Method using a nonlinear optical technique for detection of interactions involving a conformational change

Abstract

A nonlinear optical technique, such as second or third harmonic or sum or difference frequency generation, is used to detect binding interactions, or the degree or extent of binding, that comprise a conformational change. In one aspect of the present invention, the nonlinear optical technique detects a conformational change in a probe due to target binding. In another aspect of the invention, the nonlinear optical technique screens candidate probes by detecting a conformational change due to a probe-target interaction. In another aspect of the invention, the nonlinear optical technique screens candidate modulators of a probe-target interaction by detecting a conformational change in the presence of the modulator.

1 . A method for screening one or more candidate binding partners for binding to a test molecule comprising: 
 (a) illuminating a sample with one or more light beams at one or more fundamental frequencies, said sample comprising said test molecule exposed to said one or more candidate binding partners; and    (b) measuring one or more physical properties of a nonlinear optical light beam emanating from said sample;    wherein a change in the value of said one or more physical properties measured in step (b) relative to a value for said one or more physical properties measured in the absence of exposure of said test molecule to said one or more candidate binding partners indicates that said one or more candidate binding partners bind said test molecule.    
     
     
         2 . The method of  claim 1 , wherein said test molecule is bound to one or more nonlinear-active labels.  
     
     
         3 . The method of  claim 2  further comprising the step of binding said labels to said test molecule prior to step (a).  
     
     
         4 . The method of  claim 2 , wherein said one or more labels are covalently attached.  
     
     
         5 . The method of  claim 2 , wherein said test molecule is non-covalently bound to a molecule that is nonlinear-active.  
     
     
         6 . The method of  claim 2 , wherein said test molecule and one of said nonlinear-active labels form a fusion protein.  
     
     
         7 . The method of  claim 2 , wherein said one or more labels comprise green fluorescent protein or derivatives or mutants of said protein that are nonlinear-active.  
     
     
         8 . The method of  claim 2 , wherein said labels are caged.  
     
     
         9 . The method of  claim 2 , wherein said labels are molecular beacon analogues.  
     
     
         10 . The method of  claim 2 , wherein ultraviolet light acts to cleave a bond between said nonlinear-active label and said test molecule.  
     
     
         11 . The method of  claim 2 , wherein the nonlinear-active properties of said labels can be changed by exposure to a chemical agent or one or more light beam.  
     
     
         12 . The method of  claim 1 , wherein said candidate binding partners are bound to one or more nonlinear-active labels.  
     
     
         13 . The method of  claim 12  further comprising the step of binding said labels to said candidate binding partners prior to step (a).  
     
     
         14 . The method of  claim 12 , wherein said one or more labels are covalently attached.  
     
     
         15 . The method of  claim 12 , wherein said candidate binding partners are non-covalently bound to a molecule that is nonlinear-active.  
     
     
         16 . The method of  claim 12 , wherein said candidate binding partner and one of said nonlinear-active labels form a fusion protein.  
     
     
         17 . The method of  claim 12 , wherein said one or more labels comprise green fluorescent protein or derivatives or mutants of said protein that are nonlinear-active.  
     
     
         18 . The method of  claim 12 , wherein said labels are caged.  
     
     
         19 . The method of  claim 12 , wherein said labels are molecular beacon analogues.  
     
     
         20 . The method of  claim 12 , wherein ultraviolet light acts to cleave a bond between said nonlinear-active label and said candidate binding partners.  
     
     
         21 . The method of  claim 12 , wherein the nonlinear-active properties of said labels can be changed by exposure to a chemical agent or one or more light beam.  
     
     
         22 . The method of  claim 1 , wherein the one or more physical properties are intensities.  
     
     
         23 . The method of  claim 1 , wherein the one or more physical properties are polarization directions.  
     
     
         24 . The method of  claim 1 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule.  
     
     
         25 . The method of  claim 1 , wherein said one or more light beams have a wavelength in the range of 10 to 10000 nanometers.  
     
     
         26 . The method of  claim 1 , which further comprises comparing the value of said physical properties measured in step (b) with the value of the physical properties measured in the absence of exposure to said one or more candidate binding partners.  
     
     
         27 . The method of  claim 1 , wherein said test molecule or said candidate binding partners are bound to at least two distinguishable nonlinear-active labels, wherein said one or more light beams are multiple light beams, and step (b) comprises measuring one or more physical properties of at least two nonlinear optical light beams emanating from said sample.  
     
     
         28 . The method of  claim 1 , wherein said test molecule is purified.  
     
     
         29 . The method of  claim 1 , wherein said test molecule is a G protein-coupled receptor (GPCR).  
     
     
         30 . The method of  claim 29 , wherein said GPCR is bound to a nonlinear-active label.  
     
     
         31 . The method of  claim 1 , wherein said test molecule is a labeled GPCR.  
     
     
         32 . The method of any claims  1 , wherein said nonlinear optical light beam is second harmonic generated light.  
     
     
         33 . The method of  claim 1 , which further comprises applying an electric field to said sample.  
     
     
         34 . The method of  claim 33 , wherein the electric field is stationary in time, time-varying, or some combination thereof.  
     
     
         35 . The method of  claim 1 , wherein said sample further comprises a bulk phase.  
     
     
         36 . The method of  claim 35 , wherein an electric field is applied to said bulk phase.  
     
     
         37 . The method of  claim 36 , wherein the electric field is stationary in time, time-varying, or some combination thereof.  
     
     
         38 . The method of  claim 1 , wherein said one or more nonlinear optical light beams are second harmonics, third harmonics, sum frequencies or difference frequencies of the one or more fundamental frequencies.  
     
     
         39 . The method of  claim 1 , wherein said test molecule is part of a cell, liposome or membrane surface.  
     
     
         40 . The method of  claim 1 , which further comprises measuring said one or more physical properties of the nonlinear optical light beam in the absence of either the test molecules or the candidate binding partners in said sample.  
     
     
         41 . The method of  claim 1 , wherein said test molecule is part of a natural or artificial membrane.  
     
     
         42 . The method of  claim 1 , wherein said one or more candidate binding partners are part of a supported membrane, liposome or a biological cell.  
     
     
         43 . The method of  claim 1 , wherein said one or more candidate binding partners are coupled or conjugated in vitro to a solid surface.  
     
     
         44 . The method according to  claim 43 , wherein said solid surface supports a phospholipid or artificial bilayer membrane.  
     
     
         45 . The method according to  claim 44 , wherein said phospholipid or artificial bilayer comprises membrane proteins.  
     
     
         46 . The method of  claim 1 , wherein said one or more candidate binding partners comprise a portion of a surface of biological cells, liposomes, vesicles, beads, metal particles, or non-metal particles.  
     
     
         47 . The method of  claim 1 , wherein said one or more candidate binding partners are patterned on a solid surface.  
     
     
         48 . The method of  claim 47 , wherein said oligonucleotides or polynucleotides are attached to regions on the surface of size nanometers to microns in dimension.  
     
     
         49 . The method of  claim 1 , wherein said one or more candidate binding partners are patterned in an array format on a solid surface.  
     
     
         50 . The method of  claim 49 , wherein said one or more candidate binding partners comprise a plurality of different oligonucleotides or polynucleotides, said different oligonucleotides or polynucleotides each comprising a different sequence and attached to a different region on a solid surface.  
     
     
         51 . The method of  claim 49 , wherein the oligonucleotides or polynucleotides are patterned in a microarray format.  
     
     
         52 . The method of  claim 1 , wherein said one or more candidate binding partners comprise a plurality of different proteins, said different proteins each comprising a different amino acid sequence and attached to a different region on a solid surface.  
     
     
         53 . The method of  claim 52 , wherein said one or more candidate binding partners comprising a different amino acid sequence are each attached to a different region on a surface of size 1 to 1000 nanometers.  
     
     
         54 . The method of  claim 52 , wherein said proteins are attached to regions on the surface of size 1 to 1000 microns.  
     
     
         55 . The method of  claim 1 , wherein said one or more candidate binding partners comprise proteins patterned in an array format.  
     
     
         56 . The method of  claim 1 , wherein the step of illuminating involves reflection, transmission, evanescent wave, multiple internal reflection, near-field optics, confocal, optical cavity, planar waveguide, fiber-optic or dielectric-slab waveguide.  
     
     
         57 . The method of  claim 1 , wherein the step of measuring involves reflection, transmission, evanescent wave, multiple internal reflection, near-field optics, confocal, optical cavity, planar waveguide, fiber-optic or dielectric-slab waveguide.  
     
     
         58 . The method of  claim 1 , wherein said one or more candidate binding partners are molecular beacon analogues.  
     
     
         59 . The method of  claim 1 , wherein said one or more candidate binding partners are attached to a surface.  
     
     
         60 . The method of  claim 59 , wherein said surface is a metal surface, a semiconductor surface, a glass surface, a latex surface, a gel substrate, a fiber-optic surface, a silica surface or a bead surface.  
     
     
         61 . The method of  claim 59 , wherein said surface is a non-planar surface.  
     
     
         62 . The method of  claim 59 , wherein said surface is chemically derivatized.  
     
     
         63 . The method of  claim 62 , wherein said surface is derivatized with a self-assembled monolayer.  
     
     
         64 . The method of  claim 62 , wherein said surface is derivatized with an organosilane.  
     
     
         65 . The method of  claim 1 , wherein the step of measuring is repeated over different periods of time.  
     
     
         66 . The method of  claim 1 , wherein said one or more candidate binding partners is attached to a self-assembled monolayer.  
     
     
         67 . The method of  claim 66 , wherein the self-assembled monolayer is in the chemical family of silanes or terminal-functional silanes.  
     
     
         68 . The method of  claim 1 , wherein the one or more physical properties of the nonlinear optical light beam measured indicate thermodynamic or kinetic properties of said binding.  
     
     
         69 . The method of  claim 1 , wherein said binding involves a chemical bond, an electrostatic force, physisorption, chemical affinity, chemisorption, molecular recognition, physico-chemical binding, hydrogen bond or hybridization process.  
     
     
         70 . The method of  claim 1 , wherein the nonlinear optical light beam is circularly polarized.  
     
     
         71 . The method of  claim 1 , wherein the sample is on an interface comprising a cell, liposome, vesicle surface, or a solid surface.  
     
     
         72 . The method of  claim 1 , wherein the sample further comprises one or more substances selected from the group consisting of decorator molecules, decorator particles, enhancers, modulators, inhibitors, molecular beacon analogues, and indicators.  
     
     
         73 . The method of  claim 1 , wherein the mode of generation, collection or detection of the nonlinear optical light beam is one or more modes selected from the group consisting of reflection, transmission, evanescent wave, multiple internal reflection, near-field optical techniques, confocal, optical cavity, planar waveguide, fiber-optic and dielectric-slab waveguide, and near-field techniques.  
     
     
         74 . The method of  claim 1 , wherein said test molecule is a drug or blocking agent.  
     
     
         75 . A method for screening one or more candidate modulator molecules for the ability to modulate an interaction between a test molecule and its binding partner comprising: 
 (a) illuminating a sample with one or more light beams at one or more fundamental frequencies, said sample comprising said test molecule exposed to (i) said binding partner, and (ii) said one or more candidate modulator molecules; and    (b) measuring one or more physical properties of a nonlinear optical light beam emanating from said sample;    wherein a change in the value of said one or more physical properties measured in step (b) relative to the value for said one or more physical properties measured in the absence of exposure to said one or more candidate modulator molecules indicates that said one or more candidate modulator molecules modulate the interaction between said test molecule and its binding partner.    
     
     
         76 . The method of  claim 75 , wherein the one or more physical properties are intensities.  
     
     
         77 . The method of  claim 75 , wherein the one or more physical properties are polarization directions.  
     
     
         78 . The method of  claim 75 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule.  
     
     
         79 . The method of  claim 75 , wherein said one or more light beams have a wavelength in the range of 10 to 10000 nanometers.  
     
     
         80 . The method of any claims  75 , wherein said nonlinear optical light beam is second harmonic generated light.  
     
     
         81 . The method of  claim 75 , wherein the step of illuminating involves reflection, transmission, evanescent wave, multiple internal reflection, near-field optics, confocal, optical cavity, planar waveguide, fiber-optic or dielectric-slab waveguide.  
     
     
         82 . The method of  claim 75 , wherein the step of measuring involves reflection, transmission, evanescent wave, multiple internal reflection, near-field optics, confocal, optical cavity, planar waveguide, fiber-optic or dielectric-slab waveguide.  
     
     
         83 . The method of  claim 75 , wherein the step of measuring is repeated over different periods of time.  
     
     
         84 . The method of  claim 75 , wherein the one or more physical properties of the nonlinear optical light beam measured indicate thermodynamic or kinetic properties of said binding.  
     
     
         85 . The method of  claim 75 , wherein said binding involves a chemical bond, an electrostatic force, physisorption, chemical affinity, chemisorption, molecular recognition, physico-chemical binding, hydrogen bond or hybridization process.  
     
     
         86 . The method of  claim 75 , wherein the sample further comprises one or more substances selected from the group consisting of decorator molecules, decorator particles, enhancers, modulators, inhibitors, molecular beacon analogues, and indicators.  
     
     
         87 . The method of  claim 75 , wherein the mode of generation, collection or detection of the nonlinear optical light beam is one or more modes selected from the group consisting of reflection, transmission, evanescent wave, multiple internal reflection, near-field optical techniques, confocal, optical cavity, planar waveguide, fiber-optic and dielectric-slab waveguide, and near-field techniques.  
     
     
         88 . The method of  claim 75 , wherein said test molecule is purified.  
     
     
         89 . The method of  claim 75 , wherein said test molecule is a G protein-coupled receptor (GPCR).  
     
     
         90 . The method of  claim 75 , wherein said GPCR is bound to a nonlinear-active label.  
     
     
         91 . The method of  claim 75 , wherein said test molecule is a labeled GPCR.  
     
     
         92 . The method of  claim 75 , which further comprises applying an electric field to said sample.  
     
     
         93 . The method of  claim 92 , wherein the electric field is stationary in time, time-varying, or some combination thereof.  
     
     
         94 . The method of  claim 75 , wherein said sample further comprises a bulk phase.  
     
     
         95 . The method of  claim 94 , wherein an electric field is applied to said bulk phase.  
     
     
         96 . The method of  claim 95 , wherein the electric field is stationary in time, time-varying, or some combination thereof.  
     
     
         97 . The method of  claim 75 , wherein said one or more nonlinear optical light beams are second harmonics, third harmonics, sum frequencies or difference frequencies of the one or more fundamental frequencies.  
     
     
         98 . The method of  claim 75 , wherein said sample further comprises one or more candidate binding partners.  
     
     
         99 . The method of  claim 98 , which further comprises comparing the value of said physical properties measured in step (b) with the value of the physical properties measured in the absence of exposure to said one or more candidate binding partners.  
     
     
         100 . The method of  claim 98 , wherein said test molecule or said candidate binding partners are bound to at least two distinguishable nonlinear-active labels, wherein said one or more light beams are multiple light beams, and step (b) comprises measuring one or more physical properties of at least two nonlinear optical light beams emanating from said sample.  
     
     
         101 . The method of  claim 98 , wherein said one or more candidate binding partners are patterned in an array format on a solid surface.  
     
     
         102 . The method of  claim 101 , wherein said one or more candidate binding partners comprise a plurality of different oligonucleotides or polynucleotides, said different oligonucleotides or polynucleotides each comprising a different sequence and attached to a different region on a solid surface.  
     
     
         103 . The method of  claim 101 , wherein the oligonucleotides or polynucleotides are patterned in a microarray format.  
     
     
         104 . The method of  claim 98 , wherein said one or more candidate binding partners comprise proteins patterned in an array format.  
     
     
         105 . The method of  claim 98 , wherein said one or more candidate binding partners are molecular beacon analogues.  
     
     
         106 . The method of  claim 98 , wherein said one or more candidate binding partners are attached to a surface.  
     
     
         107 . The method of  claim 106 , wherein said surface is a metal surface, a semiconductor surface, a glass surface, a latex surface, a gel substrate, a fiber-optic surface, a silica surface or a bead surface.  
     
     
         108 . The method of  claim 106 , wherein said surface is a non-planar surface.  
     
     
         109 . The method of  claim 106 , wherein said surface is chemically derivatized.  
     
     
         110 . The method of  claim 109 , wherein said surface is derivatized with a self-assembled monolayer.  
     
     
         111 . The method of  claim 109 , wherein said surface is derivatized with an organosilane.  
     
     
         112 . A method for detecting a conformational change in a test molecule upon binding of the test molecule to a binding partner comprising: 
 contacting said test molecule with one or more candidate binding partners, wherein the test molecule or the one or more candidate binding partners is labeled with a nonlinear-active moiety that is not native to the test molecule or the one or more candidate binding partners, respectively;    (a) illuminating said contacted test molecule with one or more light beams at one or more fundamental frequencies; and    (b) measuring one or more physical properties of a nonlinear optical light beam emanating from said sample;    wherein a change in the value of said one or more physical properties measured in step (b) relative to the value for said one or more physical properties measured in the absence of said one or more candidate binding partners indicates that at least one of said one or more candidate binding partners bind to said test molecule and that said binding induces a conformational change in said candidate binding partners, in said test molecule, or in both said candidate binding partners and said test molecule.    
     
     
         113 . The method of  claim 112 , wherein the one or more physical properties are intensities.  
     
     
         114 . The method of  claim 112 , wherein the one or more physical properties are polarization directions.  
     
     
         115 . The method of  claim 112 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule.  
     
     
         116 . The method of  claim 112 , wherein said one or more light beams have a wavelength in the range of 10 to 10000 nanometers.  
     
     
         117 . The method of  claim 112 , which further comprises comparing the value of said physical properties measured in step (b) with the value of the physical properties measured in the absence of exposure to said one or more candidate binding partners.  
     
     
         118 . The method of  claim 112 , wherein said test molecule or said candidate binding partners are bound to at least two distinguishable nonlinear-active labels, wherein said one or more light beams are multiple light beams, and step (b) comprises measuring one or more physical properties of at least two nonlinear optical light beams emanating from said sample.  
     
     
         119 . The method of  claim 112 , wherein said test molecule is purified.  
     
     
         120 . The method of  claim 112 , wherein said test molecule is a G protein-coupled receptor (GPCR).  
     
     
         121 . The method of  claim 120 , wherein said GPCR is bound to a nonlinear-active label.  
     
     
         122 . The method of any claims  112 , wherein said nonlinear optical light beam is second harmonic generated light.  
     
     
         123 . The method of  claim 112 , which further comprises applying an electric field to said sample.  
     
     
         124 . The method of  claim 123 , wherein the electric field is stationary in time, time-varying, or some combination thereof.  
     
     
         125 . The method of  claim 112 , wherein said sample further comprises a bulk phase.  
     
     
         126 . The method of  claim 125 , wherein an electric field is applied to said bulk phase.  
     
     
         127 . The method of  claim 126 , wherein the electric field is stationary in time, time-varying, or some combination thereof.  
     
     
         128 . The method of  claim 112 , wherein said one or more nonlinear optical light beams are second harmonics, third harmonics, sum frequencies or difference frequencies of the one or more fundamental frequencies.  
     
     
         129 . The method of  claim 112 , wherein said one or more candidate binding partners are patterned in an array format on a solid surface.  
     
     
         130 . The method of  claim 129 , wherein said one or more candidate binding partners comprise a plurality of different oligonucleotides or polynucleotides, said different oligonucleotides or polynucleotides each comprising a different sequence and attached to a different region on a solid surface.  
     
     
         131 . The method of  claim 129 , wherein the oligonucleotides or polynucleotides are patterned in a microarray format.  
     
     
         132 . The method of  claim 112 , wherein said one or more candidate binding partners comprise proteins patterned in an array format.  
     
     
         133 . The method of  claim 112 , wherein the step of illuminating involves reflection, transmission, evanescent wave, multiple internal reflection, near-field optics, confocal, optical cavity, planar waveguide, fiber-optic or dielectric-slab waveguide.  
     
     
         134 . The method of  claim 112 , wherein the step of measuring involves reflection, transmission, evanescent wave, multiple internal reflection, near-field optics, confocal, optical cavity, planar waveguide, fiber-optic or dielectric-slab waveguide.  
     
     
         135 . The method of  claim 112 , wherein said one or more candidate binding partners are molecular beacon analogues.  
     
     
         136 . The method of  claim 112 , wherein said one or more candidate binding partners are attached to a surface.  
     
     
         137 . The method of  claim 136 , wherein said surface is a metal surface, a semiconductor surface, a glass surface, a latex surface, a gel substrate, a fiber-optic surface, a silica surface or a bead surface.  
     
     
         138 . The method of  claim 136 , wherein said surface is a non-planar surface.  
     
     
         139 . The method of  claim 136 , wherein said surface is chemically derivatized.  
     
     
         140 . The method of  claim 139 , wherein said surface is derivatized with a self-assembled monolayer.  
     
     
         141 . The method of  claim 139 , wherein said surface is derivatized with an organosilane.  
     
     
         142 . The method of  claim 112 , wherein the step of measuring is repeated over different periods of time.  
     
     
         143 . The method of  claim 112 , wherein said one or more candidate binding partners is attached to a self-assembled monolayer.  
     
     
         144 . The method of  claim 143 , wherein the self-assembled monolayer is in the chemical family of silanes or terminal-functional silanes.  
     
     
         145 . The method of  claim 112 , wherein the one or more physical properties of the nonlinear optical light beam measured indicate thermodynamic or kinetic properties of said binding.  
     
     
         146 . The method of  claim 112 , wherein said binding involves a chemical bond, an electrostatic force, physisorption, chemical affinity, chemisorption, molecular recognition, physico-chemical binding, hydrogen bond or hybridization process.  
     
     
         147 . The method of  claim 112 , wherein the sample further comprises one or more substances selected from the group consisting of decorator molecules, decorator particles, enhancers, modulators, inhibitors, molecular beacon analogues, and indicators.  
     
     
         148 . The method of  claim 112 , wherein the mode of generation, collection or detection of the nonlinear optical light beam is one or more modes selected from the group consisting of reflection, transmission, evanescent wave, multiple internal reflection, near-field optical techniques, confocal, optical cavity, planar waveguide, fiber-optic and dielectric-slab waveguide, and near-field techniques.  
     
     
         149 . The method of  claim 112 , wherein a nonlinear-active label is covalently bound to said test molecule or to said one or more candidate binding partners.  
     
     
         150 . The method of  claim 112 , wherein a nonlinear-active label is covalently bound to a molecule that is noncovalently bound to said test molecule or to said one or more candidate binding partners.  
     
     
         151 . The method of  claim 112 , which further comprises comparing the value of said one or more physical properties measured in step (b) relative to the value of the one or more physical properties measured in the absence of said one or more candidate binding partners.  
     
     
         152 . A method for detecting the degree or extent of the conformational change induced by binding between a test molecule and one or more candidate binding partners comprising: 
 (a) contacting said test molecule with one or more candidate binding partners, wherein the test molecule or the one or more candidate binding partners is labeled with a nonlinear-active moiety that is not native to the test molecule or the one or more candidate binding partners, respectively;    (b) illuminating said contacted test molecule with one or more light beams at one or more fundamental frequencies; and    (c) measuring one or more physical properties of a nonlinear optical light beam emanating from said sample;    wherein the extent of the change in the value of said one or more physical properties measured in step (c) relative to the value for said one or more physical properties measured in the absence of said one or more candidate binding partners indicates the degree or extent of the conformational change that said binding induces.    
     
     
         153 . The method of  claim 152 , wherein the one or-more physical properties are intensities.  
     
     
         154 . The method of  claim 152 , wherein the one or more physical properties are polarization directions.  
     
     
         155 . The method of  claim 152 , wherein said test molecule is nonlinear-active in the absence of an exogenous nonlinear-active label bound to the test molecule.  
     
     
         156 . The method of  claim 152 , wherein said one or more light beams have a wavelength in the range of 10 to 10000 nanometers.  
     
     
         157 . The method of  claim 152 , which further comprises comparing the value of said physical properties measured in step (b) with the value of the physical properties measured in the absence of exposure to said one or more candidate binding partners.  
     
     
         158 . The method of  claim 157 , wherein said test molecule or said candidate binding partners are bound to at least two distinguishable nonlinear-active labels, wherein said one or more light beams are multiple light beams, and step (b) comprises measuring one or more physical properties of at least two nonlinear optical light beams emanating from said sample.  
     
     
         159 . The method of  claim 152 , wherein said test molecule is purified.  
     
     
         160 . The method of  claim 152 , wherein said test molecule is a G protein-coupled receptor (GPCR).  
     
     
         161 . The method of  claim 160 , wherein said GPCR is bound to a nonlinear-active label.  
     
     
         162 . The method of any claims  152 , wherein said nonlinear optical light beam is second harmonic generated light.  
     
     
         163 . The method of  claim 152 , which further comprises applying an electric field to said sample.  
     
     
         164 . The method of  claim 163 , wherein the electric field is stationary in time, time-varying, or some combination thereof.  
     
     
         165 . The method of  claim 152 , wherein said sample further comprises a bulk phase.  
     
     
         166 . The method of  claim 165 , wherein an electric field is applied to said bulk phase.  
     
     
         167 . The method of  claim 166 , wherein the electric field is stationary in time, time-varying, or some combination thereof.  
     
     
         168 . The method of  claim 152 , wherein said one or more nonlinear optical light beams are second harmonics, third harmonics, sum frequencies or difference frequencies of the one or more fundamental frequencies.  
     
     
         169 . The method of  claim 152 , wherein said one or more candidate binding partners are patterned in an array format on a solid surface.  
     
     
         170 . The method of  claim 169 , wherein said one or more candidate binding partners comprise a plurality of different oligonucleotides or polynucleotides, said different oligonucleotides or polynucleotides each comprising a different sequence and attached to a different region on a solid surface.  
     
     
         171 . The method of  claim 169 , wherein the oligonucleotides or polynucleotides are patterned in a microarray format.  
     
     
         172 . The method of  claim 152 , wherein said one or more candidate binding partners comprise proteins patterned in an array format.  
     
     
         173 . The method of  claim 152 , wherein the step of illuminating involves reflection, transmission, evanescent wave, multiple internal reflection, near-field optics, confocal, optical cavity, planar waveguide, fiber-optic or dielectric-slab waveguide.  
     
     
         174 . The method of  claim 152 , wherein the step of measuring involves reflection, transmission, evanescent wave, multiple internal reflection, near-field optics, confocal, optical cavity, planar waveguide, fiber-optic or dielectric-slab waveguide.  
     
     
         175 . The method of  claim 152 , wherein said one or more candidate binding partners are molecular beacon analogues.  
     
     
         176 . The method of  claim 152 , wherein said one or more candidate binding partners are attached to a surface.  
     
     
         177 . The method of  claim 176 , wherein said surface is a metal surface, a semiconductor surface, a glass surface, a latex surface, a gel substrate, a fiber-optic surface, a silica surface or a bead surface.  
     
     
         178 . The method of  claim 176 , wherein said surface is a non-planar surface.  
     
     
         179 . The method of  claim 176 , wherein said surface is chemically derivatized.  
     
     
         180 . The method of  claim 179 , wherein said surface is derivatized with a self-assembled monolayer.  
     
     
         181 . The method of  claim 179 , wherein said surface is derivatized with an organosilane.  
     
     
         182 . The method of  claim 152 , wherein the step of measuring is repeated over different periods of time.  
     
     
         183 . The method of  claim 152 , wherein said one or more candidate binding partners is attached to a self-assembled monolayer.  
     
     
         184 . The method of  claim 183 , wherein the self-assembled monolayer is in the chemical family of silanes or terminal-functional silanes.  
     
     
         185 . The method of  claim 152 , wherein the one or more physical properties of the nonlinear optical light beam measured indicate thermodynamic or kinetic properties of said binding.  
     
     
         186 . The method of  claim 152 , wherein said binding involves a-chemical bond, an electrostatic force, physisorption, chemical affinity, chemisorption, molecular recognition, physico-chemical binding, hydrogen bond or hybridization process.  
     
     
         187 . The method of  claim 152 , wherein the sample further comprises one or more substances selected from the group consisting of decorator molecules, decorator particles, enhancers, modulators, inhibitors, molecular beacon analogues, and indicators.  
     
     
         188 . The method of  claim 152 , wherein the mode of generation, collection or detection of the nonlinear optical light beam is one or more modes selected from the group consisting of reflection, transmission, evanescent wave, multiple internal reflection, near-field optical techniques, confocal, optical cavity, planar waveguide, fiber-optic and dielectric-slab waveguide, and near-field techniques.  
     
     
         189 . The method of  claim 152 , wherein a nonlinear-active label is covalently bound to said test molecule or to said one or more candidate binding partners.  
     
     
         190 . The method of  claim 152 , wherein a nonlinear-active label is covalently bound to a molecule that is noncovalently bound to said test molecule or to said one or more candidate binding partners.  
     
     
         191 . The method of  claim 152 , which further comprises comparing the value of said one or more physical properties measured in step (b) relative to the value of said one or more physical properties measured in the absence of said one or more candidate binding partners.  
     
     
         192 . The method of claims  1 , wherein said one or more candidate binding partners are not attached to a surface.  
     
     
         193 . The method of claims  75 , wherein said one or more candidate binding partners are not attached to a surface.  
     
     
         194 . The method of claims  112 , wherein said one or more candidate binding partners are not attached to a surface.  
     
     
         195 . The method of claims  152 , wherein said one or more candidate binding partners are not attached to a surface.