristics can be produced according to the procedures taught in the present invention.

Example 5. This example describes further optimization of physical properties using GMSC sol-gels in which glycerol has been partially substituted with either ethylene glycol (EG) or polyethylene glycol (PEG). Ethylene glycol (EG) was evaluated as a substitute for glycerol in mixtures in which the ratio between glycerol and EG had been varied, but the molar ratio between total glycerol and EG had remained constant relative to other reagents. The sol-gel monoliths were prepared by the procedure described in the previous example; were cured for two weeks at 4 ° C and a relative humidity of 50% and their% shrinkage was determined as shown in Table 3. The percentage of shrinkage is defined as the mean of the decrease in length and diameter against to original dimensions. The monoliths used for the determination of shrinkage had no protein / fluorophore. For F measurements, the samples listed in Table 1 containing GGBP-H6 H152 NBD (from Example 3) were prepared as described below.

ES 2 380 922 T3

Table 3. Mean% of contraction and AF of the sol-gel matrix after 2 weeks

<td>Sol-gel matrix</td><td>Average% shrinkage</td><td>F (Glucose 10mM)</td>

<td>1. Solution (NBD of GGBP-H6 H152 0.8-1.2uM)</td><td>Not applicable</td><td> 1,53</td>

<td>2. TEOS</td><td> 35,95 +/- 0,24</td><td> 1,39</td>

<td>3. GMSC-TEAO</td><td> 8,01 +/- 0,19</td><td> 1,57</td>

<td>4. GMSC-PMSA at 15% by weight and TEOS; 0.145 mol% MTMOS</td><td> 3,99 +/- 0,27</td><td></td>

<td>5. EG / GMSC 1% -TEOS</td><td> 3,10 +/- 0,17</td><td> 1,53</td>

<td>6. EG / GMSC 5% -TEOS</td><td> 2,48 +/- 0,15</td><td> 1,47</td>

<td>7. EG / GMSC at 10% -TEOS</td><td> -</td><td> 1,37</td>

<td>8. EG / GMSC at 20% -TEOS</td><td> -</td><td> 1,34</td>

The 1% and 5% EG / GMSC sol-gels (entries 5 and 6 respectively of Table 3) were found to have significantly lower% shrinkage than either the TEOS-only sol-gels or the sol - GMSC modified TEOS gels (entries 2 and 3 respectively of Table 3 above). Polyethylene glycol (PEG) was also qualitatively evaluated as a partial substitute for glycerol in similar proportions in GMSC sol-gels and produced monoliths with favorable surface properties and rubbery flexibility. In summary, partial replacement of either ethylene glycol (EG) or polyethylene glycol (PEG) with glycerol in GMSC sol-gels provides improvements in physical properties, eg, minimizes shrinkage and reduces surface cracking. These sol-gel matrices containing binding protein were found to have similar or better performance than protein in solution.

Example 6. Trapping of binding proteins in GMSC sol-gels containing functionalized silicone additives (FSA) and polymers. The present example describes the addition of organic polyol and polymer additives to optimize cGMS sol-gels to entrap binding proteins in order to both maintain and enhance their spectral properties after binding to ligands. Binding proteins were labeled with a fluorophore (as described in Example 3). Protein solutions were added during the final hydrolysis / gelation step described above to produce final protein concentrations of 2-4pM in the sol-gel. Polymer additives and functionalized silicone additives (FSAs) were purchased from Sigma-Aldrich Chemicals (St. Louis, MO). The polymeric additives were evaluated in amounts of between 0 and about 30% by weight. The FSAs were evaluated as additives added to the GMSC sol-gels in amounts of 0 to 0.6 mol%. Thus, rotary evaporation of the GMSC reagent to remove methanol from its storage solution was followed by reconstitution in water in a 1: 1 weight ratio. To a 400 µl aliquot of this mixture, 800 µl of buffer (HEPES, PBS, or Tris) with premixed water-soluble polymeric additive was added along with any FSA-modified GMSC. A mutated binding protein was then added in solution and after mixing well, 100 µl of the mixture was placed in a 96-well microplate (Falcon White Flat Bottom Plates, Product # 35-3941, BD Labware, NJ) . The sol-gel containing microplates were cured for 12-18 hours at 4 ° C and 50% relative humidity. GMSC-BIS was prepared by the same procedure as TEOS-based GMSC, but with replacement of the oxide (bis (3-methyldimethoxysilyl) propyl) polypropylene by TEOS. GMSC-MTMOS and GMSC-ATEOS were prepared in a similar manner, except that the hydrolysis was carried out with either 10% acid or no acid in the hydrolysis step, respectively, compared to the GMSC procedure based on TEOS. Fluorescence emission was measured with a Varian Cary Eclipse scanning fluorometer with a microwell plate adapter (Varian Instruments, Victoria, Australia). Excitation was at 475 nm and emission was recorded at 500 to 600 nm, commonly controlling the maximum fluorescence peak of the emission. The widths of the slots were 5 nm for excitation and 10 nm for emission. Individual Io determinations were made for each well and 100 µl of a ligand solution (1mM maltose in the case of MBP S337C) were added and If readings were obtained from which the AF values ​​were calculated. Proteins trapped in modified sol-gel showed a higher initial fluorescence (Io) in the absence of ligand compared to equivalent concentrations of the same protein in solution. Figure 2 shows the fluorescence emission before and after the addition of glucose for the NBD of GGBP His6 H152 in H152-optimized sol-gel and in solution. The Io spectra were normalized to a maximum of 1.0. The figure shows an approximately 2-3 fold increase in aF obtained for optimized sol-gel matrices containing binding protein when exposed to analyte compared to protein in solution. Thus, after optimization of the sol-gel formulations of each protein, an increase in FA was observed. It should be noted that the emission maximum can be shifted for samples of protein-reporter group trapped in sol-gel compared to those found in solution. Furthermore, these modified sol-gel matrices provide better physical properties as shown in entry 4 of Table 3. Table 4 shows an approximate range of formulation components that provide a best response for each of the individual proteins tested.

ES 2 380 922 T3

Table 4. Optimized sol-gel formulations for NBD-GGBP His6 H152C, NBD-GGBP His6 A213C / L238C and NBD-MBP-S337C

<td></td><td colspan="2">NBD-GGBP H152C</td><td colspan="2">NBD-GGBP A213C / L238C</td><td colspan="2">NBD-MBP S337C</td>

<td></td><td></td><td>Interval</td><td></td><td>Interval</td><td></td><td>Interval</td>

<td>Polymeric additive</td><td>PMSA</td><td>14-16% by weight</td><td>PMSA</td><td>4-5% by weight</td><td>PMSA</td><td>14-16% by weight</td>

<td>FSA additive</td><td>I rent</td><td>0.13-0.16 mole%</td><td>I rent</td><td>0.01-0.03 mol%</td><td>Amine</td><td>0.01-0.03 mol%</td>

<td>Tampon</td><td>Tris</td><td></td><td>PBS</td><td></td><td>PBS</td><td></td>

<td>PH range</td><td></td><td> 7,3-7,5</td><td></td><td> 7,4-7,7</td><td></td><td> 7,4-7,7</td>

<td>Kd (mM) [value in solution]</td><td colspan="2"> 0,36 [0,07]</td><td colspan="2"> 2,2 [6]</td><td colspan="2"> -</td>

<td>AF (increase versus dissolution) [sugar provocation]</td><td colspan="2">2.93x [10mM]</td><td colspan="2">2.36x [100mM]</td><td colspan="2">2.53x [0.1mM]</td>

<td colspan="7">(GGBP = glucose / galactose binding protein; MBP = maltose binding protein; NBD = M (acetoxy) ethyl) -M-methyl) amino-7-nitrobenzoxadiazole)</td>

In the formulation optimization experiments described above, Design-Expert 6.0.5 (Stat-Ease, Inc., Minneapolis, MN) was used to create various Designs of Experiments (DdE). Among the other variables of the formulation that were optimized in each DdE were the type of buffer (HEPES, PBS and T ris) and the pH (from 6.6 to 7.8). Surprisingly, the constituents of the formulations and the optimal concentration ranges were quite different for each protein. However, in all cases, substantial performance improvements were obtained for the optimized formulations compared to either performance in solution or performance in unmodified sol-gels.

Example 7. The present example describes the entrapment of GGBP H152C in a UV cross-linked hydrogel matrix and the effect of the matrix on the change in fluorescence and binding affinity. In this Polysciences Inc. SbQ-PVA experiment, 100 ul of PBS buffer was added and mixed for one hour on a rotary mixer. 80 ul of this solution was then mixed with 20 ul of labeled protein. The final protein concentration was determined spectroscopically at 0.15 mg / ml. After mixing, aliquots were placed in 96-well plates and dried in a chamber maintained at 20% humidity for 12 h, after which they were cured with UV light. Wells containing matrix encapsulated protein were challenged with 2 µl of 10mM glucose and compared to matrix-free protein solution having equivalent protein loading. Figure 3 shows the ability of the mutated protein matrix to respond to the analyte in a manner and with a sensitivity equivalent to that obtained in solution. The Kd of the trapped protein was comparable to that obtained in solution.

Example 8. The present example describes the immobilization of a biosensor of the present invention in a dialysis membrane matrix and the ability of the matrix to provide continuous and reversible readings. Using a Varian Eclipse fluorimeter with a fiber optic connection, GGBP L238C / A213C protein (2pM in PBS buffer) entrapped with a dialysis membrane having a 3,500 dalton molecular cutoff was fixed to the distal end of the fiber. Solutions were prepared containing PBS buffer, 2mM and 20mM glucose in PBS buffer. With the probe in the PBS solution, the readings were recorded at 0.02 second intervals of the emission wavelength of 521 nm, after which the fiber was inserted into the glucose solutions. Placing the fiber in the buffer only solution again produced the initial signal. Figure 4 represents multiple alternating cycles between the buffer and glucose solutions, demonstrating the reversibility of the biosensor trapped in a permeable matrix in a physiological range. Similar results were observed with the sol-gel trapped samples, demonstrating applicability for continuous use.