WO2000070343A2

WO2000070343A2 - G-protein coupled receptor based biosensors and sense replication systems

Info

Publication number: WO2000070343A2
Application number: PCT/US2000/013160
Authority: WO
Inventors: Luke V. Schneider; William F. Stahl, Iv
Original assignee: Repliscent, Inc.
Priority date: 1999-05-14
Filing date: 2000-05-11
Publication date: 2000-11-23
Also published as: AU4847600A; WO2000070343A3

Abstract

G-protein coupled receptor (GPCR) based sensors and sense replication systems are described. Methods for detecting and discriminating a stimulus corresponding to, or capable of mimicking and/or replacing a G-protein signal transduction system; and methods for mapping, transmitting and reproducing a selected stimulus at a remote location are described. The invention is useful in the electronic, telecommunications and entertainment industries, chemical (including contraband detection, food, flavors and fragrances) biological, medical and diagnostic techniques, as well as for drug discovery.

Description

G-PROTEIN COUPLED RECEPTOR (GPCR BASED BIOSENSORS AND SENSE REPLICATION SYSTEMS

Technical Field

The present invention relates generally to G-protein coupled sensory biochemistry. Particularly, the invention relates to methods for detecting and discriminating a stimulus corresponding to, or capable of mimicking and/or replacing a G-protein signal transduction system; and methods for mapping, transmitting and reproducing a selected stimulus at a remote location.

Background of the Invention Active biological sensors, such as sniffer dogs used for contraband detection and neuron on a chip (Fromherz, P. and A. Stett, Phys. Rev. Lett., 1995, 75(8): 1670- 1673; and Fromberz, P., et al, Science,l99l, 252:1290) or whole cell biosensors (Simpson, M. L., et al, Trend Biotechnol., 1998, 16:332-338) being developed have been shown to be responsive to chemical exposures 6 orders of magnitude below that of other artificial nose detectors. (Johnston, J.M., et al, Paper presented at the 5^th Intl Symp on the Analysis and Detection of Explosives, Washington, DC (Dec 4-8, 1995); and David Walt, Tufts University, quoted in: Photonics Spectra, p31 (Nov., 1996)). However, these current chip-based biological sensors try to infer discrimination from a single measurable macrocellular response (e.g., the change in the electrical firing pattern of a neuron). There is only one indicator for a multiplicity of chemicals, making these sensors virtually useless for chemical identification. Sniffer dogs must be trained to identify specific scents and have a limited detection repertoire. All active biological sensors must be kept alive to work, which limits the conditions under which the sensor can be fielded and the useful life of the sensor. (See Simpson supra).

Current odor detection methods and devices use sensors that operate by direct detection of chemicals at the sensor surface, therefore they are unable to amplify the signal. Detection is normally accomplished either by electrochemical or optical methods. (See e.g., U.S. Patent Nos. 4,770,027; 4,884,435; 5,090,232; 5,177,994; 5,541,851; 5,623,212; 5,675,070; 5,918,257; 5,928,609; 6,022,748 and 6,017,440). Spectroscopic methods, utilizing differences in light absorption or fluorescence are used to identify and quantify specific chemicals in controlled environments (e.g., where a background environmental signal is available for comparison). Similarly, different electrode materials and cyclic voltametry can be used to identify specific chemicals in controlled environments by electrochemical systems. Because of limitations in the intrinsic ability of these sensors to discriminate specific chemicals in complex backgrounds, chemical discrimination is usually enhanced by coupling sensors to an additional selective element, such as adsorbent polymers, chemical sensitive dyes, permeability selective membranes and chromatographic separations. (See Dickinson T. A., et al, Trends Biotechnol, 1998, 16(6):250-8 and Walt, D. R., et al, Biosens. Bioelectron., 1998, 3(6):697-9). Because these sensors directly detect the odorant chemical itself, a sufficient mass of odorant molecules must be accumulated to reach the detection threshold. This problem significantly restricts the sensitivity of these systems.

However, problems associated with reliable correlation and quantification of food (e.g., texture tests, concentrations of specific chemicals) to a human panel presents a challenge for conventional artificial nose technologies (Pan Demetrakakes, Food Processing, p95 (May 1996)). Many volatile chemicals entirely change their effect on flavor as their concentration changes. For example, hexanal, a substance formed as meats and other foods oxidize, smells like new-mown grass at low concentrations, and like turpentine at high concentrations. A human sensory panel is unlikely to describe this effect consistently.

Additionally, volatile chemicals cannot be directly related to smell and/or taste because food components are often perceived differently in combinations, suggesting that various odorant and/or flavarant molecules may compete for the same receptors (see Pan Demetrakakes, supra) . Thus even when individual volatile chemicals are identified and measured, their additive effect may be entirely different. For example, when sugar, salt, citric acid and caffeine are blended in the right proportions, the resulting compound is utterly tasteless. Therefore although each substance can be detected by chemical testing, blindfolded tasters presume they are drinking plain water.

Current artificial nose and other analytical sensors (e.g., microphones, photocells, photodiodes, strain gages, accelerometers, and the like) also lack the dynamic range exhibited by natural sensory systems. To detect over the dynamic range exhibited by mammalian sensory systems, arrays of sensors adapted to produce a monotonic response over limited overlapping ranges are required. Most analytical sensors exhibit dynamic ranges of 2-5 decades. However, the human eye and ear, the dog nose, and chemotactic response in bacteria remain sensitive to small changes in environmental stimuli, even when backgrounds of the stimuli change over up to 9 decades (Koshland, D. E., et al., Science, 217:220 (1982)). The ability of natural sensory systems to adapt to changes in background concentrations has not been duplicated so far in a single analytical sensor.

U.S. Patent Nos. 5,234,566 and 5,736,342 describe biosensors, such as coded ion channel biochips, that mimic the operation of ion channels responsible for transmission of impulses between neurons, wherein the biosensors are activated by cAMP in olfactory neurons. An electroosmotic gradient is established across a lipid membrane containing a selective ion channel, and the opening of the ion channel is regulated by a chemical-specific receptor (e.g., coupled to an olfaction receptor protein). The electroosmotic gradient is maintained and the current through the membrane is monitored; an increase in current corresponds to an opening of the ion channels. However, this system has several disadvantages: (1) the ion permeability of the membrane varies with temperature and absorbed volatile organic contaminants; (2) quantitative measurements are unreliable since the signal decays rapidly over time once the ion channel opens; and (3) the integrity of artificial lipid membranes is difficult to maintain in vitro.

Signal amplification techniques have been used extensively for detection and diagnostic purposes, e.g. the use of enzymes and polymerase chain reaction (PCR) in immunodiagnostics. However, lack of appropriate signal amplification techniques that enhance the sensitivity of chemical detection limit the effectiveness of existing artificial nose sensors. Thus, there is a need for improved and cost-effective biosensors that mimic both the sensitivity and selectivity of human olfaction. Summary of the Invention

The present invention relates generally to G-protein coupled sensory biochemistry. Particularly, the invention relates to systems and methods for detecting, discriminating and transmitting to a remote location a stimulus corresponding to, or capable of mimicking and/or replacing a G-protein signal transduction system; and methods for mapping, transmitting and reproducing a selected stimulus at a remote location. Further, the invention provides an improved, efficient and cost-effective sensor that mimics both the sensitivity and selectivity of human olfaction.

In one aspect, the invention relates to a system to detect, transmit and reproduce a selected stimulus comprising:

(a) a biosensor that mimics and/or replaces a biological signal transduction system, wherein the biosensor detects interaction of the selected stimulus and the signal transduction system, and measures a signal resulting from the interaction, wherein the measured signal provides information identifying the selected stimulus; (b) a mathematical coordinate system capable of codifying the information identifying the stimulus, and electronically recording and transmitting the codified information; and

(c) an emission device capable of transposing the codified information to deliver and reproduce the selected stimulus at a location remote in space or time. In preferred embodiments, the stimulus may be a physical or a chemical stimulus; preferably a stimulus that mimics natural senses of sight, hearing, smell, taste and/or touch. The system further comprises a biosensor comprising a biochemical element capable of mimicking and/or replacing a G-protein signal transduction system, and producing a secondary messenger; and a detector for detecting said secondary messenger. In preferred embodiments, a plurality of the homologous biochemical elements are arranged in an array, wherein the array comprises a plurality of discrete receptor proteins arranged in a spatially defined and a physically addressable manner, and in a manner suitable for conducting multiple assays to detect the affinity of a stimulus to the receptor proteins to determine a chemical coordinate space of said stimulus.

The mathematical coordinate system comprises an electronic signal proportional to the measured signal. The codified information comprises an electronic signal corresponding to relative amount of stimulant entities to be combined and transmitted by the emission device in order to reproduce said selected stimulus. The emission device comprises an array of a plurality of stimulant entities, wherein the stimulant entities are combined and delivered in an appropriate proportion to reproduce the selected stimulus. In preferred embodiments, the stimulant entities are odorant molecules or flavarant molecules, and the emission device reproduces odors or flavors.

In additional embodiments, the invention relates to a method of detecting the presence and/or amount of a target stimulus in an analyte, comprising: (a) providing an analyte suspected of containing the target stimulus;

(b) contacting an aliquot of said analyte suspected of containing said target stimulus with a plurality of receptor proteins; and

(c) detecting the affinity of said receptor proteins for the target stimulus to determine the presence and/or amount of said target stimulus in said analyte. In preferred embodiments, the receptor proteins comprise G-protein coupled receptors, and are present in a biosensor, wherein the biosensor mimics and/or replaces a biological signal transduction system; the target stimulus mimics and/or replaces natural senses of sight, hearing, smell, taste, and/or touch, wherein the target stimulus comprises a mixture of one or more stimulant entities. In certain embodiments, the method further comprises detecting the presence of a target stimulus exhibiting a biological activity using a high throughput screening assay. In preferred embodiments, the target stimulus comprises a mixture of one or more stimulant entities comprising a therapeutic or a diagnostic agent. In alternative embodiments, the method further comprises purifying the target stimulus by affinity purification, wherein the target stimulus comprises a mixture of one or more stimulant entities.

In alternative embodiments, the invention relates to a method for mapping a stimulus in a mathematical coordinate space comprising:

(a) detecting the affinity of the stimulus with a plurality of receptor proteins, wherein the stimulus comprises a mixture of one or more stimulant entities;

(b) measuring the intensity with which the mixture of stimulant entities binds to said receptor protein to determine a chemical coordinate space; and (c) transposing the chemical coordinate space to a digital or an analog coordinate space.

In preferred embodiments, the invention relates to a method for reproducing a selected odor or flavor at a remote location comprising: (a) providing a plurality of odorant or flavarant molecules,

(b) mapping an odor or flavor by relative amount of each odorant or flavarant molecule to be combined in order to reproduce the selected odor or flavor; and

(c) delivering an appropriate proportion of the odorant or flavarant molecules to reproduce said selected odor or flavor according to the algorithm defined in Equation 7.

In preferred embodiments the odorant and/or flavarant molecules are delivered by an emission device comprising an ink jet printer, a pneumatic nebulizer, an ultrasonic nebulizer or an electrostatic printer. These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.

Brief Description of the Figures

Figure 1 is a schematic of an optical microwell array detector (biochip reader) suitable for use with the present artificial nose system.

Figure 2 is a schematic of human olfactory biochemistry. A single binding event at the olfaction receptor results in multiple copies of cAMP produced by the associated adenylate cyclase enzyme. The GTP/GDP ratio regulates the sensitivity of the signal transduction system through the associated G-protein. The cAMP produced is detected by enzymatic or immunodiagnostic methods.

Figure 3 shows the predicted variation in the fractional receptor activation (R^*/R_τ) as a function of receptor protein affinity for the odorant molecule (K₀R_T) and the relative odorant molecule concentration (O_τ/R_τ).

Figure 4 shows the predicted dynamic range of fractional olfactory adenylate cyclase activation (AC^*/AC_T) as a function of the relative GTP/GDP concentration ratio and fractional activation of the odorant receptor protein (R^*/R_τ). Figures 5A-5C show an example of the use of the biochip reader, illustrated in Figure 1, with a chemiluminescent assay. Figure 5 A shows the performance of the biochip reader for the measurement of chemiluminescent assay kinetics in a 250 nL microwell array; Figure 5B depicts the reaction representing the chemiluminescent assay kinetics; and Figure 5C depicts the relative rate of decomposition representing the chemiluminescent assay kinetics.

Figure 6 shows the adjustment of the actual dynamic range of an olfactory GPCR assay by varying the GTP/GDP ratio in the assay mixture.

Figure 7 illustrates a computerized driver system for adjusting quantities of individual odorant molecules delivered from an ink jet printer modified for emission of odors.

Description of the Invention

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, and medicine, including diagnostics, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Fieser and Fieser's Reagents for Organic Synthesis, Wiley & Sons, New York, 1991, Volumes 1-15; Rodd's Chemistry of Carbon Compounds, Elsevier Science Publishers, 1989, Volumes 1-5 and

Supplementals; Organic Reactions, Wiley & Sons, New York, 1991, Volumes 1-40; Solid-Phase Synthesis, Blossey, E. C. and Neckers, D. C. Eds. 1975; Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual; DNA Cloning, Vols. I and II (D. N. Glover ed.); Oligonucleotide Synthesis (M. J. Gait ed.); and Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds.).

A. Definitions

In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below. It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a receptor" includes a mixture of two or more such receptors, "an odorant molecule" includes a mixture of two or more odorant molecules, and the like.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. The following terms are intended to be defined as indicated below.

As used herein, the term "stimulus" refers to a sensory and cellular signaling system, including intracellular and extracellular signaling systems, occurring in mammals, including humans. The stimulus may be a physical or a chemical stimulus corresponding to, or capable of mimicking and/or replacing G-protein signal transduction systems. For example, a stimulus mimics natural senses of sight, hearing, smell, taste, and/or touch.

As used herein, the term "physical stimulus" refers to a stimulus comprising a mixture of one or more stimulant entities such as light, sound, temperature, pressure and the like. The physical extracellular stimuli light, sound, temperature and pressure correspond to the natural senses of sight, hearing and touch, respectively.

As used herein, the term "chemical stimulus" refers to a stimulus comprising a mixture of one or more stimulant entities such as an odorant molecule, a flavarant molecule, and the like. The chemical extracellular stimuli, odor and flavor correspond to the natural senses of smell and taste, respectively; wherein the type and/or concentration of an odorant or a flavarant molecule determines the odor or flavor, respectively.

As used herein, the term "stimulant entities" refers to a molecule or force capable of having a physical or extracellular stimulus, as defined above, either by itself or in combination with other stimulant entities. Examples of stimulant entities capable of having physical stimuli include, but are not limited to, light, sound, temperature, pressure and vibrations. Preferably, physical stimuli are within the electromagnetic sensory range of mammals. In more preferred embodiments, light stimuli have a wavelength ranging from about 200 nm to about 1 micron; temperature stimuli generate a surface temperature at the biosensor in the range of about -5°C to about 95°C; sound stimuli have a frequency ranging from about 1 Hz to about 1000 Hz; vibration stimuli have a frequency ranging from about 10 Hz to about 10,000 Hz; and pressure stimuli have a force at the surface of the detector between about 0.01 psi to about 1000 psi. Examples of stimulant entities capable of having chemical stimuli (interchangeably referred to as "stimulant molecules") include, but are not limited to odorant molecules, flavarant molecules (including odorant and flavarant molecules that impart undesired odor or flavor) and pseudo-scents, explosives, contraband drugs such as controlled substances, drugs of abuse and narcotics, hormones, therapeutic agents, diagnostic agents, extracellular metabolites, e.g., metabolites (e.g., glucose) that induce a biochemical response in a cell, viruses and antigens. Preferably, chemical stimuli have a concentration ranging from about 15 moles/liter to about 1 mole/liter in a liquid phase at the surface of the biosensor. More preferably, chemical stimuli have a concentration ranging from about 12 moles/liter to about 0.001 moles/liter in the liquid phase at the surface of the detector. When contacted with a liquid phase, chemicals present in a vapor or a gas phase, equilibrate with dissolved molecules in the liquid phase. Receptor proteins, such as olfactory or taste receptor proteins, are modified using standard molecular biology techniques. The modified receptor proteins are used to detect a variety of organic chemicals, including chemicals that do not have a discernable odor or flavor.

As used herein, the term "odorant molecule" refers to a molecule capable of having a scent/odor, either by itself or in combination with other odorant molecules. As used herein, the term "odor" refers to a combination of one or more odorant molecules that correspond to or mimic the natural sense of smell (e.g., a sensation in the nose).

As used herein, the term "flavarant molecule" refers to a molecule capable of having a flavor, either by itself or in combination with other flavarant molecules. As used herein, the term "flavor" refers to a combination of one or more flavarant molecules that correspond to or mimic the natural sense of taste (e.g., a sensation on the tongue). Examples of flavors include salty, sour, tangy, piquant, zesty, spicy, savory, sweet, bitter and umami.

An "antigen" is defined herein to include any substance that may be specifically bound by an antibody molecule. An "immunogen" is an antigen that is capable of initiating lymphocyte activation resulting in an antigen-specific immune response. As used herein, the term "biological signal transduction system"refers to a system wherein an extracellular stimulus, including a physical and a chemical stimulus as described above, is converted to a secondary messenger capable of relaying information to other intracellular mechanisms (e.g., ion channels, sigma factors that alter gene expression, and allosteric regulators of enzyme activity). In preferred embodiments, the signal transduction system is a G-protein signal transduction system. As illustrated in Figure 2, the G-protein signal transduction system is characterized by a complex of three proteins located in or attached to the cell membrane: (1) a G-protein coupled receptor (GPCR) that is specific for a chemical or physical extracellular stimulus; (2) an enzyme (such as adenylate cyclase) that produces a secondary messenger inside the cell in response to activation of the GPCR; and (3) a GTP binding protein (G-protein) that mediates the interaction between the GPCR and enzyme.

As used herein, the term "biosensor" refers to a sensor that corresponds to, mimics and/or replaces a biological signal transduction system, as described above. The biosensor detects the interaction of a selected stimulus, as described above, and a signal transduction system, and measures the resulting signal to provide information identifying the selected stimulus.

As used herein, the term "cAMP" refers to cyclic adenosine monophosphate (cAMP). The amount of c AMP produced is proportional to the amount of stimulant entity, e.g., an odorant molecule, bound to the associated receptor, e.g. an olfactory receptor.

As used herein, the term "GTP/GDP ratio" refers to the ratio of the amount of GTP to the amount of GDP present in an array element of a biosensor. The GTP/GDP ratio regulates the sensitivity of the signal transduction system through the associated G-protein. Changes in the GTP/GDP ratio between otherwise identical array elements of the biochemical sensor are used to alter the dynamic range of the sensor.

As used herein, the term "activation of the receptor" refers to conformational changes in the receptor protein that are induced by the presence or interaction with a physical stimulus or the binding of a chemical stimulus. Such activation causes the receptor protein to induce physical or chemical changes in other proteins of the signal transduction system, ultimately resulting in the production of a secondary messenger. For example, binding of a stimulant entity, such as an odorant or a flavarant molecule, to the receptor protein is an equilibrium process represented as a function of the total stimulant entity concentration (O_τ) and receptor protein concentrations (R_τ) as described in Equation 4. A single binding event at the receptor results in multiple copies of a secondary messenger, such as cAMP, produced by the associated enzyme, such as adenylate cyclase. The GTP/GDP ratio, as described above, regulates the sensitivity of the signal transduction system through the associated G-protein. The secondary messenger, e.g., cAMP, produced is detected by a detector, as described in greater detail below.

As used herein, the term "biochemical element" refers to a concerted set of associated proteins comprising (1) a receptor site; (2) an enzymatic site that produces a secondary messenger only upon stimulant entity activation or binding to the receptor; and, (3) optionally, a allosteric regulation site capable of modulating the interaction between stimulant activation of the receptor site and secondary messenger enzyme activity. The biochemical element is capable of converting an extracellular stimulus in a biological signal transduction system to a secondary messenger, as described above, thus further amplifying the measured signal. In preferred embodiments, the biochemical element is capable of mimicking and/or replacing a G- protein signal transduction system. A biochemical element comprises (1) a G-protein; (2) a G-protein coupled receptor protein corresponding to the G-protein; and (3) an enzyme corresponding to the G-protein, wherein the enzyme produces the secondary messenger in response to stimulation of the receptor. Examples of biochemical elements include homologous biochemical elements comprising olfactory receptor proteins, receptor proteins of the visual cortex, taste receptor proteins, and the like. Homologous biochemical elements are defined as members of a class of receptors associated with a particular sense, such as olfactory receptor proteins, that interact with the same G-protein and enzyme producing the same secondary messenger. In preferred embodiments, homologous receptors exhibit multiple, e.g., seven transmembrane sequences with about 50-100 % sequence homology, preferably 65-100%, more preferably 75-100% and even more preferably 80-100% sequence homology; and extramembrane sequences that may exhibit little or no sequence homology and determine the specificity of the receptor protein. A biochemical element has a dynamic range which can be adjusted by varying the concentration or ratio of allosteric regulators; a dynamic range is the concentration range of a stimulant entity over which the amount or rate of secondary messenger production is proportional to the concentration of the stimulant entity. In preferred embodiments, the dynamic range of G-protein signal transduction systems is varied by adjusting the GTP/GDP ratio and/or GTP concentration. Preferably, the GTP/GDP molar ratio is varied between about 1000:1 and 1:1000 and the GTP concentration is varied between about 0.001 millimolar and 1 molar. In certain embodiments, biochemical elements are sensitized, by genetic engineering methods, to stimuli undetectable by natural senses. Genetic engineering methods involve altering the protein sequences of the variable stimulant receptor sites of the receptor protein by random point mutation, homologous recombination, error prone polymerase chain reaction amplification, and other methods commonly used in combinatorial biology. As used herein, a "detector" refers to a device for detecting the presence and/or concentration of a desirable characteristic. Particularly, the detector measures the concentration of the secondary messenger, as described above. Examples of a detector include, but are not limited to, a spectroscopic detector, a radiochemical detector, an electrochemical detector and an amplifying biochemical assay. Examples of an amplifying biochemical assay include, but are not limited to, an immunoassay, a protein kinase assay and a membrane ion channel assay.

The term "analyte" refers to a sample derived from a variety of sources such as from drugs, veterinary materials, herbicides, pesticides, perfumes, scents, odors, hormones, food stuffs, environmental materials, a biological sample or solid, such as tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro cell culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components). As used herein, the term "biological activity" refers to activity that reduces, inhibits, prevents and/or alleviates the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

As used herein, the term "mapping" refers to the process of transposing coordinates from a chemical coordinate space of a stimulant entity, e.g. odorant or flavarant molecule, such as found in an array on a biochip, to a digital or an analog coordinate space, and is used to integrate the coordinates with the emission site in order to transmit the stimulus, e.g., odor or flavor, electronically to a remote site.

As used herein, the term "mathematical coordinate system" refers to a system comprising an electronic signal proportional to the measured signal. The mathematical coordinate system is capable of (1) codifying the information in the measured signal, wherein the codified information comprises an electronic signal corresponding to relative amount of stimulant entities to be combined and transmitted by an emission device in order to reproduce a selected stimulus; and (2) electronically recording and transmitting the codified information.

As used herein, the term "chemical coordinate space" refers to an N-dimensional mathematical matrix of numerical values that uniquely represent a stimulant entity, preferably a chemical. In certain embodiments, the number of matrix dimensions are determined by the number of different receptor proteins used in the biosensor. The numerical value at each matrix location is proportional to the amount of stimulant entity interacting with the receptor. In alternative embodiments, the number of matrix dimensions are determined by the number of different stimulant entities used in an emitter device to mimic the stimulus and the numerical value at each matrix location is proportional to the relative or absolute amount of specific stimulant entities needed to mimic the stimulus.

As used herein, the term "digital or an analog coordinate space" refers to the representation of the mathematical coordinate system as an electronic signal suitable for analog (e.g., by amplitude or frequency modulation) or digital transmission. Digital transmission is preferentially in binary, hexadecimal, or ASCII formats. As used herein, the term "emission device" refers to a device comprising an array of a plurality of stimulant entities, wherein the device is capable of converting the codified information to combine and deliver the stimulant entities in an appropriate proportion in order to reproduce the desired stimulus. Examples of an emission device include, but are not limited to, an ink jet printer, a pneumatic nebulizer, an ultrasonic nebulizer and an electrostatic printer.

As used herein, the term "array" is defined as a collection of separate receptors, each arranged in a spatially defined and a physically addressable manner. The number of receptors that can be deposited on an array will largely be determined by the surface area of the substrate, the size of a receptor and the spacing between receptors.

As used herein, the term "screening" refers to determining the presence and/or biological activity of a target stimulus in an analyte. For example, a target stimulus may be screened for efficacy or toxicity for a certain biological indication.

As used herein, the term "ScentScan™ Technology" refers to a technology to detect odor. ScentScan™ is a unique artificial scent technology wherein mammalian, preferably human, olfactory receptors are linked to signal transduction/amplification cascade as the basis of odor detection.

As used herein, the term "TasteScan™ Technology" refers to a technology to detect flavor. TasteScan™ is a unique artificial taste technology wherein mammalian, preferably human, taste receptors are linked to signal transduction/amplification cascade as the basis of flavor detection. As used herein, the term "ScentSpace™ Technology" refers to a quantitative mathematical coordinate system that enables electronic recording, dissemination and/or digital transmission of odors. ScentSpace™ technology is a system for representing odors as a set of mathematical coordinates, wherein odorant molecule libraries are screened for molecules to determine various combinations of the odorant molecules that mimic and/or reproduce other odors, and the proportion of odorant molecules required to match a specific odor coordinate, wherein two or more odorant molecules are combined according to an algorithm.

As used herein, the term "TasteSpace™ Technology" refers to a quantitative mathematical coordinate system that enables electronic recording, dissemination and/or digital transmission of flavors. TasteSpace™ technology is a system for representing flavors as a set of mathematical coordinates, wherein flavarant libraries are screened for molecules to determine various combinations of the flavarant molecules that mimic and/or reproduce other flavors, and the proportion of flavarant molecules required to match a specific flavor coordinate, wherein two or more flavarant molecules are combined according to an algorithm.

As used herein, the term "ScentEmit™ Technology" refers to a technology for scent reproduction. ScentEmit™ technology is a scent reproduction technology and a delivery system wherein the individual odorant molecules or odors are mixed in different concentrations to replicate other scents.

As used herein, the term "TasteEmit™ Technology" refers to a technology for taste reproduction. TasteEmit™ technology is a taste reproduction technology and a delivery system wherein the individual flavarants or flavors are mixed in different concentrations to replicate other flavors.

B. General Methods

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or process parameters as such may, of course, vary. It is also to be understood that the terminology and examples used herein are for the purpose of describing particular embodiments of the invention only, and are not intended to be limiting. For example, similar devices and/or systems can be derived for any of the natural senses, such as sight, hearing, smell, taste and touch.

Although a number of compositions and methods similar or equivalent to those described herein can be used in the practice of the present invention, the preferred materials and methods are described.

The present invention relates generally to G-protein coupled sensory biochemistry. Particularly, the invention relates to systems and methods for detecting, discriminating and transmitting to a remote location a stimulus corresponding to, or capable of mimicking and/or replacing a G-protein signal transduction system; and methods for mapping, transmitting and reproducing a selected stimulus at a remote location. Biological signal transduction systems are ubiquitous in the mammalian body.

In general, the system converts an extracellular stimulus, including a physical and a chemical stimulus as described above, to a secondary messenger capable of relaying information to other intracellular mechanisms. In preferred embodiments, the signal transduction system is a G-protein signal transduction system.

G-protein signal transduction systems are ubiquitous in sensory and cellular signaling systems in mammals, including humans. The G-protein coupled receptor (GPCR) system forms the biochemical basis for the natural senses, such as sight, hearing, smell, taste and touch; and various intercellular signaling systems in the body, such as hormonal systems, and the like. In a GPCR system an extracellular stimulus, including (1) a physical stimulus such as light, sound, temperature, pressure and the like, and (2) a chemical stimulus, such as an odorant or a flavarant molecule is converted to an intracellular chemical signal capable of relaying information to other intracellular mechanisms (e.g., ion channels, sigma factors that alter gene expression, or allosteric regulators of enzyme activity). The physical extracellular stimuli comprises a mixture of one or more stimulant entities, such as light, sound, temperature and pressure, and correspond to the natural senses of sight, hearing and touch, respectively. The chemical extracellular stimuli comprise a mixture of one or more stimulant entities such as, odorant and flavarant molecules, and correspond to the natural senses of smell and taste, respectively; wherein the type and/or concentration of an odorant or a flavarant molecule determines the odor or flavor, respectively. As illustrated in Figure 2, the G-protein signal transduction system is characterized by a complex of three proteins located in or attached to the cell membrane: (1) a G-protein coupled receptor (GPCR) that is specific for a chemical or physical extracellular stimulus; (2) an enzyme (such as adenylate cyclase) that produces a secondary chemical messenger inside the cell in response to activation of the GPCR; and (3) a GTP binding protein (G-protein) that mediates the interaction between the GPCR and enzyme.

The only mammalian senses, particularly human senses, not currently mimicked by a man-made sensor are smell and taste. The lack of a robust sensor that mimics human olfaction and/or tongue has inhibited the development of systems that can transmit and reproduce the sense of smell and taste, respectively. The present invention is based on the discovery of methods for mimicking G-protein signal transduction systems, wherein a biosensor detects the affinity of a stimulus to GPCR receptor proteins (determine a chemical coordinate space of said stimulus). Accordingly, the present invention provides methods and devices for mimicking and/or replacing the sensory systems of mammals.

In one aspect, the invention relates to a system to detect, transmit and reproduce a selected stimulus comprising (a) a biosensor; (b) a mathematical coordinate system; and (c) an emission device.

The biosensor mimics and/or replaces a biological signal transduction system, wherein the biosensor detects the affinity of a selected stimulus to signal transduction system, measures the resulting signal and provides information identifying the stimulus. In particular, the biosensor detects the concentration and/or rate of production of the secondary messenger, thus biochemically amplifying the stimulus.

In preferred embodiments, the biosensor further comprises a biochemical element, i.e., a concerted set of associated proteins comprising (1) a receptor site; (2) an enzymatic site that produces a secondary messenger only upon stimulant entity activation or binding to the receptor; and, (3) optionally, a allosteric regulation site capable of modulating the interaction between stimulant activation of the receptor site and secondary messenger enzyme activity. The biochemical element is capable of converting an extracellular stimulus in a biological signal transduction system to a secondary messenger, as described above, thus further amplifying the measured signal.

In more preferred embodiments, the biochemical element is capable of mimicking and/or replacing a G-protein signal transduction system. A biochemical element comprises (1) a G-protein; (2) a G-protein coupled receptor protein corresponding to the G-protein; and (3) an enzyme corresponding to the G-protein, wherein the enzyme produces the secondary messenger in response to stimulation of the receptor. Examples of biochemical elements include homologous biochemical elements comprising olfactory receptor proteins, receptor proteins of the visual cortex, taste receptor proteins, and the like. Homologous receptors are defined as members of a class of receptors associated with a particular sense, such as olfactory receptor proteins, that interact with the same G-protein and enzyme producing the same secondary messenger. In preferred embodiments, homologous receptors exhibit seven transmembrane sequences with about 50-100 % sequence homology, preferably 65- 100%o, more preferably 75-100%) and even more preferably 80-100%> sequence homology; and extramembrane sequences that may exhibit little or no sequence homology and determine the specificity of the receptor protein. A biochemical element has a dynamic range which can be adjusted by varying the concentration or ratio of allosteric regulators; a dynamic range is the concentration range of a stimulant entity over which the amount or rate of secondary messenger production is proportional to the concentration of the stimulant entity. In preferred embodiments, the dynamic range of G-protein signal transduction systems is varied by adjusting the GTP/GDP ratio and/or GTP concentration. Preferably, the GTP/GDP molar ratio is varied between about 1000:1 and 1 : 1000 and the GTP concentration is varied between about 0.001 millimolar and 1 molar. In certain embodiments, biochemical elements are sensitized, by genetic engineering methods, to stimuli undetectable by natural senses. Genetic engineering methods involve altering the protein sequences of the variable stimulant receptor sites of the receptor protein by random point mutation, homologous recombination, error prone polymerase chain reaction amplification, and other methods commonly used combinatorial biology.

In preferred embodiments, a plurality of the homologous biochemical elements are arranged in an array, wherein the array comprises a plurality of discrete receptor proteins arranged in a spatially defined and a physically addressable manner, and in a manner suitable for conducting multiple assays to detect the affinity of a stimulus to the receptor proteins to determine a chemical coordinate space of the stimulus. The receptor proteins may be identical or may differ from each other in the relative ratio of guanosine di- and tri-phosphate (GTP:GDP ratio). The biosensor has an improved dynamic range, wherein each element of the array quantitates the stimulus over a broad concentration range of concentration of stimulant entities. In an alternative embodiment, homologous GPCRs that operate through a common G- protein and enzyme, for example olfactory receptor proteins and receptor proteins of the visual cortex, may be spatially segregated in separation elements on a biosensor in order mimic the discrimination ability of a mammalian sensory system. In another embodiment, homologous GPCRs are produced by combinatorial biology methods (see, e.g., U.S. Patent Nos. 5,279,952; 5,223,408 and 5,093,257) to form a biosensor capable of detecting and discriminating stimuli poorly or inadequately detected or discriminated by natural mammalian sensory systems. For example, GPCRs specific for chemicals that lack a discernable odor, electromagnetic wavelengths outside the visible spectrum, and sounds outside the normal auditory range.

The mathematical coordinate system comprises an electronic signal proportional to the measured signal. The mathematical coordinate system is capable of (1) codifying the information in the measured signal, wherein the codified information comprises an electronic signal corresponding to the relative amount of stimulant entities to be combined and transmitted by an emission device in order to reproduce a selected stimulus; and (2) electronically recording and transmitting the codified information.

The detected stimuli can be uniquely mapped upon the mathematical coordinate system/space. Such quantitative mapping of the detector output improves the ability to record, transmit, and reproduce the stimuli. The quantitative mapping system, based on the sensory biochemistry of mammals, improves the existing systems for nonbiological sensors (e.g., the Red-Green-Blue color space standard for displays, the Cyan-Magenta- Yellow-Black color space standard for printing, and the amplitude and frequency modulated standards for sound). The quantitative mapping system wherein the biosensor is based on GPCR, is used to represent more detailed information which can be directly correlated to mammalian sensory system. Preferably, the invention defines a quantitative mathematical coordinate system for various odors and tastes.

The emission device transposes the codified information to deliver and reproduce the selected stimulus at a location remote in space or time. The emission device comprises an array of a plurality of stimulant entities, wherein the stimulant entities are combined and delivered in an appropriate proportion to reproduce the selected stimulus. In preferred embodiments, the stimulant entities are odorant molecules or flavarant molecules, and the emission device reproduces odors or flavors. Examples of an emission device include, but are not limited to, an ink jet printer, a pneumatic nebulizer, an ultrasonic nebulizer and an electrostatic printer. In preferred embodiments, the invention provides methods for improved detection and discrimination of extracellular stimuli (pressure, sound, light, odors, tastes) in an artificial sensor technology; wherein the technology utilizes a specific GPCR and its coupled biochemistry, through the secondary messenger, to affect detection of specific stimulus. In more preferred embodiments, the invention relates to an artificial nose or tongue system that mimics the sensitivity and selectivity of the human nose and tongue, and provides for scent and taste replication, respectively. Such a complete system for odor and flavor detection, electronic dissemination, and replication enables a broad spectrum of revolutionary new products and services. Thus, the invention is useful for odor detection (ScentScan™) and taste detection (TasteScan™); digital transmission (ScentSpace™ and TasteSpace™), and remote reproduction of odors (ScentEmit™) and taste (TasteEmit™). Further, the invention provides an improved, efficient and cost-effective biosensor that mimics both the sensitivity and selectivity of human olfaction and taste.

In an alternative embodiment, the invention relates to a system for detecting the presence and/or amount of a target stimulus in an analyte comprising a biosensor, wherein the biosensor mimics and/or replaces a biological signal transduction system, and the target stimulus comprises a mixture of one or more stimulant entities. In certain embodiments, the invention relates to a high throughput method for screening an analyte for the presence of a target stimulus exhibiting a biological activity. In preferred embodiments, the high throughput assay uses the G-protein coupled receptor (GPCR) based biosensors and sense replication systems for screening samples. In alternative embodiments, the invention relates to a method for affinity purification of a target stimulus, wherein the target stimulus comprises a mixture of one or more stimulant entities. For example, the high throughput assay is used to characterize efficacy and toxicity of a therapeutic agent. In preferred embodiments, the GPCR- stimulant entity binding, more preferably olfactory and taste GPCR, is used as an affinity reagent for affinity purification. Alternatively, homologous GPCRs produced using various combinatorial biology methods are used as affinity receptors to detect stimulant entities, e.g., molecules that are generally not detected or discriminated by natural olfactory and taste GPCRs.

In additional embodiments, the invention relates to a method of detecting the presence and/or amount of a target stimulus in an analyte, wherein the affinity of the receptor proteins for the target stimulus is detected using a detector. Examples of a detector include, but are not limited to, a spectroscopic detector, a radiochemical detector, an electrochemical detector and an amplifying biochemical assay. Examples of an amplifying biochemical assay include, but are not limited to, an immunoassay, a protein kinase assay and a membrane ion channel assay. In preferred embodiments, the receptor proteins comprise G-protein coupled receptors; the target stimulus mimics and/or replaces natural senses consisting of sight, hearing, smell, taste, and touch, wherein the target stimulus comprises a mixture of one or more stimulant entities.

In another embodiment, the invention provides a system for mapping a stimulus in a mathematical coordinate space, wherein the affinity of the stimulus to a receptor protein is detected and measured and the chemical coordinate space is determined. The chemical coordinate space is then transposed to a digital or an analog coordinate space. The chemical coordinate space is an N-dimensional mathematical matrix of numerical values that uniquely represent a stimulant entity, preferably a chemical. In certain embodiments, the number of matrix dimensions are determined by the number of different receptor proteins used in the biosensor. The numerical value at each matrix location is proportional to the amount of stimulant entity interacting with the receptor. In alternative embodiments, the number of matrix dimensions are determined by the number of different stimulant entities used in an emitter device to mimic the stimulus and the numerical value at each matrix location is proportional to the relative or absolute amount of specific stimulant entities needed to mimic the stimulus. A digital or an analog coordinate space is the representation of the mathematical coordinate system as an electronic signal suitable for analog (e.g., by amplitude or frequency modulation) or digital transmission. Digital transmission is preferentially in binary, hexadecimal, or ASCII formats. In another embodiment, the invention provides a method for mapping specific stimulant entities, e.g., odorant and/or flavarant molecules, in a delivery device in a manner that determines the appropriate concentration of each stimulant entity required to recreate a mapped stimulus, such as odor and/or taste. In additional embodiments, the invention relates to an improved device for replicating a stimulus at a remote location. In certain embodiments, the invention defines the composition and concentration of various stimulant entities, e.g., odorant and/or flavarant molecules, required to recreate a selected stimulus, such as odor and/or taste. Emitter devices to deliver such stimulant entities, include, but are not limited to ink jet printers, pneumatic nebulizers, ultrasonic nebulizers, and or electrostatic printers.

In additional embodiments, the invention relates to a complete system to detect, transmit, and reproduce an odor or taste. Preferably, the selected odors comprise narcotics, contraband drugs, fragrances, pseudo-scents, explosives and the like. Preferably, the selected flavor comprises salty, sour, tangy, piquant, zesty, spicy, savory, sweet, bitter and umami. In another aspect, the invention relates to a system for quality control and modification of cosmetics, foods, and beverages wherein the amount of specific odorant and/or flavarant molecules above or below a desired range of values are identified.

Odor detection (ScentScan™ Technology)

ScentScan™ is a unique artificial scent technology wherein human olfactory receptors are linked to signal transduction/amplification cascade as the basis of odor detection. Thus, the technology uses human olfactory biochemistry to greatly enhance the sensitivity and selectivity of artificial nose systems.

In another embodiment, the technology can incorporate olfactory receptors and G-protein coupled signal transduction cascades of non-human mammals able to discriminate or more sensitively detect odors not discernable by the human nose. In an alternative embodiment, the technology can incorporate homo logs of mammalian, including human, olfactory GPCRs, produced by combinatorial biology approaches, to detect and discriminate odorant molecules not normally discerned by a mammalian nose.

Olfactory biochemistry may be provided in a microwell biochip array using available micro fabrication techniques; thus lowering the cost of manufacture and improving detection speed. Each well in the array may contain a single olfactory receptor protein. All microwells contain the same signal transduction biochemistry coupled to a homogeneous fluorescence-based cAMP assay. Figure 1 exemplifies one embodiment of the invention, wherein the biosensor comprises an array of microwells coupled to a fluorescence-based cathode coupled detection (CCD) system. Each grid location in the biosensor contains an odor receptor protein, wherein activation of the receptor triggers a G-protein mediated signal transduction cascade (similar to the neuron biochemistry of the olfactory epithelium illustrated in Figure 2), ending in catalytic production of cyclic adenosine monophosphate (cAMP), wherein the amount of cAMP produced is proportional to the amount of odorant molecule bound to the receptor and the GTP/GDP ratio at the given location. Since each odorant molecule binding event produces multiple copies of cAMP, the odor signal is amplified by the detector, thus improving the sensitivity of the system as compared to currently available methodology. The GTP/GDP ratio regulates the sensitivity of the signal transduction system through the associated G-protein. The cAMP produced is detected by enzymatic or immunodiagnostic methods. The dynamic range of the biosensor is adjustable by the GDP/GTP ratio in the microwell.

The sensitivity of the system is compared to the reported sensitivity of the human nose (Table 1). The ability of the system to discriminate different scents is determined by the ability to quantify the vapor concentration of various odorant molecules exposed to the biochip, both alone and in combination.

Table 1 Human Detection Limits for Various Odorant Molecules¹

1 Wray, T.K., Environmental Solutions, p 3O (June 1, 1995).

The above described chemical compounds, also referred to as "odorant molecules (R^*)," may be present in various combinations in order to provide the desired scent. For example, odorant molecules can be combined to obtain a variety of wine aromas as illustrated in the wine aroma wheel developed by Noble (A. C. Noble, Dept. of Viticulture & Enology, University of California Davis, CA). Accordingly, the present invention provides olfactory binding and biochemical signal amplification that mimics in vivo activity. The known human olfactory receptors are membrane proteins; the protein may be embedded in a lipid membrane or micelle for proper functioning. Lipid micelles have been used previously for the crystallization of membrane proteins for tertiary structure determination by X-ray, electron, and neutron diffraction. (See Schmidt-Krey I., et al, J. Struct Biol, 123:87- 96 (1998); Boekema E. j., et al, J Struct Biol, 1998, 123:67-71; Smith B.S., et al., Acta Crystallogr D Biol Crystallogr, 1998, 54(Pt 4):697-79). Crosslinked Langmuir- Blodgett (LB) films have also been developed to mimic the cell membrane in other membrane based biosensor systems. Competitive immunoassays (Ogreid D., et al, Eur. J Biochem., 1989, 181:19-31) and cAMP-dependent protein kinase assays (Flockhart, D.A. and Corbin, J.D., C.R.C Crit. Rev. Biochem., 1982, 12:133) have been used previously for quantitative measurement of cAMP levels. However, these assays have not previously been coupled to olfactory systems, particularly in a homogeneous system.

The olfactory receptor, G-protein and adenylate cyclase enzyme may also be produced by tissue culture, preferably in a neuronal cell line. The cells wherein the proteins are integrated into the membranes are lysed, separated and resuspended in an appropriate buffer system and deposited in an array. The buffer system contains ATP, GTP and GDP necessary to activate the sensory element.

Olfactory biochemistry is analogous to many other G-protein mediated signal transduction systems that are a primary focus for drug discovery efforts. Therefore, it is possible to adapt the system for high throughput screening applications to facilitate drug discovery efforts. Other senses, i.e. sight, hearing, taste and touch, are also mediated by similar

G-protein signal transduction systems. In alternative embodiments, analogous to the olfactory biosensor, artificial sight, hearing, taste and touch sensors are produced using biosensor array with the corresponding GPCR biochemistry. Olfactory Receptor Proteins Homology searches and chromosome mapping suggest that as many as 500 to

1 ,000 olfactory receptor proteins may exist in the human genome. (Sullivan S.L., et al, Proc Natl Acad Sci (USA), 1996, 93:884-88). However, this number includes both the receptors of the main olfactory epithelium (MOE) of the nasal cavity and the neuroepithelium of the vomeronasal organ (VNO). (See Berghard A., et al, Proc Natl Acad Sci (USA), 1996, 93:2365-69). Most odors are recognized by the main olfactory system, which ultimately sends nerve fibers to higher sensory centers in the cortex. This area of the brain is associated with the conscious perception of odors, measured thoughts, and behavior. The VNO system appears to detect pheromones. The VNO transmits this olfactory information via a separate nerve fiber pathway to the emotive regions of the brain, the amygdala and hypothalamus. Therefore, the MOE olfactory proteins are responsible for our conscious sense of smell and have the most applicability to scent detection, transmission and replication.

The estimate of up to 1,000 different olfactory proteins also includes possible redundant genes or polymorphisms due to duplication of large sections of chromosomes, a possible defense against genetic loss of the sense of smell. Others have suggested that the number of substantively different olfactory receptor proteins may actually number fewer than 100, each specific only for a class of chemical compounds (Bozza T. C. and Kauer I. S., JNeurosci, 1998, 18:4560-69). Scent recognition is based on the brain interpreting the relative amount of chemical bound to each type of receptor. Malnic et al. have demonstrated a high degree of sensory overlap between different neurons and their receptor proteins in the MOE. (Malnic, B., et al., Cell, 1999, 96:713-723). In theory, only 13 truly independent binary receptors would be sufficient to discriminate 10,000 different chemicals (i.e., 2¹³ combinations). By analogy, human sight requires only 4 receptors.

The gene coding for the human olfactory G-protein (G_oIf) that mediates signal transduction between the receptors and adenylate cyclase (AC) has been identified and a complete sequence is available. (Zigman, J.M., et al, Endocrinology, 1993,

133:2508-25141. A number of adenylate cyclase enzymes have also been identified, (Stengel, D., et al, Hum. Genet., 1992, 90 (1-2):126-130; Hellevuo, K., et al, Biochem. Biophys. Res. Commun., 1993, 192:311-318; Parma, J., et al., Biochem. Biophys. Res. Commun., 1991, 179:455-462; Villacres, E.C., et al, Genomics, 1993, 16:473-478; Yoshimura, M. and Cooper,D.M., PNAS (USA),1992, 89:6716-6720; Nomura, N., et al, DNA Res., 1994, 1:27-35; Defer, N., et al, FEBSLett., 1994, 351:109-113) but only AC II and III have been implicated in olfaction. (See Berghard A., et al, Proc Natl Acad Sci (USA), 1996, 93:2365-69). Since G_0]f is known, this can be used to isolate additional olfaction receptors and AC_olf by affinity techniques, such as the Ciphergen SELDI technology (Young, J., paper presented at the CHI Genomics Opportunities conference, San Francisco, CA (Feb. 1415,1998), or the yeast two- hybrid system (U.S. Patent No. 5,986,055).

The genes coding for a number of olfactory receptors, (Buck, L. and Axel, R., Cell, 1991, 65:175-187) the olfactory G- protein, (Jones, D.T. and Reed, R.R., Science, 1989, 244:790-795) and olfactory adenylate cyclase (Bakalyar, H.A. and Reed, R.R., Science, 1990, 250:1403-1406) have been identified in the rat. These genes, as well as human analogs thereto, such as human AC_olf, are useful herein. In Vitro Biochemistry

In the present invention, the biochemical signal transduction cascade is made to work in vitro in a manner that mimics its in vivo activity. An olfaction assay using a recombinant insect system measures the production of Inosyl-3-phosphate and cAMP. (See Raming, K., et al, Nature, 1993, 361:353-356; Knipper et al., FEISS

Lett., 324:147-152 (1993) and Restrepo, et al., Am. J. Physiol, 264:C906-11 (1993)).

The known human olfactory receptor proteins are purported to contain numerous transmembrane sequences. The extracellular portion of the protein is believed to contain the odorant-binding site. The intracellular portion of the receptor is believed to contain the olfactory G-protein binding domain. For some proteins, proper assembly and functioning may require that the protein be embedded in a lipid membrane or the transmembrane sequences be encased in a lipid micelle.

Lipid micelles have been used previously for the crystallization of membrane proteins for tertiary structure determination by X-ray, electron, and neutron diffraction. (Schmidt-Krey I., et al, J Struct Biol.,1998, 123:87-96; Boekema, E. J., et al, J Struct Biol, 1998, 123 :67-71 ; and Smith B.S., et al, Ada Crystallogr D Biol Crystallogr, 1998, 54(Pι 4):697-99). In these techniques it is common to dialyze the membrane proteins against surfactants or phospholipids. The surfactants bind to and stabilize the structure of the hydrophobic transmembrane regions of the proteins. The tertiary structure of the protein is thus stabilized sufficiently to allow crystallization. Presumably, this structural stabilization will also stabilize the in vivo activity of the protein. The use of surfactant or lipid micelles may allow the receptor proteins to maintain their normal binding and regulatory activity in solution in the microwells and would provide the simplest method of manufacturing an artificial nose biosensor based on this technology.

Alternatively, a complete membrane system may be recreated in order to mimic in vivo activity of the olfactory biochemistry. Langmuir-Blodgett (LB) films have been used previously to mimic the structure and function of biological membranes. However, the intrinsic stability of LB membranes has restricted their widespread use as biomimetic systems. Crosslinked LB membranes for biosensor applications have been used. (See e.g., U.S. Patent Nos. 5,622,872; 5,342,692; 5,087,952 and 4,859,538). In this system, proteins are embedded in a lipid bilayer consisting of at least some proportion of unsaturated fatty acids. The unsaturated fatty acids are crosslinked by ultraviolet light after the LB film is formed on an aqueous surface. Proteins embedded in the crosslinked film typically retain some or all of their normal biological activity. Added stability can be obtained by preparing the LB film on the surface of an aqueous gel. Furthermore, acrylamide or agarose gels may be cast in each microwell to subsequently form an LB film on the surface of these gels. The gel should have negligible effect on the rest of the assay.

As described above, the olfactory receptor, G-protein and adenylate cyclase enzyme may also be produced in tissue culture, preferably in a neuronal cell line. The cells wherein the proteins are integrated into the membranes are lysed, separated and resuspended in an appropriate buffer system and deposited in an array. The buffer system contains ATP, GTP and GDP necessary to activate the sensory element.

The homogeneous detection of the cAMP produced by the olfactory biochemistry may be detected using methods such as, (1) immnunoassays using radio- immunoassays (RIA), enzyme-linked immunoassays (EIA), or fluorescent immunoassays (FIA) detection methods; and (2) activity assays based on cAMP- dependent protein kinases (PKAs). (See Flockhart, D.A. and Corbin, J.D., C.R.C. Crit. Rev. Biochem., 1982, 12:133, and Nagai, et al., Nature Biotech, 18:313-316 (2000)). Since the cAMP molecule is too small to perform a sandwich immunoassay, competitive immunoassays can be used for cAMP detection. Competitive immunoassays for use for cAMP detection include the two following formats. The first format involves the use of fluorescently labeled antibodies which compete for cAMP bound to the optical surface of the microwell and cAMP generated in solution by the activity of the adenylate cyclase enzyme. As cAMP accumulates in solution due to odorant molecule binding at the receptor protein, the equilibrium antibody binding will shift from that bound to the bottom of the microwell to that free in solution. A second competitive immunoassay format involves the use of fluorescent- cAMP conjugates. In this approach the antibody is bound to the optical surface of the microwell and the competition is between fluorescently labeled and unlabeled cAMP (produced by adenylate cyclase). This approach requires the identification (or development) of a fluorescent cAMP analog that retains its immunological activity. The commercially available 8-(4-chlorophenylthio)-cAMP (Sigma Chemical Co.) provides a precedent for this approach. (Ogreid D., et al, Ewr. J Biochem., 1989, 181 : 19-31).

Preferably, cAMP-dependent PKAs are used for cAMP detection. A large number of such PKAs are commercially available. In the absence of cAMP many PKAs exhibit negligible activity. As cAMP accumulates, the activity of the PKA increases. One such fluorescent PKA assay system has been previously reported, (Wright D. Ε., et al, PNAS (USA), 1991, 78:6048) and is commercially available through Sigma Chemical Co. Nagai recently reported another suitable PKA assay (see Nagai, infra). Since the PKA is itself an enzyme, this approach provides another level of signal amplification.

Detection Speed and Amplification Potential

Despite the paucity of identified GCPR receptor proteins, the biochemistry of G-protein coupled signal transduction systems is fairly well understood. The receptor proteins appear to be transmembrane proteins that interact with an enzyme, e.g., adenylate cyclase (AC_olf), through an associated G-protein, e.g., an olfactory G- protein (G_olf). Binding of an odorant molecule at the receptor causes the activation of the enzyme (AC_olf), and the catalytic production of cAMP or a secondary messenger, e.g., Inosyl-3-P. The G-protein, which mediates the transduction of the binding signal between the receptor and the enzyme (AC_olf), is itself regulated by the guanidine triphosphate (GTP) and diphosphate (GDP) ratio in the cell. By varying the GTP/GDP ratio, the cell is able to adjust the number of odorant molecule binding events (i.e., occupied receptors) that are needed to activate the enzyme (AC_olf).

The production of cAMP by AC appears to obey Michaelis-Menton kinetics - (Equation 1), where K_AC and k are the Michaelis and rate constants, respectively.

AC * + ATP < ^KAC >[AC • ATP]— → AC * + cAMP _drc_AM_Pl _ AC_T]ATP] dt <k_AC +[ATP]) (1)

AC is activated by the binding of an activated G_olf protein with the equilibrium constant KG (Equation 2).

The G-protein alternately binds the GDP and GTP, but can only be activated when GTP is bound.

Assuming the GTP and GDP concentrations are large compared to the concentration of the G_olf protein (i.e., the concentration of free G_olf protein is negligible), then the concentration of activatable olfactory G-protein (G^GTP _olf) is related to the total G-protein concentration (G^τ _olf) and the GTP/GDP concentration ratio (Equation 3).

Activation of the olfactory G-protein requires the binding of an olfactory receptor that has itself been activated by the binding of an odorant molecule (R^*). Odorant molecule binding to the receptor protein is an equilibrium process:

K_r

R + O* →>R

that can be represented as a function of the total odorant molecule and receptor protein concentrations (O_τ and R_τ, respectively),

which is mathematically identical to the binding of an antibody and its antigen.

Assuming that the activated receptor protein and GTP/GDP binding sites on the G_olf protein are independent of one another, then the concentration of activated G_olf protein (G^* _olf) is determined by the equilibrium expression:

Equations 1 through 5 provide a model for olfaction biochemistry that are used to design a biosensor and predict its performance. Equation 1 shows that in an abundance of ATP, the rate of cAMP formation is directly proportional to the concentration of activated adenylate cyclase. Thus the fraction of activated adenylate cyclase is proportional to the amount of cAMP produced over a given time period and is directly proportional to the concentration of the odorant molecule. As illustrated in Figure 3, olfactory receptors operate analogously to antibodies. The fraction of receptors binding any specific odorant molecule depends on the relative concentrations of the odorant molecule and receptor as well as the affinity of the receptor for the odorant molecule. However, unlike antibodies, the signal generated by the binding of an odorant molecule to a receptor protein must be transduced to the olfactory adenylate cyclase enzyme for detection to occur.

This signal transduction is mediated by the G-protein and is modulated by the relative GTP/GDP concentration ratio, as illustrated in Figure 4. The absolute concentration of the active receptor and the olfactory G-protein also determine the activation level of the adenylate cyclase (Figure 4). From the limit imposed by the GTP/GDP ratio going to infinity, the optimum G-protein concentration can be predicted from Figure 4 to be about:

which is independent of the odorant molecule concentration. The 1:1 binding relationship between adenylate cyclase and the G-protein suggests that the concentration of adenylate cyclase should be the same as that of the G-protein used. The dynamic range of the biochemistry is readily adjusted by varying the GTP/GDP concentration ratio (Figure 4). For each GTP/GDP ratio the fractional adenylate cyclase activation varies linearly with the activated receptor concentration over about 2 orders of magnitude. Therefore, three replicate microwells with different GTP/GDP ratios creates a biosensor able to precisely track the odorant molecule concentration over at least 6 orders of magnitude.

The olfactory receptor, G_olf, and adenylate cyclase proteins are thought to be associated with each other in the cell membrane, making the transduction of binding signals to the adenylate cyclase enzyme very fast. This implies that the kinetics of cAMP production is the rate limiting step for odor detection in vivo. These sensory proteins similarly associate in vitro in olfactory cillia extracts. Detection kinetics may become diffusion limited (similar to antibody-antigen reactions), which can require more than 30 min to reach equilibrium in a 96 well plate format. (The antibodies, Pierce Chemical, Rockford, IL). Ekins and Chu have shown that smaller assay formats can greatly reduce the time required to reach equilibrium (Ekins, R. P., and F. Chu, Trends Biotechnol, 1994, 12:89-94). In preferred embodiments, the biosensors are miniaturized.

Biosensor and Biochip Construction

The biochip reader shown in Figure 1 has been used for the detection of upconverting phosphor fluorescent and chemiluminescent diagnostic assays and in combinatorial chemistry applications. Figures 5A-5C show an example of the use of the biochip reader, illustrated in Figure 1, with a chemiluminescent assay. Figure 5 A shows the performance of the biochip reader for the measurement of chemiluminescent assay kinetics in a 250 nL microwell array; Figure 5B depicts the reaction representing the chemiluminescent assay kinetics; and Figure 5C depicts the relative rate of decomposition representing the chemiluminescent assay kinetics. A similar reader has been used for as a fluorescence-based artificial nose (see, e.g., U.S. Patent No. 5,320,814).

Preferably, the biochip consists of a two part microarray. The bottom of the array is a glass fiber optic wafer (Schott Glass). The use of a fiber bundle as the bottom surface prevents optical crosstalk between microwells on the top surface, allowing maximum miniaturization of the well volume and packing density on the microwells in the array. This system also allows all the microwells to be interrogated simultaneously rather than serially, which is critically important for making ratiometric inter-well comparisons with a rate assay system. The microwells themselves are formed by fixing a top layer with uniform holes to the surface of the fiber optic wafer. While the bottom surface is glass, the sides of the microwell can be manufactured from almost any material using plasma etching, wet etching, laser drilling, or other microfabrication processes. The two surfaces may be attached by sonic welding or with adhesives.

Similar microwell arrays have been fabricated for miniature immunodiagnostic assays (Figure 1). These arrays may be fabricated with dimensions as small as 100 μm per side and 0.1 mm deep (as shown in the detail inset in Figure 1), allowing the construction of a 100 x 100 array of 1 nL microwells on a 1 cm² chip. This 10,000 well array is sufficient to handle any of the predicted number of olfactory proteins.

The biochemical reagents are microdeposed into the microwells on the grid. A 1 nL microwell requires that the reagents be reproducibly deposited at a 50 pL volume. This is within the capabilities of current inkjet printing technology. The commercially-available BioDot™ deposition system is capable of reproducible reagent deposition at 10 nL level, which allows 100 nL microwells (a 10 x 10 grid array on a 1 cm² chip). Parallel biological reagent deposition by electrostatic printing at the 75 pL level has also been demonstrated. Accordingly, the system may include a camera-like device with a disposable biochip of receptor elements that measure odor, flavor, light, sound or pressure in direct contact with the biochip, a recorder that records the coordinates measured by the biochip reader, a mapping system that allows the user to record the biochip signals as coordinates in a mathematical space. Additional systems include a printer-like system with replaceable odor or flavor cartridges, which operates much like an inkjet printer ejecting appropriate quantities of odorant and/or flavarant molecules to mimic any scent or taste based on its coordinates.

Electronic dissemination/digital transmission (ScentSpace™ Technology) ScentSpace™ technology is a system for representing odors as a set of mathematical coordinates. ScentSpace™ technology can be used to screen odorant molecule libraries to determine various combinations of the odorant molecules that mimic and/or reproduce other odors, and the proportions of odorant molecules required to match a specific odor coordinate, wherein two or more odorant molecules are combined according to an algorithm. Thus, this technology establishes the standard for digital and analog scent communications, such as the standards for the internet, television and radio broadcasting, video games, and multimedia, CD and DVD devices. The ScentSpace™ technology is analogous to the Red-Green-Blue (RGB) and Cyan- Yellow-Magenta-Black (CYMB) systems. The RGB standard is the basis of all color image transmission, wherein any color can be defined by its unique coordinates in color space and replicated by mixing the primary colors, red, green, and blue. The CYMB color space is directly analogous to the RGB color space and allows print reproduction of color images.

The ScentSpace™ technology uses the olfactory detection technology as described above. First, the specific receptors activated by each odorant molecule are identified, followed by the determination of the level to which the odorant molecules activate each receptor. The odors are mapped as coordinates and specific odorant molecules are combined to replicate a set of coordinates. Thus, this technology can be used for electronic dissemination and/or digital transmission of odors.

In certain embodiments, similar mathematical coordinate systems are established for other human senses, such as sight, hearing, taste and touch, utilizing artificial GPCR sensors mimicking the corresponding senses. In alternative embodiments, similar mathematical coordinate space is established for mapping activity, such as efficacy, toxicity, and the like, of a variety of substances, including, but not limited to chemical composition, therapeutic agents, diagnostic agents and drugs, using the technology of the invention. For example, chemical compositions can be screened by detecting the interaction of the composition with various GPCR signal systems present in mammals, including humans.

Electronic dissemination and/or digital transmission of odors can be accomplished by multiple formats. For example, in one format the spatial dimensions can be defined by different classes of olfactory receptors wherein the coordinates represent the relative activity elicited by an odorant molecule for each of the receptor classes. In another format, the spatial dimensions can be defined by the individual ScentEmit™ odorant molecules, wherein the coordinates represent the relative proportions of each odorant molecule required to replicate the scent.

Scent Reproduction (ScentEmit™ Technology

ScentEmit™ technology is a complete scent reproduction technology wherein a set of individual odorant molecules are combined with an aerosol delivery system wherein the individual odorant molecules are mixed in different concentrations to replicate any desired scent. As described above, human olfactory receptors linked to signal transduction/amplification cascade can be used to detect odor detection (ScentScan™ technology). Further, the quantitative determination of the relative amounts of individual odorant molecules that bind to separate olfactory proteins, provides a method to map odors in coordinates and to combine specific odorant molecules to mimic any coordinate in the ScentSpace™ to construct a "scentprint" system able to reproduce any odor. The scent reproduction technology is analogous to an ink jet printer with disposable ink cartridges, wherein a control device determines the amount and order of ink ejection from each cartridge. In alternative embodiments, similar technology is used to replicate taste. Aerosol Generator ScentEmit™ odorant molecule delivery can be accomplished by several modes. Any of the standard methods for liquid aerosolization can be used to deliver odorant molecules from ScentEmit™ products. In one embodiment, the delivery system comprises a disposable cartridge wherein specific odors are mapped at specified coordinates on the cartridge. Based on the ScentSpace™ technology, a control device determines the coordinates, the appropriate proportion of specific odorant molecules to be delivered, and the order of emission of the odors from each cartridge. In preferred embodiments, the inkjet-like format is used for personal and small scale ScentEmit™ products, such as electrical and/or electronic connections, e.g. computer, stereo and television connection, plug-in room deodorizers, and the like. In an alternative embodiment, the delivery system comprises electrostatic printing, liquid nebulizers, including pneumatic and ultrasonic nebulizers, and the like. In preferred embodiments, a liquid nebulizer is used for larger scale ScentEmit™ products, such as products used in public forums like movie theaters, point-of-purchase displays, and the like. Odorant Molecules/Reagents Using the odor detection technology (ScentScan™) and odor transmission technology (ScentSpace™) described above, thousands of compounds can be scanned in a combinatorial fashion to identify individual odorant molecules; map scent coordinates; and determine the minimum subset necessary for complete odor reproduction. Thus the technology can be used to reproduce various odors, such as fragrances, pseudo-scents (for animal training and contraband detection) such as pseudo scents for cocaine, heroin, marijuana, explosives, dead bodies, "trauma and fear" formulation for search-and-rescue dogs, and the like. In alternative embodiments, similar technology is used to replicate taste.

Utility The system described above has a wide variety of uses including uses in the entertainment industry (e.g., film, television, video games and music), in advertising at the point of purchase (e.g., in store displays) or in electronic medium (e.g., television, radio, and internet), uses in professional and amateur photography and video, uses in high end telecommunications (e.g., video conferencing and the internet).

Additional applications include: affinity separation reagents and drug screening systems. The development of chemical affinity techniques may lower the cost of chemical purification, particularly flavors and fragrances still derived from natural sources. Another application is for the purification of beverages (e.g., beer and wine) to remove unpalatable odors sometimes developed in these products. In this application, the biochip can be used to target receptor proteins that bind to an unwanted odorant or flavarant molecule, and the corresponding GPCR can be used for affinity removal of the odorant or flavarant molecule, respectively, from the final product. The odor detection technology is based on a G-protein mediated signal transduction cascade. A variety of cellular signaling systems used for drug discovery and agricultural product development are based on similar G-protein/secondary messenger-mediated signal transduction systems. Therefore, homogeneous secondary messenger detection biochips can be used as a high throughput screening tool for drug discovery and agricultural product development.

Further, the artificial nose system can be used for the construction of new and improved artificial sensors able to better mimic the other human senses, i.e. sight, hearing, taste and touch. Additionally, use of G-protein mediated signal transduction systems can be used in the treatment of sensory deficit disorders and to improve the quality of life of the aged and disabled (the loss of smell in the elderly is believed to contribute to decreased appetite and malnutrition). The ability to replicate scents using compounds generally recognized as safe (GRAS) also has both medical and contraband detection applications. For example, animals may be trained using contraband detection. (Allen, W., St. Louis Dispatch, p ID (June 10, 1996)). These include pseudo scents for cocaine, heroin, marijuana, explosives, dead bodies and a "trauma and fear" formulation for search-and-rescue dogs. These scents eliminate the need to use the real substances during training, eliminating the need for regulatory controls and the risks that the real chemicals may pose to the dogs and their human handlers. Other studies have linked specific odors to senses of well-being and the alleviation of stress, (Studies funded by the Olfactory Research Fund, (New York, NY) and have showed how odors can dramatically affect consumer spending habits. (Scent and spending studies, Laughingwell and Associates, Chicago, IL).

Additionally, the artificial nose technology can be used to establish odor-based standards. For example, ScentSpace™ can be the standard for digital and analog scent communications and recording; ScentSpace™ can be the universal scent- communication standard such as television and radio broadcasting, digital and analog recording, and the internet.

TasteSpace™ can be the standard for digital and analog taste communications and recording; TasteSpace™ can be the universal taste-communication standard such as television and radio broadcasting, digital and analog recording, and the internet.

EXAMPLES The following examples are illustrative in nature, and are not intended to limit the scope of the present invention in any manner.

Example 1 Olfactory Protein Synthesis The rat genes coding for an olfactory receptor protein, olfactory G-protein (Pevsner, J. et al., Science, 1988, 241:336-3391. and olfactory adenylate cyclase, are cloned into an appropriate yeast or tissue culture system, using standard techniques. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual; DNA Cloning, Vols. I and II (D. N. Glover ed.). A receptor protein from rat olfactory receptor genes for which the corresponding odorant molecule affinities is determined, is selected to demonstrate in vivo sensitivity.

Each purified protein is tested for retention of biological activity. The affinity of receptor protein to a corresponding odorant molecule is evaluated similar to the method used for antibody-antigen affinity determinations. The receptor-odorant molecule complex is purified and odorant molecule concentrations are determined by gas chromatography. Since the receptors are transmembrane proteins, and may lose activity upon separation from the cell membrane, the affinity assays are conducted with ghost cells and purified receptors reconstituted in a lyposome or micelle system. Purified G-protein activity is assessed from its affinity for GTP. Adenylate cyclase is mixed with the active G-protein and a whole cell lysate of the cloned receptor protein culture (to ensure the presence of functional membrane bound receptor), and the adenylate cyclase activity is determined.

Example 2 cAMP Assay Development A cAMP assay system is optimized as follows. The protein kinase assay described by Wright is adapted to a 96 well microliter plate format (Wright D. E., et al, PNAS (USA), 1981, 78:6048). The sensitivity and dynamic range of the protein kinase assay is compared to the competitive cAMP immunoassay. (Ogreid, D., et al, Eur. J Biochem., 1989, 181:19-31). Each assay must be robust to the presence of adenylate cyclase, guanidine nucleotide phosphates, and the olfactory proteins. The most sensitive cAMP assay is selected for further work.

Example 3

Test Chamber Assembly A system to generate and contain controlled odorant molecule atmosphere is required for development and testing of the olfactory receptors in artificial nose technology. An ultrasonic nebulizer is used to generate a test atmosphere with appropriate dilution through critical flow orifices. Similar systems have been used for testing industrial hygiene monitors. (Parish, H., SRI International, Menlo Park, CA, personal communication (1996)). Odorant molecule concentrations are verified by purge trap collection and gas chromatographic analysis.

Example 4 Olfactory Assay Development

An in vitro olfactory assay mimicking an in vivo system is developed. The proteins produced in Example 1 are used to develop the assay in a 96 well microliter plate format. First, adenylate cyclase, G-protein and olfactory receptors are mixed together in standard buffer systems, and tested against an odorant molecule known to have affinity for the receptor. Alternatively, the receptor protein is dialyzed against various surfactant solutions to stabilize its structure in the assay. In another system, a thin lipid layer is deposited on the surface of a microwell with emuedded olfactory proteins (to mimic a cell membrane). Second, cyclic-AMP (cAMP) is quantified using the assay as described in Example 2, supra.

Example 5 Sensitivity Demonstration The ability of the olfactory and cAMP assays to quantitatively track the aerosol concentration of an odorant molecule in a test chamber as compared to odorant molecule detection sensitivity observed in vivo is assessed. Different aerosol concentrations of an odorant molecule are generated in the test chamber and verified by purge trap/GC. At least nine replicate olfactory assays are conducted in parallel at each aerosol concentration. Similarly, assays are conducted with at least nine different GTP/GDP ratios to test the dynamic range of the system. Microtiter plates containing replicate olfactory assays are exposed to the environment of the test chamber for varying times (from 0.5 to 15 min) to determine the kinetics of the olfactory biochemistry. The temperature of the chamber and assays are varied to determine the temperature sensitivity of the olfactory system. Olfactory protein and nucleotide concentrations are optimized based on these kinetic determinations to obtain the maximum possible sensitivity in the shortest period of time. Example 6 Homogeneous Assay Development A combined homogeneous olfactory and cAMP assay is developed by adjusting the buffer composition such that both the olfactory and cAMP assays function simultaneously. Alternative cAMP protein kinases may be used to provide adequate results or if the olfactory proteins act as substrates for the protein kinase. Homogeneous assays are also developed for additional olfactory receptors produced in Example 4, supra. Since the G-protein and adenylate cyclase are identical in each assay, a single homogeneous buffer system will suffice for all assays, with buffer for individual olfactory receptors.

Example 7 Synthesis of additional Olfactory Proteins Additional mammalian, including human, olfactory receptor proteins are cloned which are specific for odorant molecules described in Example 1. The quality of the purified proteins produced and their affinity for target odorant molecules is evaluated as described in Example 1, supra.

Example 8 Specificity Demonstration

To assess the specificity of the system, 96-well plates are prepared with up to 18 replicate wells for each of the 5 receptor proteins. Each of the 5 receptor proteins are isolated in their own assay wells. Different GTP/GDP ratios are used for each set of assays, as described in Example 5, supra, such that at least 3 replicates of each assay are maintained on each microliter plate.

Each of the test plates are exposed to different odorant molecule concentrations. The ability of the system to discriminate each odorant molecule is determined from the relative fluorescence of the wells containing the receptor specific for that odorant molecule, as compared to the fluorescence for other receptors. Odorant molecules are combined in different concentrations (i) to assess the affinity of a receptor for more than one odorant molecule, and (ii) to determine the ability of the system to quantitate the concentration of individual odorant molecules present in a combination.

Example 9 Chip Reader Development A chip reader, as shown in Figure 1 , is assembled. The system utilizes a time gated laser induced fluorescence wherein detection is accomplished by a cathode coupled device (CCD) for simultaneous measurement of relative fluorescence in each of the microwells.

Example 10 Biochip Construction Biochips are constructed from compatible materials using appropriate microfabrication processes. (See, e.g., U.S. Patent Nos. 6,017,696; 5,320,814; and 5,143,854). The bottom surface of the biochip is made of a glass fiber optic array. The sides of the wells may be constructed from many different materials. Various coating chemistries may be used to minimize assay artifacts caused by protein adsorption to the biochip surfaces. The biochips will include between 100 and 10,000 microwells per cm².

Example 11 Reagent Deposition Process Development A microdeposition process for filling a microwell array on the biochip is as follows. Previous work has shown that the commercially-available BioDot™ technology provides sufficient reproducibihty and mechanical accuracy for use with 100 nL (100 grid) biochip arrays. This technology is based on serial deposition, one reagent and one well at a time. An inkjet printer may be used to reproducibly deposit 50-70 pL of antibodies and DNA probes with a mechanical precision of less than 10 μm, sufficient to allow construction of 1 nL microwell (10,000 grid) biochips. Alternatively, electrostatic printing may be used for high speed parallel reagent deposition. A combination of electrostatic printing and inkjet technology may be used for reagent deposition. Example 12 Biochip System Demonstration Five different rat olfactory receptors are deposited on biochips in an array similar to that described in Example 8, supra. The biochips are subjected to a test atmosphere as described in Example 8, supra.

Example 13 Human Olfactory Adenylate Cyclase Identification Both adenylate cyclase II and III have been implicated in human olfaction. The adenylate cyclase associated with main olfactory epithelium (MOE) is identified, cloned, and produced. The gene for human olfactory G-protein for MOE has been identified and is available.

Example 14 Human Olfactory Protein Synthesis

At least 5 human olfactory receptor proteins are produced, the human olfactory G-protein and adenylate cyclase enzyme, by cloning known genes into a suitable host as described in Example 1, supra. Quality control of the purified proteins is conducted as described in Example 1, supra.

Example 15 Homogeneous Human Olfactory Assay Development The homogenous assay developed in Example 6, supra, is adapted for the human olfactory proteins produced in Example 14, supra, using the 96-well microliter system, as described above.

Example 16 Human Biochip Synthesis The biochip system described in Example 12, supra, is modified (e.g., the microdeposition process is altered), and adapted to human olfactory assays, as described in Example 15, supra. Example 17 Reader Optimization The biochip reader described in Example 9, supra, is optimized based on the results obtained in Example 12, supra. Modifications may include optimizing (1) the optical train for optimal throughput, (2) the time gating to diminish background autofluorescence artifacts, and (3) the excitation laser intensity.

Example 18 Olfactory Tissue Preparation The olfactory tissue preparation of the rat olfactory cilia was performed as described by Shirley et al. (Shirley, S. G., et al, Biochem. J, 1986, 240:605-607). The temperature was maintained at 0-4° C throughout the process and ah solutions were incubated on ice. The olfactory epithelia were dissected from a freshly sacrificed rat according to Anholt's method (Anholt, R. et al., J. Neurosci., 1986, 6:1962-1969). The excised tissue was bathed in ice-cold Ringer's solution supplemented with deoxyribonuclease enzyme (0.01 %>) and MgCl₂ (2.2 mM) for 2 min with gentle agitation to remove mucus from the tissue; the tissue was re-bathed with fresh solution. The tissue was rinsed with a phosphate buffer (0.9 %> NaCI and 2 mM ethylene glycol tetraacetic acid (EGTA, 2 mM), pH 7.0), transferred to the buffer solution; and sonicated (10 sec in 2 sec intervals) to detach the cilia from the tissue. The sonicated solution was pooled and the residue was removed by centrifugation (30 min at 1,000 g and 4° C). The suspension was re-centrifuged (90 min at 350,000 g and 4° C) to isolate the cilia. The pellet was suspended in phosphate buffer (1 ml). The protein concentration of the final cilia preparation (50 μg/ml) was determined using a protein assay kit (Coomassie Plus Protein Assay Reagent Kit, Pierce) and bovine serum albumin standard. The cilia preparation was stored at -80° C for later use.

Example 19 Odorant Molecule Stimulation Reaction

The olfactory cilia prepared according to Example 18, supra, were stimulated with (+)-carvone according to the following modification of the procedure described by Boekhoff (Boekhoff, I., et al., EMBO J, 1990, 9:2453-2458) to determine the kinetics of secondary messenger (cAMP) production. The purified cilia solution and the reaction buffer were equilibrated to 37° C in a heating block prior to initiating the reaction. The reaction was conducted at 37° C. Each reaction was initiated by adding a quantity of 60 μl of purified cilia solution (50 μg/ml) to 300 μl quantity of reaction buffer, containing:

0.9 % NaCI

10 mM Ethylene glycol tetraacetic acid (EGTA) 2.5 mM MgCl₂ 1 mM ATP

1 mM dithiotheritiol (DTT)

1 mM (+)-carvone (odorant molecule)

0-1 mM GTP

0-100 mM GDP

A 48 μl quantity of the reaction buffer was immediately removed and quenched by addition to an ice-cold 10 wt%> aqueous trichloroacetic acid (TCA) solution. Additional 48 μl aliquots were removed at various incubation times between 0 and 60 min and similarly quenched in 10%> TCA.

Example 20 cAMP Determination The quenched reaction samples from Example 19, supra, were extracted with water-saturated diethyl ether (5 x 4 volumes). Residual diethyl ether was evaporated (60° C) prior to determination of the cAMP concentrations.

The cAMP concentration of each extracted solution was determined using the BioTrak RPN 225 cAMP enzyme immunoassay kit (Amersham Pharmacia Biotech, Inc., Piscataway, NJ). Aliquots of each extracted sample were diluted 100:1 with an assay buffer supplied with the assay kit to eliminate the influence of the reaction solution.

The cAMP concentration was determined by the absorbance at 450 nm after quenching with sulfuric acid(1.0 M) after 60 min, as described in the kit instructions. The cAMP concentration was determined from a standard curve prepared over the range 0.04 to 2.56 μM of cAMP. The rate of cAMP production was determined by a linear regression within the stimulation reaction time between 0 to 20 min. Results determined for the time points measured in Example 19, supra, linear regressions and standard deviations are shown in Table 2

Table 2 cAMP assay results for Example 19

(Reaction conditions- GTP(1 mM) and GDP (0.1 mM))

Effect of GTP and GDP on Olfactory Stimulation

Several olfactory stimulation reactions were conducted with the olfactory cilia prepared in Example 18, supra, by the procedure described in Example 19, supra, with different GTP and GDP concentrations used in the reaction buffer. The resulting cAMP production rates (and standard deviations) were determined as described above, and are illustrated in Figure 6. The accumulation of cAMP was not seen in the stimulation reaction without GTP.

Example 21 An Olfaction-Based Coordinate Space for Digital Transmission of Odors A coordinate space that can be used for the digital representation, transmission, and reproduction of odors is described in this example. This space is based on mapping the relative intensity (or affinity) with which a mixture of chemicals comprising odorant molecules bind to individual olfactory proteins. Each olfactory receptor protein is represented as a unique coordinate in space. According to Sullivan, the maximum number of olfactory receptor proteins is less than 1000 (Sullivan S. L., et al., Proc Natl Acad Sci (USA), 1996, 93:884-888). Thus the unique coordinate of each olfactory receptor protein can be digitally represented as a number between 0 and the maximum number of olfactory receptor proteins, i.e., a number between 0 and 1024 (2¹⁰ bits).

The intensity (or affinity) with which the mixture of chemicals comprising odorant molecules binds to each of these olfactory receptors is also represented as a digital number. The accuracy with which an odorant molecule can be represented depends on the number of digital increments used to map the intensity of the olfactory receptor protein interaction. This interaction can be divided into 16, 254, 512, 1024, or more digital levels. Therefore, it is possible to accurately represent any odorant molecule by a map consisting of 1024 levels (2¹⁰ bits) for each of 1024 coordinates, or 1024 x 1024 bits (131 kb) of digital information.

Example 22 An Emitter-Based Coordinate Space for Digital Transmission of Odors A coordinate space that can be used for digital representation, transmission, and reproduction of odors is described in this Example. This space is based on mapping an odor by the relative amounts of other odors that must be combined to recreate the mapped odor. Based on the aroma wheel developed by Noble (see Noble, supra) a minimal set of defined odors might include 12, 29, 94 or more different mixtures of chemicals, such as might be placed into individually-addressable cartridges of a scent emitter device.

Each of these 94 odors may be represented digitally as a coordinate between 0 and 128 (2⁷). The relative amount of each of these odors that must be combined to recreate another odor can be represented as an intensity level. The ability to accurately replicate any other odor is limited by the number of such intensity levels, with 16 levels being less accurate than 256, which is less accurate than 512, which is less accurate than 1024 (or more) levels. Thus, it is possible to accurately represent any odor by a map consisting of 1024 levels (2^!0 bits) for each of 128 coordinates, or 128 x 1024 bits (16 kb) of digital information. Example 23 An Algorithm for Odor Reproduction Any of the digital sets of odor coordinates described in Examples 21 and 22 provide templates by which any number of other similarly mapped odors can be combined to optimally reproduce the first mapped odor. Assuming that the sum of the quantities of all odors to be mixed equals 1, the fraction of this total that will be provided by any given odor (i) can then be represented by f_;. The mapped intensity of each odor (i) at each coordinate (j) is represented by M_g, and that of the original odor is represented by N_j. Therefore, the relative amounts (Q of each odor (i) needed to replicate the original odor can be determined by minimization of the function (Err), as described in Equation 7:

within the constraint that:

∑f_f = 1 i

Example 24 Emitter Device An emitter device was constructed from a commercially available color ink jet printer (HP Deskjet 612C, Hewlett Packard, Palo Alto, CA). Holes were drilled in each of the four chambers (top and bottom) of the ink cartridge in the printer. A syringe was used to remove the ink from the chambers of the cartridge, and each chamber was refilled with a different extract. The extracts can be commercially available, such as food flavors and the like, or the can be prepared from raw materials. For example, a sage extract was prepared by heating (50°C, 1 hour) rubbed sage leaves (1 gram) in 50%> aqueous ethanol (50 ml), followed by filtration of the solution to yield the sage extract.

Cinnamon was deposited in the black ink chamber, sage in the magenta chamber, vanilla in the yellow chamber, and orange in the cyan chamber. The standard printer driver in the operating system of the computer was programmed to control relative amounts of each odorant molecule emitted from each chamber. The user interface (Figure 7) comprised slider controls and definable system settings for (1) volume range emitted by each chamber (cyan, yellow, magenta and black settings), and (2) overall volume emitted at a given time (horizontal repeat and number of rows). A hot-plate was inserted in the printing surface adjacent to/facing the printer cartridge. A fan was attached to the back of the printer to blow the scent towards the user. The paper detection sensor was disabled.

Source Code

// main.cpp

// Rustan Leino, 4 March 2000

#include "main.h" #include "resource.h"

// global variables

HINSTANCE hinst = NULL;

HWND hwndMain = NULL; char szAppName[] = "Scent UI";

MAIN WINDOW * mainWindow = NULL;

// function prototypes

LRESULT CALLBACK WindowProc(HWND hwnd, UINT uMsg, WPARAM wParam, LPARAM lParam); int ErrorBox(LPCTSTR lpText); int MsgBox(LPCTSTR lpText);

LRESULT CALLBACK AboutDlgProc(HWND hwnd, UINT msg, WPARAM wParam, LPARAM lParam);

// WINDOW

WINDOW: :WINDOW(HWND hwnd) { _hwnd = hwnd;

SetWindowLong(_hwnd, GWLJ SERDATA, (LONG)this); } WINDOW: :WINDOW() { iwnd = NULL;

}

WINDOW: :~WιNDOW() {

}

bool WINDOW: :create(LPCTSTR IpClassName, // pointer to registered class name LPCTSTR lpWindowName, // pointer to window name DWORD dwStyle, // window style int x, int y, int nWidth, int nHeight,

HWND hWndParent) { // handle to parent or owner window _hwnd = Create Window(lpClassName, lpWindowName, WS_CHILD I WS VISIBLE | dwStyle, x, y, nWidth, nHeight, hWndParent, NULL, // menu hinst, NULL); // window-creation data if (_hwnd = NULL) { return false;

}

SetWindowLong(_hwnd, GWLJJSERDATA, (LONG)this); return true;

}

void WINDOW: :setld(int id) {

SetWindowLong(_hwnd, GWL ID, id); }

void WINDOW: :setText(char * psz) { SetWindowText(_hwnd, psz); }

void WINDOW: :setTextNumber(int n) { char szBuf[20]; wsprintf(szBuf, "%d", n); setText(szBuf);

}

void WINDOW: :invalidate() {

InvalidateRect(_hwnd, NULL, TRUE); }

LRESULT WINDOW: :sendMsg(UINT uMsg, WPARAM wParam, LPARAM IParam) { return SendMessage(_hwnd, uMsg, wParam, IParam);

}

// STATIC TEXT

STATIC TEXT: :STATIC_TEXT() : WINDOWO

{ }

bool STATIC_TEXT::create(char * pszText, int x, int y, int nWidth, int nHeight, HWND hWndParent) { return WINDOW: :create(" STATIC", pszText,

SSJLEFT, x, y, nWidth, nHeight, hWndParent); // SCROLLBAR

SCROLLBAR: :SCROLLBAR() : WINDOWO { }

bool SCROLLBAR: :create(int x, int y, int nWidth, int nHeight, HWND hWndParent) { return WINDOW: :create(" SCROLLBAR", "",

SBS_HORZ I SBS_BOTTOMALIGN, x, y, nWidth, nHeight, hWndParent);

}

void SCROLLBAR: :setlnfo(int min, int max, int page, int pos) {

SCROLLINFO sif; sif.cbSize = sizeof(sif); sif.fMask = SIF_RANGE | SIF_PAGE | SIF_POS; sif.nMin = min; sif.nMax = max; sif.nPage = page; sif.nPos = pos;

SetScrollInfo _hwnd, SB_CTL, &sif, TRUE);

}

void SCROLLBAR: :setPos(int pos) { SCROLLINFO sif; sif.cbSize = sizeof(sif); sif.fMask = SIF POS; sif.nPos = pos;

SetScrollInfo hwnd, SB_CTL, &sif, TRUE);

}

int SCROLLBAR: :getPos() {

SCROLLINFO sif; sif.cbSize = sizeof(sif); sif.fMask = SIF_POS;

GetScrollInfo(_hwnd, SB_CTL, &sif); return sif.nPos;

}

void SCROLLBAR: :onScroll(int nScrollCode, int nPos) { }

// ADJUSTER SCROLLBAR

ADJUSTER_SCROLLBAR::ADJUSTER_SCROLLBAR( ADJUSTER * adjOwner) : SCROLLBAR0 , _adjOwner(adj Owner)

{ }

bool ADJUSTER_SCROLLBAR::create(int x, int y, int nWidth, int nHeight,

HWND hWndParent) { if (! SCROLLBAR: :create(x, y, nWidth, nHeight, hWndParent)) { return false;

} setlnfo(0, 109, 10, 0); return true; } void ADJUSTER_SCROLLBAR::onScroll(int nScrollCode, int nPos) { int n = getPos(); switch (nScrollCode) { case SB_BOTTOM: // Scrolls to the lower right. n = 0; break; case SB_ENDSCROLL: // Ends scroll. return; case SB_LINELEFT: // Scrolls left by one unit. n~; break; case SBJLINERIGHT: // Scrolls right by one unit. n++; break; case SB_PAGELEFT: // Scrolls left by the width of the window. n -= 10; break; case SB_PAGERIGHT: // Scrolls right by the width of the window. n += 10; break; case SB THUMBPOSITION: // Scrolls to the absolute position. The current // position is specified by the nPos parameter, n = nPos; break; case SB_THUMBTRACK: // Drags scroll box to the specified position. The

// current position is specified by the nPos parameter, n = nPos; break; case SB_TOP: // Scrolls to the upper left. n = 100; break; } if(n < 0) { n = 0; } else if (100 < n) { n = 100; } setPos(n);

_adj O wner->onNew Value(n) ;

}

// BUTTON

BUTTON: :BUTTON() : WINDOW0

{ }

bool BUTTON: :create(char * pszText, int x, int y, int nWidth, int nHeight, HWND hWndParent) { return WINDOW: :create("BUTTON", pszText,

BS_PUSHBUTTON | BS_CENTER, x, y, nWidth, nHeight, hWndParent); }

// DIALOG WINDOW

DIALOG_WINDOW::DIALOG_WTNDOW(HWND hwnd) : WINDOW0

{ _hwnd = hwnd; // setting _hwnd this way will prevent setting GWLJJSERDATA HWND DIALOG_WINDOW::getItem(int n) { return GetDlgItem(_hwnd, n); }

void DIALOG_WINDOW::setText(int id, char * psz) { DIALOG_CONTROL c _hwnd, id); c.setText(psz); }

void DIALOG_WINDOW::setTextNumber(int id, int n) { DIALOG_CONTROL c(_ hwnd, id); c.setTextNumber(n); }

void DIALOG_WINDOW::getText(int id, char * psz, int cb) { GetWindowText(getItem(id), psz, cb);

}

int DIALOG_WINDOW::getTextNumber(int id) { char szBuf[40]; getText(id, szBuf, sizeof(szBuf)); int n = 0; for (int i = 0; '0' <= szBuf[i] && szBuf[i] <= '9'; i++) { n = 10*n + szBufIi] - '0';

} return n;

}

// DIALOG CONTROL DIALOG_CONTROL: :DIALOG_CONTROL(HWND hwnd) : WINDOWO

{ _hwnd = hwnd; // setting Jiwnd this way will prevent setting GWLJJSERDATA }

DIALOG_CONTROL::DIALOG_CONTROL(HWND hwndDialog, int id) : WINDOWO

{ Jxwnd = GetDlgItem(hwndDialog, id); // setting _hwnd this way will prevent setting GWLJJSERDATA

}

// COMBO

COMBO: :COMBO(HWND hwndDialog, int id)

: DIALOG_CONTROL(hwndDialog, id)

{

}

int COMBO: :addString(char * psz) { return sendMsg(CB_ADDSTRING, 0, (LPARAM)psz);

}

int COMBO: :selectString(char * psz) { return sendMsg(CB_SELECTSTRLNG, -1, (LPARAM)psz); }

// MAΓN_WΓNDOW

MAIN_WINDOW::MAιN_W NDOW(HWND hwnd) : WINDOW(hwnd) LRESULT MAIN_WINDOW::onCreate() { return 0;

}

LRESULT MAIN_WINDOW::onCommand(WPARAM wParam, LPARAM IParam)

{ return DefWindowProc(_hwnd, WM COMMAND, wParam, IParam);

}

void MAιN_WιNDOW::onPaint(HDC hdc) {

DefWindowProcC hwnd, WM_PAJNT, (WPARAM)hdc, 0); }

// ADJUSTER

ADJUSTER: : AD JUSTER() : JextNameO , _sb(this) , JextValue() , _value(0)

{ }

bool ADJUSTER: :create(char * pszName, int x, int y, HWND hwndParent) { int nHeight = 16; if (! extName.create(pszName, x, y, 70, nHeight, hwndParent)) { return false;

} if (!_sb.create(x+70+20, y, 300, nHeight, hwndParent)) { return false;

} if (! JextValue.create("0%>", x+70+20+300+20, y, 70, nHeight, hwndParent)) { return false; } return true;

}

void ADJUSTER: :setName(char * pszName) { JextName.setText(pszName);

}

void ADJUSTER: :onNewValue(int nValue) { value = nValue; char szBuf[20]; wsprintf(szBuf, "%d%%", nValue); JextValue . setText(szBuf) ;

}

int ADJUSTER: :getValue() { return _value; }

// SETTINGS

char SETTINGS : :_aszNames[4] [MAX_NAME_LENGTH] ; COLORREF SETTINGS ::_col[4]; char SETTINGS : :_szDeviceName[ 1024] ; DWORD SETTINGS ::_nHorizontalRepeat; DWORD SETTINGS ::_cRows;

void SETTINGS ::load() { lstrcpy(_aszNames[0], "cinnamon"); _col[0] = RGB(0, 0, 0); lstrcpy(_aszNames[l], "sage"); _col[l] = RGB(0, 255, 255); lstrcpy(_aszNames[2], "vanilla");

_col[2] = RGB(255, 0, 255); lstrcpy( _aszNames[3], "orange"); _col[3] - RGB(255, 255, 0); lstrcpy(_szDeviceName, "Display"); nHorizontalRepeat = 1 ;

_cRows = 30;

HKEY hkSoftware = NULL; HKEY hkLeinoSoftware = NULL; HKEY hkScentUI = NULL;

LONG err = RegOpenKeyEx(HKEY_CURRENT_USER, "Software",

0L, KEY_ALL_ACCESS, &hkSoftware); if (en != ERROR_SUCCESS) { return;

} err = RegOpenKeyEx(hkSoftware, "LeinoSoftware",

0L, KEY_ALL_ACCESS, &hkLeinoSoftware); if (en != ERROR_SUCCESS) { RegCloseKey(hkSoftware); return;

} err = RegOpenKeyEx(hkLeinoSoftware, "ScentUI",

0L, KEY_ALL_ACCESS, &hkScentUI); if (err != ERROR_SUCCESS) {

RegCloseKey(hkLeinoSoftware); RegCloseKey(hkSoftware); return; }

char data[sizeof(_szDeviceName)]; DWORD type;

DWORD cb = MAX_NAME_LENGTH; err = RegQueryValueEx(hkScentUI, "NameO", 0, &type, (BYTE*)data, &cb); if (err = ERROR_SUCCESS && type = REG_SZ && lstrlen(data) > 0) { lstrcpy(_aszNames[0], data); } cb = MAX_NAME_LENGTH; err = RegQueryValueEx(hkScentUI, "Namel", 0, &type, (BYTE*)data, &cb); if (err = ERROR_SUCCESS && type = REG_SZ && lstrlen(data) > 0) { lstrcpy(_aszNames[l], data); } cb = MAX_NAME JLENGTH; err = RegQueryValueEx(hkScentUI, "Name2", 0, &type, (BYTE*)data, &cb); if (err = ERROR_SUCCESS && type = REG_SZ && lstrlen(data) > 0) { lstrcpy(_aszNames[2], data); } cb = MAX_NAME_LENGTH; err = RegQueryValueEx(hkScentUI, "Name3", 0, &type, (BYTE*)data, &cb); if (err = ERROR_SUCCESS && type = REG_SZ && lstrlen(data) > 0) { lstrcpy(_aszNames[3], data); } cb = sizeof(_szDeviceName); err = RegQueryValueEx(hkScentUI, "Device", 0, &type, (BYTE*)data, &cb); if (err = ERROR_SUCCESS && type = REG_SZ) { lstrcpy(_szDeviceName, data); }

COLORREF col; cb = sizeof(col); err = RegQueryValueEx(hkScentUI, "ColorO", 0, &type, (BYTE*)&col, &cb); if (err = ERROR_SUCCESS && type = REGJDWORD) { _col[0] = col; } cb = sizeof(col); en = RegQueryValueEx(hkScentUI, "Colorl", 0, &type, (BYTE*)&col, &cb); if (err = ERROR_SUCCESS && type = REG_DWORD) { _col[l] = col; } cb = sizeof(col); err = RegQueryValueEx(hkScentUI, "Color2", 0, &type, (BYTE*)&col, &cb); if (err = ERROR_SUCCESS && type = REGJDWORD) { _col[2] = col; } cb = sizeof(col); err = RegQueryValueExφkScentUl. "Color3", 0, &type, (BYTE*)&col, &cb); if (err = ERROR_SUCCESS && type = REG_DWORD) { _col[3] = col; }

DWORD value; cb = sizeof(_nHorizontalRepeat); err = RegQueryValueEx(hkScentUI, "HorizontalRepeat", 0, &type, (BYTE*)&value, &cb); if (err = ERROR_SUCCESS && type = REG_DWORD) { nHorizontalRepeat = value;

} cb = sizeof(_cRows); err = RegQueryValueEx(hkScentUI, "NumRows", 0, &type, (BYTE*)&value,

&cb); if (err = ERROR_SUCCESS && type = REG_DWORD) { _cRows = value; }

RegCloseKey(hkScentUI); RegCloseKey(hkLeinoSoftware);

RegCloseKey(hkSoftware);

}

void SETTINGS: :save() { HKEY hkSoftware = NULL;

HKEY hkLeinoSoftware = NULL; HKEY hkScentUI = NULL;

DWORD dwDisposition; LONG err = RegCreateKeyEx(HKEY_CURRENT_USER, "Software",

0, NULL, REG_OPTION_NON_VOLATILE, KEY ALL ACCESS, NULL,

&hkSoftware, &dwDisposition); if (err != ERROR_SUCCESS) { return;

} err = RegCreateKeyEx(hkSoftware, "LeinoSoftware",

0, NULL, REG_OPTION_NON_VOLATILE, KEY ALL ACCESS, NULL, &hkLeinoSoftware, &dwDisposition); if (err != ERROR_SUCCESS) { RegCloseKey(hkSoftware) ; return;

} err = RegCreateKeyEx(hkLeinoSoftware, "ScentUI",

0, NULL, REG_OPTION_NON VOLATILE, KEY_ALL_ACCESS, NULL, &hkScentUI, &dwDisposition); if (err != ERROR_SUCCESS) { RegCloseKey(hkLeinoSoftware); RegCloseKey(hkSoftware); return;

}

RegSetValueEx(hkScentUI, "NameO", 0, REG_SZ,

(BYTE*)_aszNames[0], strlen(_aszNames[0])+l); RegSetValueEx(hkScentUI, "Namel ", 0, REG_SZ,

(B YTE*)_aszNames[ 1 ] , strlen(_aszNames[ 1 ])+ 1 ) ; RegSetValueEx(hkScentUI, "Name2", 0, REG_SZ,

(BYTE*)_aszNames[2], strlen(_aszNames[2])+l); RegSetValueEx(hkScentUI, "Name3", 0, REG_SZ, (BYTE*)_aszNames[3], strlen(_aszNames[3])+l);

RegSetValueEx(hkScentUI, "Device", 0, REG_SZ,

(BYTE*)_szDeviceName, strlen(_szDeviceName)+l);

RegSetValueEx(hkScentUI, "ColorO", 0, REG_DWORD,

(BYTE*)&_col[0], sizeof(COLORREF)); RegSetValueEx(hkScentUI, "Colorl", 0, REGJDWORD,

(BYTE*)&_col[l], sizeof(COLORREF)); RegSetValueEx(hkScentUI, "Color2", 0, REG_DWORD, (BYTE*)&_col[2], sizeof(COLORREF));

RegSetValueEx(hkScentUI, "Color3", 0, REGJDWORD, (BYTE*)&_col[3], sizeof(COLORREF));

RegSetValueEx(hkScentUI, "HorizontalRepeat", 0, REGJDWORD, (BYTE*)&_nHorizontalRepeat, sizeof(_nHorizontalRepeat));

RegSetValueEx(hkScentUI, "NumRows", 0, REG_DWORD, (BYTE*)&_cRows, sizeof(_cRows)); RegCloseKey(hkScentUI); RegCloseKey(hkLeinoSoftware) ; RegCloseKey(hkSoftware); }

int SETTINGS ::dialogBox(HWND hwndParent) { return DialogBox(hinst, MAKELNTRESOURCE(IDD_SETTINGS), hwndParent, (DLGPROC)DlgProc);

}

void SETTINGS ::setColorNumber(HWND hwndDialog, int idRed, int idGreen, int idBlue, COLORREF col) {

DIALOG VLNDOW dialog(hwndDialog); dialog. setTextNumber(idRed, GetRValue(col)); dialog. setTextNumber(idGreen, GetGValue(col)); dialog.setTextNumber(idBlue, GetBValue(col));

}

COLORREF SETTINGS ::getColorNumber(HWND hwndDialog, int idRed, int idGreen, int idBlue) {

DIALOGJvVTNDOW dialog(hwndDialog); int r = dialog.getTextNumber(idRed); int g = dialog.getTextNumber(idGreen); int b = dialog.getTextNumber(idBlue); if (r < 0) { r = 0; } else if (256 <= r) { r = 255; } if (g < 0) { g = 0; } else if (256 <= g) { g = 255; } if (b < 0) { b = 0; } else if (256 <= b) { b = 255; } return RGB(r, g, b); }

LRESULT CALLBACK SETTINGS ::DlgProc(HWND hwnd, UINT msg, WPARAM wParam, LPARAM IParam) { switch (msg) { case WMJNITDIALOG: {

DIALOG_WTNDOW dialog(hwnd); dialog. setText(IDC_NAMEO, _aszNames[0]); dialog.setText(IDC_NAMEl, _aszNames[l]); dialog.setText(IDC_NAME2, _aszNames[2]); dialog.setText(IDC_NAME3, _aszNames[3]); setColorNumber(hwnd, LDC_RED0, IDC_GREEN0, IDC_BLUE0, _col[0]) setColorNumber(hwnd, IDC_RED1, IDC_GREEN1, IDC_BLUE1, _col[l]) setColorNumber(hwnd, IDC_RED2, IDC J3REEN2, IDC_BLUE2, _col[2]) setColorNumber(hwnd, IDC_RED3, IDCJ3REEN3, IDC_BLUE3, _col[3]) dialog.setTextNumber(IDC_HORIZ_REPEAT, _nHorizontalRepeat); dialog.setTextNumber(IDC_NUM_ROWS, _cRows);

COMBO combo(hwnd, IDC_DEVICE_COMBO); combo. addStringC'Display"); combo. setText(_szDeviceName);

// enumerate printers

DWORD cbNeeded; DWORD cPrinters; if (!EnumPrinters(PRiNTER_ENUM_LOCAL, NULL, 1, NULL, 0, &cbNeeded, &cPrinters) && GetLastErrorO != ERROR JNSUFFICIENT BUFFER) { char szBuf[128]; wsprintf(szBuf,

"Call to get EnumPrinters information failed with error code %>ld", GetLastEπor()); ErrorBox(szBuf); return TRUE; } BYTE * pBuffer = new BYTE[cbNeeded]; if (pBuffer = NULL) {

EπorBox("Failed to allocate EnumPrinters buffer"); return TRUE; } if (!EnumPrinters(PRιNTER_ENUM_LOCAL, NULL, 1, pBuffer, cbNeeded, &cbNeeded, &cPrinters)) { char szBuf[128]; wsprintf(szBuf, "Call to get printer information failed with error code %>ld",

GetLastEπor()); EπorBox(szBuf); delete pBuffer; return TRUE; }

PRINTER JNFOJ * pil = (PRINTER JNFOJ *)pBuffer; for (DWORD i = 0; i < cPrinters; i++) { combo. addS tring(pil [i].pName);

} delete pBuffer; return TRUE; }

case WM_COMMAND: switch (LOWORD(wParam)) { case IDOK: {

DIALOG_WINDOW dialog(hwnd); dialog.getText(IDC_NAME0, _aszNames[0], MAX_NAME_LENGTH); dialog.getText(IDC_NAMEl, _aszNames[l], MAX_NAME_LENGTH); dialog.getText(IDC_NAME2, _aszNames[2], MAX_NAME_LENGTH); dialog.getText(IDC_NAME3, _aszNames[3], MAX_NAME_LENGTH); _col[0] = getColorNumber(hwnd, IDC_RED0, IDC_GREEN0, IDC_BLUE0); _col[l] = getColorNumber(hwnd, IDC_RED1, IDC_GREEN1, IDC_BLUE1) _col[2] = getColorNumber(hwnd, LDC_RED2, IDC_GREEN2, IDC_BLUE2) _col[3] = getColorNumber(hwnd, IDC_RED3, IDC_GREEN3, IDC_BLUE3) nHorizontalRepeat = dialog.getTextNumber(IDC JIORIZ REPEAT); if (_nHorizontalRepeat < 1) { nHorizontalRepeat = 1 ;

}

_cRows = dialog.getTextNumber(IDC_NUM_ROWS); if(_cRows < l) { _cRows = 1 ;

} dialog.getText(IDC_DEVICE_COMBO, _szDeviceName, sizeof(_szDeviceName)) ;

EndDialog(hwnd, TRUE); return 1;

} case IDCANCEL:

EndDialog(hwnd, FALSE); return 1; default: break;

} break;

} return 0;

}

// SCENT MAIN WINDOW

SCENT_MAIN_WINDOW::SCENT_MAIN_WΓNDOW(HWND hwnd) : MALN_WINDOW(hwnd)

{ LRESULT SCENT_MALN_WINDOW::onCreate() { if (MATN_WrNDOW::onCreate() = -l) { return -1;

} for (int i = 0; i < 4; i++) { if (!_adj[i].create(SETTINGS::_aszNames[i], 10, 10+i*40, _hwnd)) { return -1; }

} if (!_button.create("Emit scent", 90, 170, 90, 30, Jrwnd)) { return -1;

} _button.setId(ID_FILE_EMITSCENT); return 0; }

void SCENT_MAIN_WINDOW::setNames() { for (int i = 0; i < 4; i++) {

_adj[i].setName(SETTINGS::_aszNames[i]); } }

LRESULT SCENT_MATN_WiNDOW: :onCommand(WPARAM wParam, LPARAM IParam) {

WORD wNotifyCode = HIWORD(wParam); // notification code

WORD wID = LOWORD(wParam); // item, control, or accelerator identifier

HWND hwndCtl = (HWND) IParam; // handle of control

switch (wID) { case ID FILE EXIT: PostQuitMessage(O) ; break; case ID_EDIT_SETTLNGS: if (SETTINGS::dialogBox(Jrwnd)) { setNames();

SETTINGS ::save();

} break; case ID_HELP_ABOUT: DialogBox(hinst, MAKEINTRESOURCE(IDD_ABOUT), hwnd,

(DLGPROC)AboutDlgProc); break; case ID_FILE_EMITSCENT: emit(); break; default: break;

} return MAιN_WιNDOW::onCommand(wParam, IParam); }

void SCENT_MATN_WINDOW::onPaint(HDC hdc) {

MAIN_WLNDOW: :onPaint(hdc); #ifdefOLD_STUFF RECT rect; if (!GetUpdateRect(_hwnd, &rect, TRUE)) { return;

}

PAINTSTRUCT ps; hdc = BeginPaint(Jτwnd, &ps); emitScent(hdc, 10, 220); EndPaint(Jτwnd, &ps); #endif }

void SCENT_MALN_WINDOW::emitScent(HDC hdc, int x, int y) { HBRUSH hBrush[4]; int values[4]; for (int i = 0; i < 4; i++) { hBrush[i] = CreateSolidBrush(SETTINGS::_col[i]); values[i] = _adj[i].getValue(); }

RECT rect; rect.left = 0; rect.top = y; rect.right = rect.left + SETTINGS : :_nHorizontalRepeat; rect.bottom = rect.top + SETTINGS ::_cRows; for (intj = 0; j < 100; j++) { for (i = 0; i < 4; i++) { if (j < values[i]) { FillRect(hdc, &rect, hBrush[i]);

} rect.left += SETTINGS ::_nHorizontalRepeat; rect.right += SETTINGS " nHorizontalRepeat;

} }

for (i = 0; i < 4; i++) { DeleteObject(hBrush[i]);

} }

void SCENT_MALN_WrNDOW::emit() { if (lstrcmpi(SETTINGS::_szDeviceName, "Display") = 0) { HDC hdc = GetDC(_hwnd); if (hdc = NULL) {

ErrorBox("Failed to obtain Device Context of main window"); return;

} emitScent(hdc, 10, 220); ReleaseDC(_hwnd, hdc); } else { HDC hdc = CreateDC(NULL, SETTINGS ::_szDeviceName, NULL, NULL); if (hdc = NULL) { char szBuf[1200]; wsprintf(szBuf, "Failed to obtain Device Context of device \"%s\"", SETTINGS : :_szDeviceName); ErrorBox(szBuf) ; return;

}

DOCINFO di; di.cbSize = sizeof(DOCINFO); di.lpszDocName = "Scent output"; di.lpszOutput = NULL; di.lpszDatatype = NULL; di.fwType = 0;

StartDoc(hdc, &di);

StartPage(hdc);

emitScent(hdc, 0, 0);

EndPage(hdc);

EndDoc(hdc); DeleteDC(hdc); } }

// the meat

int PASCAL WinMain(HINSTANCE hlnstance, HINSTANCE hPrevInstance, LPSTR IpszCmdLine, int nCmdShow) { // Register the window class for the main window, if (! hPrevInstance) { WNDCLASS wc; wc.style = 0; wc.lpfnWndProc = (WNDPROC) WindowProc; wc.cbClsExtra = 0; wc.cbWndExtra = 0; wc.hlnstance = hlnstance; wc.hlcon = LoadIcon(hInstance, MAKEιNTRESOURCE(ICO_SCENT)); wc.hCursor = LoadCursor((HLNSTANCE) NULL, IDC_ARROW); wc.hbrBackground = GetStockObject(WHITE_BRUSH); wc.lpszMenuName = MAKEιNTRESOURCE(ID_MENU); wc.lpszClassName = "ScentMainWndClass"; if (!RegisterClass(&wc)) {

ErrorBox("RegisterClass failed"); return FALSE;

} }

hinst = hlnstance; // save instance handle SETTINGS ::load();

// Create the main window. hwndMain = Create Window("ScentMainWndClass", szAppName, WSJDVERLAPPED WINDOW, CWJJSEDEFAULT, CW USEDEFAULT,

CWJJSEDEFAULT, CW USEDEFAULT, (HWND) NULL, (HMENU) NULL, hinst, (LPVOID) NULL); // If the main window cannot be created, terminate

// the application, if (IhwndMain) {

ErrorBox("CreateWindow failed"); return FALSE; }

// Show the window and paint its contents.

ShowWindow(hwndMain, nCmdShow);

Update Windo w(hwndMain) ;

// Start the message loop.

MSG msg; while (GetMessage(&msg, (HWND) NULL, 0, 0)) { TranslateMessage(&msg) ; DispatchMessage(&msg);

}

// Return the exit code to Windows, return msg.wParam;

LRESULT CALLBACK WindowProc(HWND hwnd, UINT uMsg, WPARAM wParam, LPARAM IParam) { switch (uMsg) { case WM_COMMAND: if (mainWindow != NULL) { return mainWindow->onCommand(wParam, IParam); } break;

case WM HSCROLL: { int nScrollCode = (int) LOWORD(wParam); // scroll bar value int nPos = (short int) HIWORD(wParam); // scroll box position HWND hwndScrollBar = (HWND) IParam; // handle of scroll bar WINDOW * window = (WINDOW *)GetWindowLong(hwndScrollBar, GWLJJSERDATA); SCROLLBAR * sb = (SCROLLBAR *)window; if (sb != NULL) { sb->onScroll(nScrollCode, nPos);

} break; }

case WM_PAINT: {

HDC hdc = (HDC)wParam; // the device context to draw in if (mainWindow != NULL) { mainWindow->onPaint(hdc); return 0;

} break;

}

case WM_CREATE: { mainWindow = new SCENT_MAIN_WINDOW(hwnd); // this storage is shamelessly never returned if (mainWindow == NULL) { return -1;

} return mainWindow->onCreate(); case WM DESTROY: PostQuitMessage(O); break;

default: break;

} return DefWindowProc(hwnd, uMsg, wParam, IParam);

}

int ErrorBox(LPCTSTR lpText) { return MessageBox(hwndMain, lpText, szAppName, MB_OK | MBJCONHAND);

}

int MsgBox(LPCTSTR lpText) { return MessageBox(hwndMain, lpText, szAppName, MB OK | MBJCONINFORMATION);

}

LRESULT CALLBACK AboutDlgProc(HWND hwnd, UINT msg, WPARAM wParam, LPARAM IParam) { switch (msg) { case WMJNITDIALOG: return TRUE;

case WM_COMMAND: switch (LOWORD(wParam)) { case IDOK: case IDCANCEL: EndDialog(hwnd, TRUE); return 1; default: break; } break;

} return 0;

} // main.h

// Rustan Leino, 4 March 2000

#ifhdef_MAIN_H_ #defme MAIN H

#include <windows.h>

// WINDOW

class WINDOW { protected:

HWND Jrwnd;

public: WINDOW(HWND hwnd);

WINDOWO; // call "create" later -WINDOWO;

bool create(LPCTSTR IpClassName, // pointer to registered class name LPCTSTR lpWindowName, // pointer to window name

DWORD dwStyle, // window style int x, int y, int nWidth, int nHeight,

HWND hWndParent); // handle to parent or owner window

void setld(int id); void setText(char * psz); void setTextNumber(int n); void invalidate();

LRESULT sendMsg(UINT uMsg, WPARAM wParam, LPARAM IParam); };

// STATIC TEXT

class STATIC_TEXT : public WINDOW { public:

STATIC_TEXT(); bool create(char * pszText, int x, int y, int nWidth, int nHeight, HWND hWndParent); };

// SCROLLBAR

class SCROLLBAR : public WINDOW { public:

SCROLLBAR0; bool create(int x, int y, int nWidth, int nHeight, HWND hWndParent);

void setInfo(int min, int max, int page, int pos); void setPos(int pos); int getPos();

virtual void onScroll(int nScrollCode, // scroll bar value int nPos); // scroll box position } ;

// ADJUSTER SCROLLBAR

class ADJUSTER;

class ADJUSTER_SCROLLBAR : public SCROLLBAR { private:

ADJUSTER * _adj Owner;

public:

ADJUSTER_SCROLLBAR(ADJUSTER * adjOwner); bool create(int x, int y, int nWidth, int nHeight,

HWND hWndParent);

/"Override*/ void onScroll(int nScrollCode, int nPos);

} ;

// BUTTON

class BUTTON : public WINDOW { public:

BUTTON0; bool create(char * pszText, int x, int y, int nWidth, int nHeight, HWND hWndParent); };

// DIALOG WINDOW

class DIALOG JVINDOW : public WINDOW { public:

DIALOG_WINDOW(HWND hwnd);

HWND getltem(int n);

void setText(int id, char * psz); void setTextNumber(int id, int n);

void getText(int id, char * psz, int cb); int getTextNumber(int id);

} ;

// DIALOG CONTROL

class DIALOG_CONTROL : public WINDOW { public:

DIALOG_CONTROL(HWND hwnd); DIALOG_CONTROL(HWND hwndDialog, int id);

} ;

// COMBO

class COMBO : public DIALOG ^ONTROL { public: COMBO(HWND hwndDialog, int id);

int addString(char * psz); int selectString(char * psz);

};

// MAIN WINDOW

class MAIN_WINDOW : public WINDOW { public:

MAIN_WINDOW(HWND hwnd);

virtual LRESULT onCreate() ; virtual LRESULT onCommand(WPARAM wParam, LPARAM IParam); virtual void onPaint(HDC hdc);

} ;

// ADJUSTER

class ADJUSTER { private: int _value; STATICJTEXT JextName;

ADJUSTER_SCROLLBAR _sb;

STATIC TEXT JextValue;

public: ADJUSTERO; bool create(char * pszName, int x, int y, HWND hwndParent);

void setName(char * pszName);

void onNewValue(int nValue); int getValue();

}; // SETTINGS

#defme MAX_NAME_LENGTH 31

class SETTINGS { private: static LRESULT CALLBACK DlgProc(HWND hwnd, UINT msg,

WPARAM wParam, LPARAM IParam); static void setColorNumber(HWND hwndDialog, int idRed, int idGreen, int idBlue,

COLORREF col); static COLORREF getColorNumber(HWND hwndDialog, int idRed, int idGreen, int idBlue);

public: static char _aszNames[4][MAX_NAME_LENGTH]; static COLORREF _col[4]; static char _szDeviceName[1024]; static DWORD nHorizontalRepeat; static DWORD _cRows;

static void load(); static void save(); static int dialogBox(HWND hwndParent); };

// SCENT MAIN WINDOW

class SCENT_MAIN_WINDOW : public MAIN_WINDOW { private:

ADJUSTER _adj [4]; BUTTON _button; void emitScent(HDC hdc, int x, int y);

public:

SCENT_MAIN_WINDOW(HWND hwnd);

/^♦override*/ LRESULT onCreate();

/^♦override*/ LRESULT onCommand(WPARAM wParam, LPARAM IParam); void onPaint(HDC hdc);

void setNames();

void emit();

} ;

#endif // _MAIN_H_

// { {NO_DEPENDENCIES } }

// Microsoft Developer Studio generated include file.

// Used by scent.rc //

#defme ICO_SCENT 2

#defme IDD_ABOUT 102

#defιne IDJVIENU 103

#define IDD_SETTINGS 104 #define IDC_NAME0 1000

#defme IDC_NAME1 1001

#defme IDC_NAME2 1002

#define IDC_NAME3 1003

#defme IDC_DEVICE_COMBO 1007 #defme IDC_RED0 1019

#defme IDC_GREEN0 1020

#defme IDC BLUE0 1021 #defιne IDC_RED1 1022 #defme IDC_GREEN1 1023

#defme IDC_BLUE1 1024

#defme IDC_RED2 1025 #defme IDC_GREEN2 1026

#defme IDC_BLUE2 1027

#defme IDC_RED3 1028 #defme IDC_GREEN3 1029

#define IDC_BLUE3 1030 #define IDC_HORIZ_REPEAT 1034

#defme IDC_NUM_ROWS 1035

#defme ID_FILE_EXIT 40002

#defme ID_HELP_ABOUT 40003

#defme ID_FILE_EMITSCENT 40004

#defme ID EDIT SETTINGS 40005

// Next default values for new objects

//

#ifdef APSTUDIO JNVOKED #ifhdefAPSTUDIO_READONLY_SYMBOLS #define _APS_NEXT_RESOURCE_VALUE 101 #defme _APS_NEXT_COMMAND_VALUE 40001 #defme _APS_NEXT_CONTROL_VALUE 1000 #defme _APS_NEXT_SYMED_VALUE 101 #endif #endif

Thus, G-protein coupled receptor (GPCR) based biosensors and sense replication systems and methods for detection, electronic dissemination/digital transmission, and remote reproduction of a stimulus corresponding to, or capable of mimicking and/or replacing a G-protein signal transduction system are disclosed. Although preferred embodiments of the invention have been described in some detail, it is understood that obvious variations can be made without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

Claims We Claim:

1. A system to detect, transmit and reproduce a selected stimulus, comprising:

(a) a biosensor that mimics and/or replaces a biological signal transduction system, wherein the biosensor detects interaction of the selected stimulus and said signal transduction system, and measures a signal resulting from said interaction, wherein the measured signal provides information identifying the selected stimulus; (b) a mathematical coordinate system capable of codifying the information identifying the stimulus, and electronically recording and transmitting said codified information; and

(c) an emission device capable of transposing the codified information to deliver and reproduce the selected stimulus at a location remote in space or time.

2. The system of claim 1 wherein the selected stimulus mimics natural senses of sight, hearing, smell, taste and/or touch.

3. The system of claim 1 wherein said stimulus comprises a physical stimulus or a chemical stimulus.

4. The system of claim 3 wherein said physical stimulus comprises light, sound, vibration, temperature or pressure.

5. The system of claim 3 wherein said chemical stimulus comprises a mixture of one or more stimulant entities comprising odorant molecules, flavarant molecules, pseudo-scents, explosives, contraband drugs, controlled substances, narcotics, hormones, therapeutic agents, diagnostic agents, extracellular metabolites, viruses or antigens.

6. The system of claim 1 wherein the biosensor further comprises a biochemical element capable of producing a secondary messenger, further amplifying the measured signal.

7. The system of claim 6 wherein said biochemical element is capable of mimicking and/or replacing a G-protein signal transduction system.

8. The system of claim 7 wherein said biochemical elements comprise a G-protein; a G-protein coupled receptor protein corresponding to said G-protein; and an enzyme corresponding to said G-protein, wherein the enzyme produces the secondary messenger in response to stimulation of said receptor; and the system further comprises a detector for detecting said secondary messenger.

9. The system of claim 8 wherein said biochemical elements are homologous, and further wherein a plurality of said biochemical elements are arranged in an array in a spatially defined and a physically addressable manner, wherein each spatial position in the array comprises one homologous element.

10. The system of claim 7 wherein the biochemical elements have a dynamic range, wherein the dynamic range is varied by adjusting GTP/GDP ratio.

11. The system of claim 9 wherein the homologous biochemical elements comprise olfactory receptor proteins, receptor proteins of the visual cortex or taste receptor proteins.

12. The system of claim 9 wherein the array comprises a plurality of discrete receptor proteins arranged in a spatially defined and a physically addressable manner, wherein said receptor proteins are arranged in a manner suitable for conducting multiple assays to detect the affinity of a stimulus to said receptor proteins to determine a chemical coordinate space of said stimulus.

13. The system of claim 6 further comprising an amplifying biochemical assay for detecting concentration of the secondary messenger.

14. The system of claim 13 wherein the amplifying biochemical assay comprises an immunoassay, a protein kinase assay or a membrane ion channel assay.

15. The system of claim 6 or claim 13 further comprising a detector for detecting the concentration of the secondary messenger, wherein the detector is a spectroscopic detector, a radiochemical detector or an electrochemical detector.

16. The system of claim 1 wherein said mathematical coordinate system comprises an electronic signal proportional to the measured signal.

17. The system of claim 1 wherein said codified information comprises an electronic signal corresponding to relative amount of stimulant entities to be combined and transmitted by the emission device in order to reproduce said selected stimulus.

18. The system of claim 1 wherein said emission device comprises an array of a plurality of stimulant entities, wherein said stimulant entities are combined and delivered in an appropriate proportion to reproduce the selected stimulus.

19. The system of claim 18 wherein the stimulant entities comprise odorant molecules or flavarant molecules, and the emission device reproduces odors or flavors.

20. The system of claim 1 wherein the biosensor comprises an array of biochemical elements, wherein said biochemical elements are sensitized to stimuli normally not detected by natural senses.

21. The system of claim 20 wherein the biochemical elements are sensitized by genetic engineering methods.

22. A method of detecting the presence and/or amount of a target stimulus in an analyte, comprising:

(a) providing an analyte suspected of containing the target stimulus; (b) contacting an aliquot of said analyte suspected of containing said target stimulus with a plurality of receptor proteins; and

(c) detecting the affinity of said receptor proteins for the target stimulus to determine the presence and/or amount of said target stimulus in said analyte.

23. The method of claim 22 wherein said receptor proteins are present in a biosensor, wherein said biosensor mimics and/or replaces a biological signal transduction system.

24. The method of claim 23 wherein said receptor proteins comprise G- protein coupled receptors.

25. The method of claim 24, wherein said receptor proteins comprise olfactory receptor proteins, taste receptor proteins or receptor proteins of the visual cortex.

26. The method of claim 22 wherein said target stimulus mimics or replaces natural senses of sight, hearing, smell, taste and/or touch.

27. The method of claim 26, wherein said target stimulus comprises a mixture of one or more stimulant entities comprising light, sound, vibrations, odorant molecules, flavarant molecules, pseudo-scents, explosives, contraband drugs, controlled substances, narcotics, hormones, therapeutic agents, diagnostic agents, extracellular metabolites, viruses or antigens.

28. The method according to claim 22, further comprising detecting the presence of a target stimulus exhibiting a biological activity using a high throughput screening assay.

29. The method of claim 28 wherein the target stimulus comprises a mixture of one or more stimulant entities comprising a therapeutic or a diagnostic agent.

30. The method according to claim 22, further comprising purifying the target stimulus by affinity purification, wherein the target stimulus comprises a mixture of one or more stimulant entities.

31. The method of claim 30 further comprising removing undesirable stimulant entities, wherein said stimulant entities comprise odorant and/or flavarant molecules imparting an undesired odor and/or flavor to an analyte.

32. A method for mapping a stimulus in a mathematical coordinate space comprising: (a) detecting the affinity of said stimulus for a plurality of receptor proteins according to the method of claim 22, wherein said stimulus comprises a mixture of one or more stimulant entities;

(b) measuring the intensity with which said mixture of stimulant entities binds to said receptor protein to determine a chemical coordinate space; and (c) transposing said chemical coordinate space to a digital or an analog coordinate space.

33. The method of claim 32, wherein said stimulant entities comprise odorant or flavarant molecules and the stimulus comprises an odor or a flavor.

34. The method of claim 33, wherein said receptor proteins comprise olfactory receptor proteins or taste receptor proteins.

35. A method for reproducing a selected odor or flavor at a remote location comprising:

(a) providing a plurality of odorant or flavarant molecules, (b) mapping an odor or flavor by relative amount of each odorant or flavarant molecule to be combined in order to reproduce said selected odor or flavor; and

(c) delivering an appropriate proportion of said odorant or flavarant molecules to reproduce said selected odor or flavor according to the algorithm defined in Equation 7.

36. The method of claim 35, wherein said odorant and flavarant molecules are delivered by an emission device comprising an inkjet printer, a pneumatic nebulizer, an ultrasonic nebulizer or an electrostatic printer.