WO1995001569A1

WO1995001569A1 - Method for detecting the presence of an analyte

Info

Publication number: WO1995001569A1
Application number: PCT/US1994/007322
Authority: WO
Inventors: Hans O. Ribi; Brian Sullivan; Herman Gaub; Joe Murdoch
Original assignee: Biocircuits Corporation
Priority date: 1993-06-29
Filing date: 1994-06-29
Publication date: 1995-01-12

Abstract

Methods for detecting the presence of an analyte in a sample are provided. The methods employ a detection system which is comprised of a polymerized polyunsaturated lipid film. The polymerized lipid film has binding molecules associated with it which are members of a specific binding pair of which the analyte is the other member. The polymerized lipid film absorbs light at one wavelength before the binding of the analyte to the binding molecule, but absorbs at a different wavelength following binding. The shift in absorption is detectable by a color shift in the polymerized lipid film. The binding event can also change the emission spectrum of the film from one intensity to another. These optical changes are used to detect the presence of an analyte in a given sample.

Description

METHOD FOR DETECTING THE PRESENCE OF AN ANALYTE

INTRODUCTION

Technical Field

The field of this invention is the detection of analytes using polymerized lipid layers.

Background As the world has become more complex and as our understanding of different phenomena has increased, there has been a concomitant need to improve methods of measuring the wide variety of substances in use today. From the clinical laboratory, there has been increasing interest in being able to measure various substances in the doctor's office, the home, at bedside, in the field, as well as other sites. With the continuously increasing number of physiologically active substances, both naturally occurring and synthetic, there has been a desire to be able to measure the substances as indicative of the health status of an individual, for therapeutic dosage monitoring, for research, and the like. The substances may be found in a wide variety of samples, ranging over numerous orders of magnitude in concentration for their dynamic ranges of activity, and further differ as to the ease with which one may detect their presence. An area which has only recently assumed substantial commercial importance and will be of increasing importance is the detection of specific nucleotide sequences. Nucleotide sequences find application in genetic counseling, forensic medicine, detection of diseases, and the like. There is, therefore, a wide diversity of opportunities to measure diverse substances from different sources with different sensitivities and for a wide range of purposes.

The methods for detection have ranged from radioactive labels, light absorption, fluorescence, chemiluminescence, agglutination, etc. Each of these methods has found application and has disadvantages as well as advantages over alternative methods. As yet, there has been no single method which has proven applicable in all situations. There is, therefore, substantial interest in devising new methods which may provide for significant opportunities in measuring compounds of interest, where the protocols, apparatus, or reagents may provide advantages over other techniques.

Relevant Literature

U.S. Patent No. 4,489, 133 describes procedures and compositions involving orderly arrays of protein molecules bound to surfactant. J. Am. Chem. Soc. (1988) 110: 7571-7572 describe methods for forming multilayer thin polymerized films. Lieser et al, (1979) 17: 1631-1644 describe the preparation, spreading behavior, multilayer formation, and polymerization phenomena of various long chain diacetylene monocarbonic acids. Bhattachargee et al. , J. Chem. Phys. (1980) 73: 1478-1480 report the effects of pH and electrolyte on the absorption and fluorescence spectra of polydiacetylenes. Chance et al. , J.Chem Phys. (1979) 71:206-211 report on the effects of increasing temperature on the optical properties of polydiacetylene films. Olmstead et al, J.Phys. Chem. (1983) 87: 4790-4792 describe the blue to pink transition of a polydiacetylene film by raising the temperature of the film. Fouassier et ah, Israel J. of Chem. (1979) 18: 227-232 describe the photochemistry of the polymerization of multilayer polydiacetylenes. Kanetake et al, J. Phvs. Soc. of Japan (1985) 54: 4014-4026,

Day et al , Israel J. of Chem. (1979) 18:325-329, Mino et al , Langmuir (1992) 8:594-598, and Mino et al , Langmuir (1991) 7:2336-2341 describe color transitions of polydiacetylene films.

SUMMARY OF THE INVENTION

Novel methods are provided for detecting the presence of an analyte in a sample using a polymerized film. The film may be lipid layers, vapor deposited, cast, cast liposomal, spun or gel phase. The polymerized lipid layer used in the method is a polymerized polyunsaturated film which is associated with binding moieties. Upon binding of an analyte, the absorbed light or stimulated emitted light of the polymerized film shifts or changes, indicating that binding of the analyte has occurred. The shift in absorbed or emitted light provides for an easy detection method for a wide variety of analytes.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods are provided for detecting the presence of analyte in a sample. The methods make use of the shift in absorbed light or change in stimulated emitted light of a polymerized layer in response to a binding event between an analyte and a ligand group associated with the layer. Central to the method is the presence of a layer comprising a polymer of polymerizable diynoic lipid monomers that absorb or emit light of one wavelength following polymerization, but upon binding of an analyte, absorb light of a different wavelength or emit light of a different wavelenght. The shift in absorbed light or emitted light is used to detect the presence of analyte in a sample.

The polymerized lipid layer may be prepared in a variety of ways, where the layer may be homogenous or heterogenous as to the number of unsaturated lipids which are involved in the polymer, the nature of the moieties bound to the lipids, and the like. Further, the polymerized layer can be in any convenient form, particularly single or multilayer films. The film used is preferably a monolayer. The polymerized layer can be formed using the method described in U.S. application serial nos. 366,651, filed 06/15/89 and 453,784, filed 12/20/89 where a novel temperature gradient technique is employed. Surfactant films may be formed on the surface of an aqueous subphase by standard technologies for lipid monolayers, vapor deposited, cast, cast liposomal, spun, or gel phase. A solution containing a monomeric surfactant composition, dissolved in an organic solvent, is applied to the subphase surface by a micro-syringe. Solvents may include hydrocarbons such as pentane, hexane, heptane, and decane. The hydrocarbons may be straight chain, branched, cyclic, or unsaturated. Solvents may include chlorocarbons such as mono-, di-, tri- or tetrachloroethane. The addition of more polar solvents such as alcohols, furans, ethers, esters, or the like may be added to enhance the solubility of the surfactant composition.

The subphase composition is one process variable which dictates the physical characteristics of the surfactant layer which is formed. The subphase can be composed of pure water, glycerol, polyethylene glycol, or other polar organic solvents miscible with water including DMF, DMSO, acetone, alcohols, ketones, furans, dioxane, ethanolamine, phenols, colloidal substances including dispersed carbon powder, alone or in combination, or the like. High boiling point solvents such as glycerol will reduce evaporation during heating, while low boiling point solvents will enhance the evaporation. Other organic solvents can be used to stabilize the surfactant film, particularly to favorably interact with the polar headgroups, linkers and ligands of the surfactant. The subphase can also contain organic or inorganic acids or bases which affect the surfactant film through ionic interactions, i.e., charge stabilization. The ionic components can include mono- and polyvalent ions and cations, and mono- and oligosaccharides. Monomeric polymerizable surfactants are spread on the subphase at a concentration ranging from .01 to 50.0 mM in spreading solvent. Typically 0.1 to 5.0 mM is most useful. Films may either be formed from a homogenous solution of polymerizable surfactants or may be formed with a mixture of polymerizable surfactants and filler surfactants which have no polymerizable groups. The surfactants may or may not have ligands bound to the polar head group of the surfactant. The filler surfactant may have an hydroxyl, polyhydroxyl or polyethylene oxide headgroup which acts to prevent non-specific adherence of biological matter. When ligand is bound to the surfactant, the mole percentage incorporation of the ligand-surfactant to the filler-surfactant will vary depending on the particular assay, generally ranging from 0.01 to 100%, more usually from 0.1-

10% and usually in the range of about 1.0 to 5%. Steric displacement can enhance protein binding, and steric hindrance can inhibit protein binding. The composition of the polar headgroup of the filler-lipid can thus provide a control mechanism for adjusting binding affinities and interactions, including responsiveness in an assay. Film formation involves applying a subphase to a surface or well. A solution containing the monomeric surfactant is applied to a precleaned (aspirated) subphase surface until the surface is substantially saturated. The aqueous medium is pre-heated to melt and disperse the surfactant, usually to a temperature of not more than about 130°C, more usually not more than about 110°C, which results in evaporation of the spreading solvent. The medium is then allowed to cool to below room temperature, usually to about 7°C. The rate of cooling, a key process variable for making highly crystalline films, is controlled by regulating the traverse rate of the subphase slide from the heating element to the cooling element. Typical traverse rates vary from .06 cm/min. to 1.0 cm/minute, usually 0.3 cm/min.

The surfactant molecule may have a single lipid chain, e.g., a diynoic acid or a plurality of lipid chains, e.g., diester glycerides, preferably a single chain, and generally not more than two lipid chains. Illustrative surfactants include ethanolamino-10,12-pentacosadiynamide,

6,8-hexadecadiynoic acid, 2-hydroxyethyl octadeca-8-lO-diynoate, eicosa-12,14- diynyl-10, 12-phosphatidyl serine, pentaeicosa-10,12-diynoic acid, tricosa-10, 12- diynoic acid, acetylene compounds with multiple diyne groups and other polymer surfactants including single acyl chain polymerizable surfactants. Various other surfactants may be present as diluents of the polymerizable surfactant. These surfactants may be naturally occurring, synthetic, or combinations thereof, and may be illustrated by laurate, stearate, arachidonate, cholesterol, bile acids, gangliosides, sphingomyelins, cerebrosides, or the like. Various functional groups may be present in the film to provide for polymerization and various optical properties, such as Forster energy transfer. For the most part, the functional groups will comprise diynes, although other polyunsaturated molecules may find use, such as activated monoynes, e.g., a- ketomonoynes.

For the most part, the hydrophobic portion of the surfactant will have a chain of at least 6 aliphatic carbon atoms, usually a straight chain of at least 8 aliphatic carbon atoms, and generally not more than a total of about 100 carbon atoms, usually not more than about 34 carbon atoms. Preferably, the number of carbon atoms will vary from about 12 to 32, more usually 23 to 30, and more preferably 24 to 29 carbon atoms. The lipid molecules will terminate in a hydrophilic moiety, cationic, anionic or neutral (nonionic) where the functionalities may include non-oxo carbonyl, e.g., carboxylic acids, esters and amides, oxo-carbonyl, such as aldehydes or ketones, oxy, such as ethers, polyethers, and hydroxyl, amino, such as primary, secondary, and tertiary amines and ammonium, phosphorus acid esters and amide, such as phosphate, phosphonate, and phosphonamide, sulfur functionalities, such as thiol, sulfonates, sulfate, and sulfonamides, and the like. Hydrophilic groups may include drugs, peptides, ligands, receptors or chromophores. Usually, the polymerizable functionality will be separated from the polar and non-polar termini by at least one carbon atom, generally from about 1 to 50 carbon atoms, more usually from about 1 to 8 carbon atoms. The polymerizable group may typically be incorporated into the hydrophobic interior of the surfactant film. The individual polymerizable groups can be spaced at regular intervals from 0-50 carbons apart, typically 0-10 carbon atoms apart, most usually joined by a bond. There can be as many of these groups in the chain as its length allows. Variations of the headgroup provide for variations in film properties, such as stability of the film, surface charge, control of interhead-group hydrogen bonding, reduction of non¬ specific binding or fluid matrix effects, and ease of chemical modifications. The hydrocarbon tail of the surfactant may also terminate in a hydrophilic group so that the surfactant is bipolar. [Sher, Justus Liebigs Ann. Chem. (1954) 589:234; and Akimoto, et §1. Angew. Chem. (1981) 2010:91].

The polymerized films used in the method can be prepared from the above lipid monomers, for the most part, using conventional techniques and employing particular conditions to achieve the layers with desired qualities. For the most part, in film formation, conventional Langmuir-Blodgett techniques may be employed. A large number of parameters are available which can be used to influence the nature of the product. These parameters include the buffer type, including pH, ionic strength, cations employed, e.g., mono- or polyvalent, composition of the surfactant, both as to the polymerizable surfactant and the nonpolymerizable surfactant, including such considerations as chain length, the nature of the polymerizable functionality, the nature of the polar head group, the manner in which the surfactant layer is formed, including concentration of surfactant and solvent, the nature of the solvent, the spreading method, the amount of surfactant employed, subphase composition, superphase composition, all of which will affect the formation of mono- or multilamellar layers. Additionally, physical parameters, such as film tension, crystallization time, temperature, humidity, traverse rates, will affect the nature of the polymerized film. The monomers, which are to make up the polymerized layer used in the subject method, after being compacted in a layer, are then polymerized employing any convenient means, e.g., ultra-violet light. Polymerization times are important for controlling the assay responsiveness of the layers, such as the films. Prolonged polymerization times (10 minutes) generally lead to non-responsive layers.

Polymerization times between 20 seconds to 5 minutes can lead to more responsive layers dependent upon radiation intensity. Typically, times ranging from 30 seconds to 2 minutes give the greatest response. The distance of the UV light source can also be a factor in formation of the polymerized layers. Distances typically range from 1 to 4 inches, usually 1-3 inches. The fluence of UV light on the surface will range from 1 mjoule/cm² to 100 mjoule/cm², more usually 30 mjoule/cm².

Other parameters, including the presence of inert gases or free radical initiators, can be used to control the polymerization rates and material responsiveness. Other initiation systems include combinations of light and light sensitive initiators, heat labile chemical initiators or the like. Such initiators are conventional and need not be described here. The activation is maintained until at least substantially complete polymerization is achieved. Polymerization may also be carried out by using electron beams, X-ray sources, synchotron radiation and the like.

Polymerization should occur in the absence of free oxygen. Accordingly, the film may be polymerized in the presence of inert gases, submerged in the subphase, or in any other environment where free oxygen is not present.

The polymerized layer can then be transferred to any convenient support for subsequent visualization of the change in absorbed or emitted light. The layer can be left in solution, placed on a fluid support, or transferred to a porous layer or tape. When the layer is transferred to a fluid support, greater flexibility may be achieved with regards to when the light is absorbed or emitted. Specifically, the polymerized groups experience a greater degree of flexibility than they do on a solid support, thus allowing for the possibility of reversible shifts in absorbed or emitted light. Transferring the layer to a solid support may limit the film's flexibility, and thus reversibility, of the color shift. However, transferring the film to a porous surface can be used to draw the sample through the layer, thus concentrating the analyte on the film surface. This may enhance binding of the analyte to the ligand on the film surface.

Transfer of the layer to the various available supports, can be accomplished through any convenient means, particularly using Langmuir-Blodgett techniques [George L. Gaines Jr.: Insoluble Monolayers at Liquid Gas Interfaces, Interscience

Publishers, I.Prigogine Editor, John Wiley and Sons (1964)]. Finally, the film can be transferred to the support before polymerization, and polymerized while on the support. Following transfer of the polymerized film to the appropriate support, the polymerized film is ready to be used to detect the presence of analyte. The ligand used in analyte detection may be bound to an independent macromolecule or to the surfactant, preferably to an independent macromolecule such as a protein, peptide, sugar, receptor, or other ligand. This binding group will be at least 0.5 kD, usually 20 kD or more and may be bound covalently or non- covalently to the support for the film. The macromolecule may be applied to the support before or after the lipid film is applied to the support. The ligand can be chemically coupled, enzymatically coupled or absorbed to the film. The ligand density will range from .01 % to 100 % of the surface area. If the ligand is bound to the surfactant layer, depending upon the desired density of the ligand bound to the surfactant, the ligand size and the ligand's physical/chemical properties, the ligand may be present in from about 0.01 to 100 mol % of surfactant, more usually at least about 0.1 mol %, and preferably at least about 1 mol %, generally not more than about 10 mol %. The mol ratio will depend on the size and nature of the ligand, whether contiguous ligands are desired in the layer, and the like. The ligands may be joined by any convenient functionality, including esters, e.g., carboxylate and phosphate, ethers, either oxy or thio, amino, including ammonium, hydrazines, polyethylene oxides, amides, such as carboxamide, sulfonamide or phosphoramide, carbons or polycarbons, combinations thereof, or the like. Specific groups may involve saccharides, both mono- and polysaccharide, including aminosaccharides, carboxy saccharides, reduced saccharides, peptides, polypeptides, nucleotides, oligonucleotides, or the like. Specific groups include zwitterions, e.g., betaine, peptides, sugars, such as glucose, glucuronic acid, 3-galactosamine, sialic acid, etc., phosphatidyl esters, such as phosphatidyl glycerol serine, inositol, etc. The ligand can be any molecule, usually a small molecule, containing a reactive group. Typical ligands could be biotin, drugs such as alkaloids, chromophores, antigens, chelating compounds, crown ethers, molecular recognition complexes, poly saccharides, polypeptides, polynucleotides, ionic groups, polymerizable groups, fluorescence quenching groups, linker groups, electron donor or acceptor groups, hydrophobic groups or hydrophilic groups. The ligand may also serve as a site which can be further chemically modified to bring about new physical features or film characteristics.

The ligands which are covalently bonded to the surfactant will normally be a member of a specific binding pair. Thus, the ligands may be varied widely, usually being molecules of less than about 2 kD, more usually less than about 1 kD. For the most part, the ligands will be considered to be receptors or haptenic, which may include small organic molecules, including oligopeptides, oligonucleotides, saccharides or oligosaccharides, or the like. However, in some situations, the ligand bound to the surfactant may be a macromolecule, usually not exceeding about 500 kD, more usually not exceeding about 200 kD. Thus, proteins, nucleic acids, or other polymeric or nonpolymeric compounds of high molecular weight may also be employed. There is also the possibility to use crown ethers which will bind to particular ions. The particular manner in which one or more surfactants may be bound to the ligand is not critical to this invention and will depend, for the most part, on convenience, ease of synthesis, stability, available functional groups, and the like. Synthetic macrocyclic complexes may be incorporated into the surfactant layer for the purpose of molecular recognition of various natural and non-natural compounds. In many cases, particular moieties will be used for a variety of purposes.

For example, biotin may be used to bind to avidin or streptavidin, where the complementary member may then be used to link a wide variety of other molecules. Various lectins may be employed to bind a variety of sugars which may be attached to molecules of interest. Specific ligands may be employed which bind to complementary receptors, such as surface membrane receptors, soluble receptors, or the like.

Of particular interest is the binding of receptor, either directly or indirectly, to the surfactant. Direct binding will usually be covalent, while indirect binding will usually be non-covalent, such as non-specific or specific adsorption. Receptors of particular interest will be antibodies, which include IgA, IgD, IgE, IgG and IgM, which may be monoclonal or polyclonal. The antibodies may be intact, their sulfhydryl bridges totally or partially cleaved, fragmented to F(ab')₂ or Fab, or the like. The intact and totally cleaved antibodies may be used to make a recombinant protein A-antibody hybrid, to be incorporated into the assay. Coupling through the antibody's oligosaccharide moiety to hydrazines can be achieved with the intact, partially and totally cleaved antibody. Maleimide linkages may be used for the intact, partially and totally cleaved antibodies, and the F(ab')₂ fragment, while the Fab fragment may be incorporated in an antibody hybrid.

Other examples for antibody coupling to polymer films will include the use of recombinant hybrid linker proteins and recombinant antibody molecules. The antibodies may be functionalized at the Fc portion to ensure the availability of the binding sites for further binding. Other receptors include naturally occurring receptors, such as viral receptors, surface membrane protein receptors, blood protein receptors, etc.

In using the polymerized lipid layer for analyte detection, the analyte containing sample can be contacted to the polymerized layer using any convenient means. The analyte containing sample may or may not have been subject to prior treatment, such as removal of cells, filtration, dilution, concentration, detergent disruption to release antigen, centrifugation, or the like.

When the analyte in the sample should be maintained in an aqueous environment, the following procedure is found to be useful and may be treated as exemplary. An aqueous medium is formed, which is normally buffered at a pH in the range of about 4 to 9, preferably from about 5 to 9. The salt concentration will generally be in the range of about 10 mM to 1 M. Illustrative buffers include phosphate, borate, barbitron, carbonate, Tris, MOPS, MES, etc. Illustrative buffer compositions include phosphate buffered saline; 138 mM NaCl, 50 mM potassium phosphate, pH 7.2; 200 mM sodium borate, pH 8.2, etc. Use of polyvalent ions is often desirable. The concentration of the multivalent cations will depend to some degree upon the nature of the cation, generally ranging from about 0.1 to 200 mM, more usually from about 10 to 100 mM and will be included in the determination of total salt concentration. The addition of detergents is often critical to reduce non-specific binding of the reagent to be coupled to the film particularly when the reagent is a protein. The amount of the detergent will depend on the nature of the protein, generally ranging from 0.001% to 10%, more usually from about 0.01 % to 2%. Where non-specific adsorption of the binding member of the film is desirable, detergent may be left out. After submersing the polymer surface in an aqueous buffer containing from about 10-140 mM NaCl, 4-40 mM tris pH 6.5-7.5, as well as any additional appropriate coupling reagents and receptors, the reaction mixture is allowed to stand for sufficient time for completion of reaction, followed by washing.

The sample may be contacted to the polymerized layer by direct injection into a reservoir buffer covering the layer, by capillary action through a shallow flow cell covering the layer, by fluid pumping through a flow cell, by gas phase adsorption and diffusion onto a wetted surface covering the layer, or the like. For detecting extremely low concentrations of analyte, for example, less than about 10^"

¹² M, the flow cell method is preferred, since it allows a large volume of sample to pass over the film surface so as to concentrate the specific binding member on the surface. At higher concentrations, a reservoir device configuration is useful, because the diffusion rate becomes less of a factor. The binding event can be either direct or distal to the polymerized layer so long as there is associated with the binding event a shift in the absorbed or emitted light of the layer. A large number of coupling pairs may be employed, where the binding may be covalent or non-covalent. Various proteins which bind specifically to a complementary ligand may be employed, such as enzymes, lectins, toxins, soluble receptors, antibodies, and the like. Illustrative proteins include DHFR, streptavidin, avidin, cholera toxin, lectins, the c-H-ras oncogene product, and nucleases. For linkages with oligosaccharides, hydrazine may be used, by itself or bound to a polymer, e.g., poly (aery lhydrazide). Alternatively, biotin, nucleotides, or other molecular recognition analogs, or the like may be used. Nucleic acids, such as ssDNA or RNA may be employed. Maleimide linkages may be employed for linking to a thiol containing molecule, which may be biotin, avidin, any ligand or binding protein, sulfhydryl containing polymer, a nucleic acid, or molecular recognition analogs. For example, an intact antibody, with a functional oligosaccharide moiety, may be cleaved with periodic acid, and the resulting aldehyde reacted with the hydrazine under reductive conditions to form a stable carbon-nitrogen bond. For providing sulfhydryl groups to react with a maleimide, the antibody may be reduced at the hinge region, partially cleaved at the hinge region, or proteolytically cleaved near the hinge region for forming a thio ether with the activated olefin. In each case, care will be taken in selecting the method of linkage to ensure that the desired sites for binding to the complementary member of the specific binding pair are available for binding. Alternatively, sulfhydryl surfactants may be attached to sulfhydryl groups on the antibody molecules.

When a molecule binds to a complementary ligand attached to the film the binding event can induce an absorbance or emission change in the film. In addition, various agents or processes can be used to enhance the optical change in the film due to the binding event. For example, the optical properties of polydiacetelyne films or materials can be changed due to pH, temperature, mechanical stress, various solvents, ionic strength or the like.

The binding event may also be distal to the polymerized layer and still be detectable by the layer. The binding event may occur in the ambient solution of the polymerized layer and result in a change in the layer's ambient conditions, e.g. a change in pH. The light absorbed or emitted by the polymerized film has been found to shift as a response to change in acidity of the ambient conditions of the film. For example, the light absorbed by a non-fluorescent blue form of polymerized film has been found to shift to a fluorescent pink form as a response to a change in acidity. Using this finding, the analyte may bind to a substrate which enzymatically converts the analyte to a composition which increases the pH of the ambient conditions. Groups which may augment the pH include phenols, esters, carboxylates, and phosphates, but not nitrates or sulphates. This method may also be used for the detection of nucleotides, where the nucleotides have such groups attached to them. The binding event may also be distal and effect the shift in absorbed or emitted light of the polymerized lipid layer where the analyte binds to the binding molecule which then binds to an antibody associated with the polymerized layer.

The method of detection focuses on the change in absorbed or emitted light of the polymerized film which results from the presence of analyte. The spectral shift is the result of the change in absorbed light of the layer. For purposes of the method, it does not matter that the absorbed or emitted light is polarized or non¬ polarized. The color shift will normally be from blue to pink. The pink form is fluorescent while the blue form is not. Other color shifts can also be used in the method. Other possible color shifts, including red to yellow, are described in the referenced literature.

The degree of absorbance shift in the polymerized layer may be proportional to the amount of analyte which is in the sample. The action of the analyte moiety binding to the film may cause a physical, chemical or electronic change in polymer/film composition. The binding event may induce or inhibit the film's ability to undergo a transition from one absorbance spectra to another. Likewise, the binding event may induce or inhibit the emission spectrum of the film. In both cases, the lateral concentration of the binding events may be proportional to the degree of optical change that the film is allowed to undergo.

The method may be varied so as to provide for signal enhancement. One form of signal enhancement focuses on the use of a polymerized multilayer film. The use of multilayers results in enhanced shifts making analyte detection easier. Alternatively, cascade enzymes which further enhance the signal may be used.

Enzymatic processes can be used to amplify the signal change that the film can undergo. For example, a large analyte molecule may initially be bound to the film. In a sandwich assay configuration, a second antibody which contains an enzymatic label may bind to a second site on the analyte. Depending on the type of enzyme, the enzyme can be used to catalyze the formation of an abundance of product molecule which in turn react with the film and propagate a signal change in the absorbance or emission of the film. One enzyme product can be utilized by another enzyme already embedded or attached to the Aim providing for an amplification cascade. In certain polymerized layers, the binding event may elicit a cooperative response, where a small amount of analyte will trigger a shift in a large portion of the layer.

Different types of assays can be designed which fall within the scope of the method, so long as the presence of analyte is detected by a shift in the absorbed light of the polymerized layer. In the case of DNA assays, single-stranded DNA is immobilized in the film using one or two points of attachment. The attachment may be covalent or non-covalent, e.g., biotin, avidin, hybridization, etc. When the sample containing a complementary strand of DNA is added, DNA duplexing leads to signal generation. In the case of a viral assay, virus capsid or envelope may bind directly to immobilized antibody or to specific viral receptors coupled to the film. Macromolecules will be assayed in a similar fashion to the viral assay. For serology, the same principle applies, but antigen is immobilized, and antibody is measured. Alpha-galactose-l,4-beta-galactose immobilized in the polymer film can bind to receptors of P. Fimbriea bacteria.

Binding of the analyte may perturb the orderly packing leading to a structural change in the polymer layer. With polyvalent antigens cocking may be achieved, where cocking the binding of the polyvalent antigen results in bending of the film with a change in conformation of the polymer which results in the shift in absorbed light.

The following experiments are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

The following experiments demonstrate shifts in emitted light of a polymerized film in response to both changes in ambient conditions and the binding of DNA.

Experiment 1. Fabrication of a Multilayer Blue Film

A glass microscope slide, 1 in. x 3 in. in area was neutralized in .22M KOH/Methanol solution for 2 hr. The slide was rinsed with Milli-Q water and dried with pure nitrogen. The slide was then made hydrophobic by treatment with dimethyl-n-octadecylchlorosilane. The slide was placed on a hot plate which was maintained at a temperature of 110°C. The hot plate was positioned next to a cold plate, maintained at a temperature of 7°C, which was separated from the hot plate by an insulating gap that was neither hot or cold. When the slide adjusted to the temperature of the hot plate, 1/6 of the slide was moved over the cold plate. HPLC water was used as the liquid subphase. The subphase was placed on the warm side of the entire suface of the slide. The lipid monomer comprising a 2mM Ethyl Morpholin Pentacosadiynoic Amide/chloroform solution, was applied in a dropwise manner to the subphase with a micro-syringe. The chloroform acted as a spreading agent to ensure even spreading of the lipid monomer over the surface of the subphase. lOOμl of the monomer were applied, which greatly exceeded the amount needed to form a monolayer over the surface and resulted in the formation of a multilayer. Because the excess concentration of monomer, multilayers were formed even though some monomer was displaced off of the slide.

The slide was then transferred from the hot plate to the cold plate at a rate of 0.3 cm/sec. Transferring the slide to the cold plate resulted in the formation of a highly uniform crystal orientation in the multilayer. The multilayer was then polymerized by irradiation with UV light (UVP, Inc. Model UVG-54 Mineralight) for 100 sec. The fluence of the light at the film surface was 30 mjoule/cm². The distance of the film from the lamp was 2.5 in. Polymerization was carried out in an oxygen free environment, which ensured the formation of the blue multilayer film.

The resultant blue multilayer film was then transferred to a clean hydrophobic glass slide which had been made hydrophobic by treatment with dimethyl-n-octadecylcholorsilane, and maintained at room temperature for subsequent use in the detection of analyte.

Example 2. Fabrication of a Monolayer Blue Film

A glass trough (dimensions of 6 in. x 6 in. x 12 in.) was filled with 2.5 L of 0.0°C Milli-Q water. As above, a 2.0 mM Ethyl Morpholin Pentacosadiynoic Amide monomer/chloroform solution was applied to the surface of the water in a dropwise fashion by use of a micro-syringe. Care was taken to add just enough lipid monomer to form a single lens over the surface of the water. In order to form a single lens, 25 μ\ were added.

A (1 in. x 3 in.) glass microscope slide was made hydrophobic by treatment with dimethyl-n-octadecylchlorosilane. The upper surface was then placed face down onto the surfactant lipid monolayer and pushed below the surface of the water. The hydrophobic portion of the surfactant lipid monomers previously on the surface adhered to the slide. Excess lipid monomer which did not adhere to the slide was aspirated off of the water surface.

The slide was then rotated by 180° under the water so that the hydrophilic groups were facing upwards. The monolayer was kept submerged in the Milli-Q water at a distance of .5 inches from the surface of the water.

Polymerization was by irradiation with a 250 UV lamp (UVP Model UVG- 54 Mineralight) for 100 sec. at a distance of 1.5 in. from the surface of the water (2.0 in. from the monolayer surface). The fluence of light at the subphase surface was at 30 mjoule/cm².

Experiment 3. Blue to Pink Color Shift of Films in Response to Change in Ambient pH

The blue films described above were observed to undergo a color shift from blue to pink in response to changes in the ambient pH of the films. The color shift was demonstrated as follows. First, a 1 in. x 3 in. slide with a blue film, as prepared above, was placed on a white background. Following, several solutions with pH values of .8, 1.2, 2.2, and 2.8 were made using 1M H₂SO₄ and the appropriate amount of Milli-Q water. The solutions of various pH values were pipetted onto the blue film and the following data was obtained.

pH Film Color Fluorescence

(at 550nm excitation)

0.8 Blue None

1.2 Blue None

2.2 Blue None

2.8 Pink Emission at : 565 nm 610-640 nm

As demonstrated in the above table, decreasing the acidity of the ambient pH resulted in a shift in the absorbed or emitted light of the polymerized layer, so that the layer shifted colors from blue to pink and became fluorescent during the transition.

Experiment 4. Color Shift in Response to Binding of DNA to Ethyl Morpholin PDA Film

An assay for the presence of DNA in a sample was run as follows.

A. Formation of the Flow Cell

A flow cell used to detect the presence of DNA in a sample was manufactured by first placing an 8-well flow cell made of double sticky silicon rubber spacer on the surface of a 2 in. x 3 in. microscope slide. The microscope slide served as the bottom of the flow cell. Following, a 1 in. x 3 in. glass slide which has the blue polymerized film, as produced above, was placed on top of the rubber spacer. Each well in the finished flow cell had a volume of 30 μl. Since the top slide was smaller than the bottom slide, a portion of each cell was exposed so that sample could be added to each well.

B. Detection of the Oligonucleotide

The flow cell was placed into an LED fluorescence monitoring unit and the background fluorescence of the cell was measured. Following, 4 of the wells were filled with 30μl samples of 2.6 x lfr⁴ M oligonucleotide/sterile water ( 21 -men 5'-

LGG CAG-TTA-TCT-GGA-AGA-TCA-3' ). The remaining 4 wells were filled with sterile water to serve as controls.

The flow cell was incubated for 20 min. at 53°C to allow for binding of the oligonucleotides to the film to occur. Following incubation, the solution was removed from each well and the flow cell was dried using highly purified nitrogen gas.

The flow cell was then placed back into the EPI fluorescence monitoring device and the fluorescence was measured. The fluorescence was found to increase in the wells which had DNA oligonucleotides present. Since the blue film does not fluoresce but the pink film does, it was concluded that a shift in the absorbed light must have occurred in response to the binding of the DNA to the film. Control wells containing no DNA showed differentially 50% lower response compared to wells containing the DNA. It is evident from the above discussion and experiments that a simple, easy to use methods for the detection of a wide variety of analytes are provided. The methods focus on the shift in the absorbed or emitted light of a polymerized layer. The shift is a direct result of the binding of an analyte to the layer. By employing a wide variety of ligands in the layer, a wide variety of analytes can be detected using the above method. The method is not limited to the detection of a visual color shift of the layer, but can also use the appearance of fluorescence on the layer.

All publications and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is claimed is:

1. A method for detecting the presence of an analyte in a sample using a

' polymerized lipid layer and a binding molecule associated with said polymerized

5 lipid layer, said binding molecule capable of binding with said analyte, said method comprising: contacting said sample to said polymerized lipid layer for sufficient time for binding of said binding molecule with said analyte; whereby the absorbed or emitted light of said polymerized lipid layer 10 changes in response to the presence of said analyte.

2. A method according to Claim 1, wherein said polymerized lipid layer is a monolayer.

15 3. A method according to Claim 1, wherein said polymerized lipid layer is a multilayer.

4. A method according to Claim 1, wherein said polymerized lipid layer is comprised of diynyl lipid monomers.

20

5. A method according to Claim 1, wherein said binding molecule is covalently attached to said polymerized lipid layer.

6. A method according to Claim 1, wherein said binding molecule is adsorbed 25 to a support, wherein said support is supporting said polymerized lipid layer.

7. A method according to Claim 1, wherein said binding molecule and said analyte bind distal to said polymerized lipid layer, so as to affect the ambient conditions of said polymerized lipid layer.

30

8. A method according to Claim 1, wherein said shift in absorbed light results in a color shift in said polymerized layer from blue to pink.

9. A method according to Claim 1, wherein said emitted light in said polymerized lipid layer changes in intensity.

10. A method for detecting the presence of an analyte in a sample using a polymerized diynyl lipid film and a binding molecule associated with said polymerized diynyl lipid film, said binding molecule capable of binding with said analyte, said method comprising: contacting said sample to said polymerized diynyl lipid film for sufficient time for binding of said binding molecule with said analyte; whereby the absorbed or emitted light of said polymerized lipid film changes in response to presence of said analyte, resulting in a color shift of said film from blue to pink or a change in intensity of said emitted light.