MXPA98005968A - Polymeric film, sample of assay and method for the direct colorimetric detection of anali - Google Patents

Abstract

A film, a test sample and a method for the direct detection of analytes are provided using observable spectral changes in monomolecular films that occur under the selective binding of analytes to the film.

Description

POLYMERIC FILM, TEST SAMPLE AND METHOD FOR DIRECT COLOR-METRIC DETECTION OF ANALYZES BACKGROUND OF THE INVENTION Field of Invention: The present invention relates to a polymeric film that is used in a test sample and a method for direct detection of small molecules, biomolecules and detectable analytes. The method and the test sample use observable spectral changes in monomolecular polymer films, which changes occur under the selective binding of a molecule, biomolecule or analyte to the polymeric film. The polymeric film comprises a lipid bilayer with an affinity ligand specific to the analyte, which layer responds to the binding of the analyte with the ligand by changes in the absorption spectrum of the light. The change is qualitatively and quantitatively detectable.

Background and related techniques.

Analytical chemical methods for the detection of most chemical and biological molecules and / or analytes are virtually unavailable due to the destruction of the characteristics of the analyte during the preparation and analysis stages and also because of, typically, the small amount of the analyte present in the test sample.

Although usable in their own right, analytical chemical methods are of limited or impractical applicability to many biological materials in which valuation may be important. Unless expensive and cumbersome gas chromatography methods are used, large amounts of analytes are generally required to perform the detection. Frequently, quantitative results are either limited or not available.

Analysis of medical-biological systems include direct microscopic observation using several classical pathological techniques and cell staining, they also have their limitations. Well-developed microbiological techniques such as culture, colony characterization and observation of nutrient and metabolic limitations are used to enhance these techniques. Although culture techniques and direct observation of tissue have served as the bulwark of medical detection processes for many years, it also has considerable limitations. The pathological analysis of patient tissues to determine the stage of development of a disease and the identification of the causative pathogens, in general, requires an invasive procedure. On the other hand, cultivating the pathogen of various body fluids or other samples takes time and is expensive.

A sudden discovery that opened the way to a new era in medicine occurred with the development of immunoassay methods. In these methods, an antibody was developed that binds specifically to a target of interest. Although expensive in both their development and production, animal antibodies typically allow a very accurate analysis of a number of analytes that have previously been virtually inaccessible in both clinical and research situations.

An important technical advance in the immunoassay was the development of monoclonal antibodies, because the antibody itself is a small molecule, it is preferred to titrate it in some way so that the binding event can be detected. a dye, fluorescent, radioactive material or other titration Conversely, "if inhibition of binding occurs between a known quantity of titrated analyte introduced and the material to be analyzed, the decrease in the signal indicates the presence of the test analyte. If the agglutination of the antibody particles is of sufficient volume and density, the formation of a precipitate can also serve to signal the presence of an analyte.

In recent years, the research and medical communities have come to rest heavily on immunoassay techniques to detect and quantify biological materials. Although successful in many aspects, the indirect nature of the immunoassay methods as well as their dependence on antibody materials results in a variety of complications, problems and limitations of the test. The development and production of antibodies remain expensive and these molecules are sensitive to changes in the environment. Also, only those materials in which the antibodies can be produced can be detected by these systems.

Many small biological molecules are notoriously difficult to test in a direct way due to the severe limitation of environmental scales that they can tolerate without losing their specific characteristics. For these molecules it has relied heavily on immunoassays. The requirement that an antibody be developed and produced for each possible objective limits the effectiveness of immunoassay methods in those applications, such as drug design development and testing.

A disadvantage of immunoassay systems is readily apparent in rapidly developing pathogens, such as influenza viruses, where the outer coat of the pathogen that is normally available for immune reactions, constantly shifts the antibody recognition elements and, therefore, it renders them immunologically unrecognizable.

Certain types of analytical chemistry techniques were optimized by immobilizing one or more of the components of a reaction. For example, if the material to be tested is present only in a small amount in a test sample, the detectable analyte may be in such a small concentration that it is beyond the detection capabilities of any normal test system. In these cases, the immobilization systems have proven to be advantageous.

Many immobilization materials are available, such as Sephadex columns. The requirements for these materials are their specific binding properties, their relatively inert reaction to other materials so that they do not themselves interact in the test reaction or interfere in some other way with the immobilization test and its regularity in the structure. provides predictable conditions in the test situation.

Classically, the immobilization has been performed on columns, liposomes or other surfaces. The use of these materials provides many advantages for a test system. For example, these materials allow easy segregation of reagents from the other test components.

In a typical immobilization scheme, the analyte is concentrated by adhesion to the immobilization material for which it has a specific affinity. The test is then performed on a surface area limited to the immobilized surface, rather than in the diffused three-dimensional arrangement of the original sample fluid. The results are concentrated in a smaller area and are more likely to be detected.

Bilayer films placed on surfaces have been used to provide the immobilization matrices. Chemical modifications of these surfaces by organic monomolecular films have recently been used in an effort to develop new materials. Molecular self-assembly techniques, such as that described by Langmuir, 3, 932, (1987), have been used to coat surfaces with a well-defined two-dimensional molecule arrangement. These films with two layers have been useful as immobilization supports for analytical reactions. Biosensors based on these films can detect molecules of diagnostic significance such as glucose and urea, as described for example in Thin Solid Films 180: 65, (1989) and 210: 443 (1992). In these cases, the classical analytical chemical systems are immobilized on the films in order to improve the reading of the test results and otherwise simplify and improve the detection capabilities of the test procedure.

Similarly, the detection of receptor-ligand interaction is performed by indirect tests such as the enzyme-linked immunosorbent assay. Although biotechnologically functionalized films have led to molecular recognition at an interface, the problem of translating the recognition event of the molecule into a measurable signal has remained problematic until the advent of the present invention.

The detection of the immobilized reaction products is generally performed by coupling the immobilized matrix to a secondary device such as an optical fiber (Colloid Interface Sci., 124: 146 (1998)), a quartz oscillator (Thin Solid Films, 210: 471). (1992)), or electrode surfaces (Chemical Letters, 627 (1990)).

Some of the analyte enlases to the immobilized matrices are detectable by the fluorescent titre where the florescence or its temperate state indicates the occurrence of a binding event. Immobilized matrices can be made in addition to a bilipid layer wherein the detection materials are embedded in the surface of the supporting bilipid layer (Advanced Materials, 3: 532 (1991)).

It is known that polydiacetylene films change color from blue to red when the temperature increases or the pH changes due to conformational changes in the conjugated backbone (Langmuir, 8: 594 (1992)). Although a goal has been reached in the research community by exploiting this feature in the detection of link events, researchers still have to develop a method that uses this phenomenon in practical applications.

Accordingly, it is highly desirable if the direct detection method can be provided for the detection of very small chemical and biological molecules present in minimal amounts. Ideally, it is advantageous to develop a technology of monomolecular film supports in a simple and unique manner such that the binding or linkage of the event causes an easily observable change in the support material that can be directly detected.

Therefore, it is a primary objective to provide a simple and reliable test sample for the detection of minute quantities of various chemical and biological analytes using a novel polymer film for direct colorimetric detection of analytes.

THE INVENTION It is an object of the present invention to provide a polymeric film, useful in a test for detection of minute quantities of chemical or biological analytes, a method of a test sample for direct colorimetric detection of small molecules of various analytes using color changes observable in a monomolecular polymer film, which occur when the molecule binds to a ligand of the polymeric bilayer film.

It is another object of the present invention to provide a test suitable for detecting the presence of minute amounts of chemical or biological molecules, directly detecting the binding event (when the analyte specifically binds to a ligand of the polymer bilayer.

It is a further object of the present invention to provide a method and a test sample for direct detection of viruses, bacteria, parasites and other pathogens or drugs, hormones, cell wall fragments, enzymes and their interactions, as well as other biologically important materials. .

It is another object of the present invention to provide a method and a test sample for the development of drugs and to improve the activity of drugs by observing their binding or competitive inhibition of natural binding events between some or all of the surfaces of the polymeric film and the ligand. Natural bioactive of the biomolecular test.

It is still another object of the invention to detect the presence of biomolecules, using visible color changes or colorimetric detection in the lipid bilayer of the polymeric film of the invention, which color changes occur as a result of the specific binding of the biomolecules to the bilayer.

It is a further object of the present invention to provide a simple, economical and reliable test sample and a test kit for qualitative and quantitative detection of small quantities of small molecules, which test sample is stable over a wide range of conditions environmental and laboratory when the analyte is mixed with a number of other materials.

It is still another object of the present invention to provide a polymerized bilayer film for direct detection of the presence of an analyte, comprising: a) a ligand that has direct affinity for the analyte or that functions as a competitive link to the analyte; b) a linear structural linker having two terminal ends, wherein the linker is attached at its first terminal end to the ligand; c) a conjugated polymer backbone to which the structural linker is attached at its second terminal end; d) targeting groups that are bonded to the surface of the conjugated polymer backbone in positions not occupied by the structural linker; Y e) a support structure; wherein the film undergoes detectable spectral modifications by binding or binding an objective analyte to the moiety or ligand moiety.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 provides a schematic view of an embodiment of the present invention.

Figure 2 is an optical micrograph of one of the inventive polymer films (Figure 2A) and the optical micrograph illustrates the colorimetric response of the influenza virus (Figure 2B).

Figure 3 shows the structures of the compounds used in film formation tested in competitive display experiments.

Figure 4 illustrates quantification of the visible absorption spectrum before and after incubation with virus.

Figure 5 is a graph of the colorimetric response of a bilayer sialolate film to increase the concentrations of an influenza virus.

Figure 6 illustrates the utility of the invention for detection of influenza virus by detecting a colorimetric response in the presence or absence of the binding inhibitor.

DEFINITIONS As they are used here: "" Analyte "means a detectable chemical molecule, a biomolecule or a portion thereof, that is detectable by a specific binding to a ligand of the polymeric film of the invention, which results in changes in the spectral characteristics of the polymeric film.

"Ligand" means a hydrophilic lipid monomer or its derivative to which the test analyte binds at a specific detection and recognition site, which can be rendered polymeric by binding the ligand through a binding arm to a thin film of two. polymerized layers. The ligand is typically specific to either the individual lozenge or a group thereof. The ligand forms a part of the detection headings of the polymeric film. The ligand is attached to a terminal end of the molecule spacer or linker and polymerized mixed with a hydrophobic matrix lipid monomer. The ligand can be monovalent or multivalent.

"Linker" or "spacer" means a molecule with a linear structure linked through a terminal end to the ligand and through the second terminal end to the base film. Specifically, the binder is attached to one of several monomers that have been polymerized in a chromatic detection element. A structural linker has a sufficient length and conformability to allow the attachment of multiple sites on the analytes. The binder can be, for example, tetraethylene glycol.

"Conjugated polymer", "polymer backbone", "conjugated polymer backbone" means a layer of a polymerized ligand and a two-layer monomer matrix assembly, capable of signaling the bond occurring at the surface of the film by a chromatic transition. The polymer skeleton can be, for example, polydiacetylene. The polymer skeleton is attached to a structural linker.

"Polymer bilayer" is made of polymerizable ligands and matrix lipid monomers, polymerized into a chromatic detection element. The binding of the analytes to the ligand bound to the polymer backbone, induces efforts within the bilayer, by changing the effective conjugation length of the polymer backbone. The lipid matrix monomers suitable for polymerization are lipid monomers. These remains include: acetylenes, diacetylenes, alkenes, thiophenes, imides, acrylamides, methacrylates, vinyl ether, malic anhydrides, urethanes, allylamines, siloxanes, or vinylpyridine, etc. Lipids containing these groups can be made into homopolymers or mixed polymers. The preferred group for use in this invention is diacetylene, because of its unique optical properties in the form of polymerized polydiacetylene. However, other polymerization groups may be used when they provide an observable change in properties under a binding or binding event.

"Lipid ligand monomers" are carbohydrates, amino acids and such other molecules having carboxylic groups.

"Lipid monomers" are easily polymerized into polymeric films by ultraviolet irradiation or other means for polymer backbone formation. The most preferred monomers are diacetylene monomers such as polydiacetylene and octadecyltrichlorosilane.

"Lipid targeting leader groups" means forming lipids, which stabilize and structurally orient the polymer bilayer to facilitate color stability until the binding of the analyte is disrupted.

"Lip or tails of lipids" means a remainder or half that serves to anchor the polymerized film to the surface of the support.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a test sample and to a method for the direct colirimetric detection of small molecules, biomolecules and analytes that bind in an interaction similar to a receptor-ligand using a novel polymeric thin film construction. The invention further relates to the polymer thin film, as well as to the method for producing the polymer films for specific detection of various analytes through their binding to a ligand wherein the ligand or its derivatives are rendered polymeric by polymeric ligand binding to through a link arm link to a thin polymerized two-layer film.

The presence of the analyte investigated detected by binding of the analyte to the ligand, is observed through changes in the spectral characteristics of the polymeric film. The polymer-ligand assembly encompasses specific molecular recognition and detection sites, all contained within a single molecular assembly.

The present invention allows for the first time, the direct detection of small chemical and biological molecules, such as pathogens and drugs, using observable spectral changes in monomolecular films. Accordingly, the present invention represents a completely new embodiment with respect to the direct detection of small molecules, biomolecules and analytes using color changes in a monomolecular film whose changes occur when these materials specifically bind to the target molecule.

The object of the invention represents a dramatic advance on both chemical and immuno assay systems, since it is capable of both quantitative and qualitative detection of small chemical molecules, biomolecules and / or identifiable analytes, which are otherwise undetectable in a simple manner , fast and practical. The present invention combines the advantages of both immunoassay and chemical analysis in a simple system.

Additionally, the test sample of the invention is useful for environmental tests by detecting several analytes in their most advantageous environmental conditions by the rigor allowed, direct analysis occurring at very narrow environmental scales. The speed and simplicity of the color change indicator of the present invention are its most marked advantages.

In general, the present invention does not require a preanalysis purification step. The results are easily readable by an untrained observer and the test can "be conducted under ambient conditions." Very mild test conditions allow the detection of small biomolecules in a closely natural state, providing information about their interactions and avoiding the risk of modification. or degradation of the analyte This feature of the present invention is due to the high specification of the ligands incorporated in the detection film Additionally, the inventive direct testing system avoids the costly, complicated and increased inaccuracies inherent in the indirect systems currently available.

I. THE POLYMERIZED BICYCLE FILM.

The polymeric film according to the invention is a multilayer assembly that allows the direct detection of the presence of a wide scale of analytes by changes in color spectral criteria.

A. PREPARATION OF THE POLYMER FILM The polymeric detection films of the invention are simple and easy to prepare.

Briefly, monomeric lipids having hydrophilic groups, such as carbohydrates, amino acids, etc., which groups are easy to bind to biological materials, are used as the ligands for detection of these materials. The monomeric matrix lipids, which have hydrophobic groups, are used to assemble and orient the film. Both lipids are mixed in a proportion of about 5-40% of ligand-matrix lipids in an organic solvent, such as chloroform, benzene, hexane, xylene and others, or in a mixture of the organic solvent with an aqueous solvent, such as a chloroform-methanol sample, preferably in a ratio of about 9: 1. The solution of ligand-matrix lipids in the solvent are added slowly, typically in small droplets, on the surface of the water, where they orient themselves by linking the hydrophilic groups to the water and projecting the hydrophobic groups out of the water. During that step, the organic solvent either evaporates or is removed leaving behind the lipids like a film of fluid placed on the surface of the water. For better film formation and performance, lipids are applied, either in high concentration over a small area of water or, typically, compressed into sandwiches by placing barriers on both sides of the lipid film. Next, the lipid monomers are polymerized by, for example, exposure to ultraviolet light, thereby forming a flotation polymer that is, on the surface, visibly blue. Then this polymer is mounted on or fastened to the separately prepared support structure, such as, for example, glass, microscopic glass, celluloid, etc. or any other firm support structure that can be covered with a hydrophobic anchoring surfactant such as octadecylsiloxane or hexamethyldisiloxane and others, wherein the siloxane group is the one that provides anchoring. The final assembly is blue.

The diacetylenic lipid monomers of the invention such as compound 1 (FIG. 1) are easily polymerized in monolayers by ultraviolet radiation to form a conjugated polydiacetylene skeleton of alternating eneine groups using common techniques (Colloid Polymer Science, 225: 36). (1977), and J. of Polymer Science, Letters to the Editor, 16: 205 (1978)). In one embodiment of the invention, a thermochromatic polydiacetylene bilayer is assembled on a support and then used for the detection process. The polydiacetylene layer is functionalized with a specific receptor ligand for the target molecule to be detected. Both quantitative and qualitative findings regarding the presence of the target material can be obtained using various embodiments of the present invention.

In one embodiment of the present invention, the bilayer is composed of a self-assembled monolayer of octadecyltrichloroxylan and a monolayer of Langzymer-Blodgett of polydiacetylene (Langmuir-Blodgett Films, Roberts., Ed., Wiley New York (1996)). The polydiacetylene layer in this case is functionalized with a sialic acid analogue. Sialic acid is the receptor-specific ligand for influenza hemagglutinin, as well as for other pathogens. The sialic acid ligand serves as a molecular recognition element.

This colorimetric technology is coupled to materials whose chemical properties can be glued together to join a variety of small organic molecules. Many organic hosts form inclusion complexes with protic and dipolar aprotic compounds. Certain inclusion compounds, or clathrates, such as compounds one and two observed in reaction scheme 1, have been shown to be highly selective sorbents for vapors from organic solvents (Angew, Chem Int. Ed. Engl., 32: 110 (1993)). For example, compound 1 has a pronounced affinity for the diosan and little affinity for butanol, acetone, methanol, 2-propanol, cyclohexane, toluene and water. The lack of affinity to cyclohexane is particularly remarkable given the similarity in chemical structure. On the other hand, compound 2 shows a pronounced affinity for 1-butanol on the same group of solvents. This interruption, combined with colorimetric detection leads to a new class of chemically sensitive materials immobilized on surfaces. The surfaces that both the clamping element and the detection element have are constructed in a simple supra-olecular assembly, and represent a novelty of the method for direct detection of a wide variety of environmental contaminants.

The clathrate-forming compounds coupled to the polydiacetylene polymer form a new class of materials that are chemically sensitive, robust and have unique optical properties. These materials offer a novel but still simple method to detect the presence of organic solvents by monitoring the color changes that occur in the film to the binding of offending compounds. No technical expertise is required to use that detector and is therefore suitable for on-site analysis, by people with little or no technical experience. The molecular level hints at why the clathrate-forming compounds of a given complex structure with a given test molecule leads to a wide variety of thin polymeric clathrate-forming films. All these things are within the scope of the invention.

B. THE COMPOSITION OF THE POLYMERIC FILM.

The inventive test sample film is composed of the base polymeric two-layer film, the surface of which contains both targeting and detection header groups. The detection header groups are composed of a ligand specific to the analyte in question which is attached to a terminal end of a linear structural linker. This linker, in turn, is attached to the base film by its second terminal end. The base film surface is also provided with lipid targeting header groups.

A schematic illustration of an embodiment of the present invention is shown in Figure 1.

Ligand (1) of receptor-binding lipid that is hydrophilic in nature is shown bound through the terminal end of its linker molecule (3) to the second lipid matrix monomer of the polymeric film which is hydrophobic in nature. Both the matrix lipid and the ligand are polymerized in a chromatic detection element (5). The chromatic detection element (5) is, through its hydrophobic side connected to a monolayer support layer (7) which is also hydrophobic and in turn is attached to a support structure, such as a microscopic slide (9). Alternatively, any surface that will house the hydrophobic sensing element (5) can be replaced by the elements (7) and (9). For example, a plastic surface may serve instead.

The polymeric film comprises a lipid ligand monomer, optionally a compound used as a linker or spacer and a matrix lipid, both polymerized in the detection polymer film having detection and orientation heading groups and this film is mounted on the system of support . 1. LIGANDO GROUP A ligand or its derivative is a hydrophilic lipid monomer to which the tested analyte is bound at a specific recognition and detection site, which can be rendered polymeric by binding the ligand through a linking arm of the linker or spacer at a thin polymerized two-layer film. Typically the ligand is specific, either to the individual analyte or to one of its groups. The ligand forms a part of the detection headers of the polymeric film and can be monovalent or multivalent.

The ligand group of the present invention, seen in Figure 1 (1), is selected from a wide variety of materials, the main criteria for that selection being that the ligand has an affinity and a specific selection for the analyte that is selected. The ligand can be targeted to a single title or to a wide variety of materials, such as when a class of materials is to be tested. Suitable ligands include peptides, carbohydrates, nucleic acids or any organic molecule that binds to the receptors. For example, all influenza strains use the same binding sites when they bind to a host receptor molecule. Therefore, the host receptor molecule is advantageously employed to screen all strains of influenza, including those that have not been characterized in any way.

Ligands are also advantageously used in the present invention when they function as competitive bonds to the analyte, for example, when a pathogen is introduced with a test material to be tested for the presence of a receptor molecule. In the absence of the receptor molecule, the pathogen binds to the bilayer of the test sample and produces a color. To the extent to which the surface of the pathogen binds to the receptor molecule introduced into the test material the binding is decreased. In this way, a receptor molecule is detected and quantified. 2. LINKER A linker or spacer can be a molecule with a separate linear structure linked through a terminal end to the ligand, and through the second terminal end to the base film or a part of the ligand or matrix. Specifically, the linker is attached to one or more monomers that have been polymerized in a chromatic detection element. A structural linker has a sufficient length and conformability to allow binding of multiple sites on the analytes The linker can be, for example, tetraethylene glycol. 3. POLYMER SKELETON.

The polymer skeleton is a layer of an assembly of two polymerized layers capable of signaling the bond that occurs on the surface of the film through a chromatic transition, visible as a change of color from blue to red. The polymer skeleton can be, for example, polydiacetylene. The polymer skeleton is bonded to the polymer bilayers.

The bilayer of the polymer is made with polymerizable monomers, preferably ligand / matrix lipids polymerized in a chromatic detection element. The binding of the analyte to the ligand bound to the polymer backbone induces stresses within the bilayer by changing the effective conjugation length of the polymer backbone.

The monomers suitable for polymerization are lipid monomers. Monomers are lipid matrix halves or moieties, such as acetylenes, diacetylenes, alkenes, thiophenes, imides, acrylamides, methacrylates, vinylether, malic anhydrides, urethanes, allylamines, siloxanes or vinylpyridinium, etc .. Lipids containing these groups They can prepare homopolymers or mixed polymers. The preferred group for use in this invention is the diacetylene group because of its unique optical properties in the polydiacetylene, its polymerized form. Most preferred monomers matrix are diacetylene monomers such as polydiacetylene and octadecyl trichloro silane, however, they can be used other polymerization groups, where these provide an observable change in properties at an event occurrence binding. The second components of the bilayer are the ligands described above.

The lipid monomers are easily polymerized to monolayers by ultraviolet radiation or by other means. 4. DETECTION OF LIPIDS AND ORIENTATION GROUPS.

The lipids are important to form and structurally orient the bilayer of the film so that the binding of the analyte results in a detectable color change. The detection is based on a structuring effect of the targeting groups that properly stabilize the physical structure of the bilayer to facilitate color stability until the binding of the analyte to the molecular recognition ligand groups occurs. The joint causes sufficient spherical disturbances or stress of the structure to result in a color change. The relative stability and rigidity engendered by the orientation lipids bind the bilayer, so that a spherical change in an area triggers a larger effect on the surface as a whole that can be easily observed.

The observed spectral changes are due to joint-induced stresses that change the effective conjugation length "of the polymer backbone. group suitable as ligand detection in the present invention are hydrophilic materials which include -CH2OH lipids, -CH2OCONHPh, -CH2OCONHEt, CH2CH (Et) OCONHPh, - (CH2) 90H, -CH2OCOPh, -CH2OCONHMe, -CH2OTs, -CH ( OH) Me; -CH2OCOR2, where R2 is n-CsHu, n-C7H15, n-C9H19 n-CnH23, n-C13H27, n-C15H3? , n-C? 7H35, Ph, PhO, or o- (H02C) C6H4, -OSO2R2, wherein R2 is Ph, p-MeC6H4, p-FC6H, p-C1C6H4, p-BrC6H4, p-MeOC6H4, m-CF3C6H4, 2-C10H7 or Me; and -C02M, where M is K, HNA or Ba / 2.

Preferred materials that can be employed as header groups in the present invention are: -CH2OCONHR2, or -CH2CONRH2, wherein R2 is Et, n-Bu, n-C6H13, n-C0H17, nC? 2H25, cyclo-C6Ha ?, Ph, p-MeC6H4, m-MeC6H4, o-ClC6H4, m- ClC6H4, p-Cl C6H4, o-MeOC6H4, 3-thienyl, Me, Et, Ph, I-C10H7 Et, Ph, EtOCOCH2, Bu0C0CH2, Me, Et, i-Pr, n-C6Hi3, EtOCOCH2, BuOCOCH2, Ph, or 2, 4 (N02) 2C6H3OCH2, CH2CH2OH.

The most preferred detection groups are selected from CH2COX, wherein X is OH, MeO or any of its salts.

Materials suitable for use as matrix orientation groups are hydrophobic lipids used to assemble and orient the film. The groups comprising the tails of the lipids are of a wide variety. The compounds which serve to anchor the polymerized film to the support surface can be those residues selected from the following: CH3-, CH30-, neo-C5HpO-, cyclo-C6HuO-, PhCH20-, p-AcC6H40-, p- BzC6H40-, p-Br C6H4C0CH20-, p- (PhCH = CHC0) C6H40-, p- (PhC0CH = CH) C6H40-, o-BZC6H4NH-, p-BZ C6H4NH-, MeOCH2CH2NH-, n-C6H13NH-, EtO- . The preferred group in this invention is the methyl group.

. MOVIE SUPPORT The support structure (9) to which the film is attached may be of a variety of materials. The material used in certain embodiments of the invention, such as in Example 1, is a microscope glass slide made hydrophobic by treatment with an appropriate surfactant such as octadecyltrichlorosilane. Any material that is a bit hydrophobic such as plastic, mica, metal, ceramic or other relatively uniform polymer surface can be used. Glass is the preferred transfer medium in this invention due to its transparency that facilitates readings in color changes. However, non-transparent materials can also be used, using a reflectance type measurement.

B. RECEIVER-UNION MOLECULES USED AS LIGANDS.

Binding receptor molecules are materials on the surface of a host cell to which a pathogen binds itself as a prelude to the infective event. The selection of those molecules as the ligand group of the present invention provides a specific recognition site for these pathogens, when these molecules tend to be highly and genetically conserved in the pathogen that has obvious criticality to pathogen survival. Different strains of the same pathogen in general will not produce a false negative result when molecules such as those of the ligand group in the present invention are selected. Receiving molecules tend to be smaller and less complex and often less hydrophobic.

Binding receptor molecules such as those described above are detectable by the present invention as a numerical increase in receptor molecules that have been recognized, identified, isolated and synthesized for a large number of pathogens.

Many of these receivers have been improved for use in various analytical and treatment systems.

A good example of the utility of the invention is the sialic acid derivative used for detection of influenza or malaria. Examples of the receptors for a certain number of pathogens have been provided in the application as table 1. All of these as well as any other ligand that falls within the scope of the invention are within the scope of the present invention.

Example 1 describes an exemplary use of sialic acid derivatives as a preferred embodiment for the use of binding receptor molecules.

II.CONDITIONS OF PROOF OF THE SAMPLE.

The test of the invention is carried out under mild test conditions, sensitive to the tested analytes. The polymeric thin film maker of the invention employs ligands and analytes that are stable or have suitable binding characteristics specifically within a range of narrow environmental or in vitro limited conditions.

The present invention meets the stringent limitations even within this narrow range of such conditions. This allows, for example, to maintain three-dimensional formations of sensitive biomolecules and biochemicals throughout the test procedure.

The test is' extremely simple because the analyte or molecule tested is put in contact with the polymeric film of the invention and the color change and its intensity are observed and measured to be quantified. Typically, this process takes only about thirty minutes.

The present invention works well even under extremely limited conditions. The conditions of the test, such as pH, salinity and temperature can be carefully controlled by retro-feeding, titration and other techniques without interfering with the accuracy or sensitivity of the analysis.

Because of this wide-scale experimental advantage of the present invention, intact cells or sensitive subcellular inclusions are tested, without disturbing their structural integrity. Very acute stages of cell development can be monitored, such as the various stages of malaria infection. Additionally, the association between several factors can be tested or monitored, even during the interaction process using the method of the present invention.

The film and method of the invention are suitable for testing very small biological molecules or other molecules for which antibodies can not be developed. These target materials include organic solvents or contaminants present at extremely low levels. There are special opportunities available for the advances made by the present inventors to examine drugs in both forensic and clinical applications. The measurement techniques applied in the present invention allow testing materials that are of a minute size or that have a small number or a single rating.

The unexpected spectral signal that is achieved with the present invention is due to a physical perturbation of the bilayer that occurs as a result of the binding event of, for example, ultivalent materials, such as viruses and cell membrane fragments, which perturbation is then detected. using the present inventive method. Therefore, multivalent materials generally elicit a particularly strong response in the test system because of the conformational changes introduced in the bilipid layer as a result of the binding, causing the physical reconfiguration of the structure.

The pre-bonding of smaller univalent analyte materials to a carrier can also prove advantages in increasing the efficiency of the present invention. For example, the analyte can be attached to a polymer or to the surface of a liposome. This will concentrate the binding event on the inventive bilipid surface to specific points, increasing the spectral modification at each point of contact. Additionally, the curved surface of the liposome to which the analyte binds will serve to entrain the peripherally bound analytes in the opposite direction from the bilipid surface and force the centrally located analytes onto the liposome on the bilipid surface. This pre-joining step then results in increased twisting, disturbance and signal generation on the two-layer surface.

The test of the invention is suitable for detecting weakly bound analytes, as well as for multivalent analytes. The multivalent property of the linked polymer ligands of the present invention provides an improved binding capacity in the case of naturally multivalent analytes. Multivalency can also be provided for analytes of limited valence prior to the test procedure to infuse them with this advantage of the present invention. The inventive exploitation of multivalence allows a specific interaction although weak to be amplified many times.

A structural linker of sufficient length and conformability aids in the attachment of multiple sites on the analyte, even when they are conformationally separated on a curved surface. As a result of these special features, the present invention can detect many ligands previously unsuitable for test evaluation.

The main criteria for effective indication of the presence of an analyte is that the surface of the indicative bilayer is sufficiently disturbed to produce the required spectral change. The binding of the analyte to an immobilization particle serves this purpose, since it concentrates the analyte in a small area and also provides a three-dimensional appearance over a relatively large area for even a small analyte.

A large variety of ligands can be employed in the present invention, allowing great flexibility in the detection of a multivalent test target. The selection of ligands is based on the most advantageous spherical and binding characteristics, rather than on the accommodation of the test system. Therefore, the most advantageous ligands are selected on the basis of factors such as hydrophobicity and hydrophilicity, dimension, position of the binding site and conflicting affinities compared to the analyte to be detected. Ligands that are advantageously employed in the present invention ide carbohydrates, peptides, nucleotides, heterocyclic compounds and other organic molecules.

The structure and morphology of the film assembling the polymerized bilayers of the invention are shown in Figure 1 as a schematic diagram of the polymerized bilayer assembly. The siloxane bonds of the lower monolayer are not illustrated. Figure 2 (A) shows an optical micrograph of the bilayer assembly sialolated between intertwined polarizers. Large domains up to 150 μM are visible. Scale: lcm = 20μM.

Initial investigations have focused on the binding of influenza virus to sialic acid as a model system for colorimetric detection. The study is reported in example 1. A lipid monomer containing a sialic acid header group bonded to a carbon, provides a molecular recognition site for the viral lectin, hemagglutinin. Figure 3 shows the lipid (11) of matrix, the lipid (13) of the sialolate and the lipid (15) of lactose used in formatting a LB film and a-NeuAc (17), β-O-NeuAc (19) compounds and a-glucose (21) used for competitive expedition experiments, as described in Example 4. The synthesis of compound (13) is reported in J. Of The American Chemistry Society, 115: 1146 (1993). A carbon glycoside was used in place of the naturally occurring oxygen glycoside to prevent hydrolysis by neuraminidase, which is also present on the surface of the virus.

III.- OBJECTIVE MATERIALS One of the advantages of the present invention is its utility for detection of a wide scale of target materials, binding or binding events and biochemical reactions susceptible to analysis using the present inventive techniques. Many of these materials previously could not be detected using the available tests. The present invention utilizes specific binding without the complications of immunoglobulin generation.

The rigor and advantages presented by the inventive test system allows the detection and quantitative evaluation of materials that have previously been unattainable because of the limitations of the methods of the prior art. The present invention has already been tested in a unique test method for accurate detection of malaria parasitic infection, as described in example 9. The development of an effective test for malaria in transitory stages has so far proven an intractable challenge for either the immunological test or analytical chemical technique methods.

IV. QUALITATIVE AND QUANTITATIVE EVALUATION.

In the present invention and in the test, several spectral changes of the bilayer are used to detect the presence or absence of target material. The skeleton of the conjugated polymer of the polymerized bilayer assembly points to a bond on the surface of the film by means of a chromatic transition. The color or other spectral transition is easily observable to the naked eye as a color change from blue to red and can be quantified by visible absorption spectroscopy.

The film was designed to undergo color transition from blue to red, only due to the receptor-ligand interactions that occur on the surface of the bilayer. The bilayer assembly incorporated both a molecular recognition site and a detection element. This simple color-based detector allows rapid quantitative detection of binding by visual inspection of the film or quantitative detection by visible absorption spectroscopy.

As shown above, the thin films of chemically functionalized polydiacetylenes of the present invention act as simple colorimetric bio-perceivers. These films are derived with a carbohydrate-based ligand that specifically binds bio-organisms such as viruses. The conjugated polymer film is initially blue. The binding of a virus or other analyte of the derived polymer causes a change in the color of the film, from blue to red. The intensity of the red color that results corresponds approximately to the amount of virus. The means for amplifying the spectral signal for quantification are well known in the art, such as scintillators, which can be advantageously employed when low levels of analyte are present. Because of the empirical nature of the signal, there are many opportunities to automate the reading of the present inventive test system.

In one embodiment of the present invention, a blue color shift is observed by test technicalities. Because of the simplicity of the reaction, that observation can easily be done by an untrained person, by doing the proper test, for example, for a domestic test. Alternatively, spectral testing equipment, known in the art, can be used to determine a change in spectral color shift beyond the limits of simple visual observation, including optical density at a wavelength, of illumination light particular.

Spectral changes outside of human vision can be effectively employed in the present invention, by the use of various spectral analyzers, such as light meters or through technical observation of the surface using various translation devices, such as infrared detectors. and ultraviolet.

Spectral signal amplification means for quantification are well known in the art, such as scintillators, which can be advantageously employed when low levels of analyte are present. Because of the empirical nature of the signal, there are many opportunities to automate the reading of the test system of the present invention.

For a more accurate quantitative measurement, the film is scanned with a visible absorption spectrometer where the relative change in intensities at 620 nm (blue) and 550 nm (red) is easily assessed as shown in Figure 4 (Table 2 ). The degree of color response is directly proportional to the concentration of the analyte. The present example moves this technology from the biodiagnostic to the reality of environmental diagnosis, exploring a new class of ligands for which there is a precedent for the union of small organic molecules. These ligands are similarly treated to polydiacetylene skeleton, which provides colorimetric detection.

U T I L I D A The present invention has wide applications for the detection of a very wide variety of analytes. These include small biomolecules, junction observation and other chemical events detecting traces of quantities of many materials.

Due to the very broad applicability, important classes of analytes are detected by the present invention, which have previously shown difficulty or impossibility of detection by prior art methods. They can detect many viruses, bacteria and proteins related to them, or infections caused by them. These include pathogens such as influenza, HIV and malaria, among others. The direct colorimetric detection by the inventive polymer films offers new possibilities of application for diagnosis and examination for new candidates of chemical products or binding ligands.

The present invention is useful for the development and examination of designer's chemical products. Normally, for tests of competitive inhibition of drug receptor molecules, radio-titrated materials are typically used. However, this process takes time and requires access and handling of radio-labeled materials. Other techniques, such as fluorescent tempering, are limited because each test is self-contained and, therefore, a great effort for examination is prohibitive for the time it takes and for the costly.

There are many advantages with respect to the genetically conserved host recognition site, which is the objective by the embodiment of the present invention. For example, a determination of a patient's exposure to the flu flow will be definitive and not limited to a particular strain, if the binding of the receptor ligand of the influenza pathogen is detected. This advantage of the present invention also avoids the need for a large number of immunological tests, since, the clinical analysis, can rest on a simple analysis. Additionally, even strains of an uncharacterized flu flow recently involved can be identified, also avoiding false negative tests.

An analogous limitation of immunoprotests occurs in well-established pathogens, such as malaria parasites. In these organisms, phases of the life cycle that would allow an immune response have to be extraordinarily limited, so that the immune response is eliminated.

The present invention exploits the binding site of the genetically conservative host to identify the pathogen. Even in comparatively large parasites, the host binding site tends to remain constant at all times throughout generations of pathogens. Additionally, parasites are usually present in the body in a large number of diverse life stages. In well-established parasites, unattainable sites often vary considerably from stage to stage, the advantage being that the host organism is unable to mount an immune response quickly enough to avoid the invasion of the parasites.

In this particular application of the present invention, several iterations of a product of a drug can be examined rapidly, for interference with infective binding by a pathogen. Table 1 provides a number of examples of the host receptor molecules that provide the pathogen binding site required for infectivity. All of these examples, along with many others, can be exploited with the present invention for drug development and optimization. Multiple exploits that can be made on a single, two-layered sheet, allow many very fine iterations of a candidate drug to be "tested," such as several levels of pH titration.Using this invention for drug testing, the common effect of cooling on expensive, individual test drug research for each sample.

The availability of large volume of economic tests will dramatically increase the speed of drug development. For this purpose, a naturally occurring transmembrane receptor (RTM) is reconstituted in a lipid bilayer wherein the lipid layer is constructed from the polymerizable monomers. This is particularly applicable to the inventive compounds that have the two triple bonds in the chain. Once the receptor is incorporated into the lipid, the lipid is irradiated to ensure the RTM is in place. The binding of small molecules to the binding site in the RTM produces a conformational change in the RTM that affects the lipids and causes a color change.

A wide variety of RTMs have been isolated. RTMs that include hormones, neurotransmitters and other receptors of physiological regulation, are particularly useful in the improvement and development of drugs using the present invention. The use of RTMs that occurs naturally in the present invention has particular applications for drug screening. The present invention has an immediate relevance in the development of new drugs, on which biological effects depend on the binding to membrane binding receptors. For example, the dopamine receptor binds to the dopamine of the natural compound. In order to employ the present invention to investigate new compounds that behave like dopamine, that is, they bind to the dopamine receptor, the dopamine receptor, used as a ligand, is exposed to the new drugs similar to dopamine. proven The performance of the drug test is indicated in Example 6 as a practical embodiment of the polymer films of the invention and their applications.

Due to the ease of the examination, which is available using the present invention, many small changes can be made in the structure of the candidate drug and analyzed immediately, they provide great speed and flexibility in the development and optimization of the drug. By looking at the area of modification that provides the greatest changes in effectiveness, the critical structures of the drug can be quickly identified. This allows a critical approach to the scope of modification of the drug, which will greatly increase the speed of development of the drug.

The basic investigation of drug interactions, optimization and new development of the drug, is also practically done with the present invention. There are drugs that can be analyzed to determine which structures are of greater importance in their therapeutic effects. These structures can be optimized and even transported to a non-active, smaller, biologically acceptable or less expensive structure. These qualities and the ability to cross the blood-brain barrier, can be conferred.

If two different drugs are available for the treatment of a disease, their structures can be analyzed to actively use the technology of the present invention. Then, their activity sites can be incorporated into a single drug. Additionally, concomitant structures that optimize activity can be appropriately placed on the new hybrid drug. Any interference in activity, can be determined and improved or eliminated before tests in animals and humans costly and slow.

Another important application of the present invention and method is the economic and exact test of infectious states and other medical conditions. For example, the levels of antibodies of a specific pathogen can be monitored easily and economically by means of the competitive inhibition of a fixed amount of pathogenic material placed in the analytical solution. Additionally, certain antibodies can be detected through their direct and specific binding to the inventive membrane.

A wide variety of biologically related materials is advantageously amenable to both quantitative and qualitative analysis, using the present invention.

The invention by several pathogens can be detected remotely, before clinical manifestations are observed. This is of a particularly critical advantage in patients with low immunity, such as in newborns, patients treated with organo-organizer chemotherapy and AIDS victims.

In pregnancy tests, the human chorionic development hormone is tested using the present invention. An elevation in luteinizing hormone announces the principle of ovulation both for confirmation of pregnancy and in use. in methods of natural birth control.

Because of the simplicity of reading, the present invention is very suitable for domestic use. For example, it allows multiple tests at low cost, necessary in natural birth control methods or for fertility tests to optimize the chances of achieving a pregnancy.

The economical multiple test capability of the present invention makes it possible through multiple cavities provided on a single two-layer sheet to provide an excellent incentive for pregnancy detection, extremely early. Pregnancy detection before the lack of A period is important, because it allows you to avoid exposure to harmful or harmful factors, which are critical to the final result, when you start the first days of pregnancy. It is also important when a pregnant woman may have been exposed to a disease that will have later or no clinical manifestation for the mother, although it can severely damage the development of the fetus she already carries. These diseases can include rubella, toxoplasmosis and other pathogens. The present invention allows a simple and economic observation of these conditions Another important application of the present invention is the monitoring of patients with chronic conditions, such as diabetes. For example, blood insulin levels can now be monitored regularly at home using the present invention. This allows diabetics to adjust their insulin delivery to make it more accurate by following insulin requirements. It also allows diabetics to quickly differentiate the early symptoms of a transient condition such as the flu flow from undue variations in insulin levels.

The present invention also allows the production of a simple domestic test of cholesterol levels, which allows patients to determine their cholesterol levels in the private home, encouraging the most reluctant to test their cholesterol level and that be warned of this information that is often critical. For patients with known hypercholesterolemia, the present invention provides an ideal means to intimately monitor the palliative effects that are achieved with the treatment. The multiple concavity test equipment that has been made possible and practical by the present invention is particularly useful for weekly or even daily monitoring of these levels.

The monitoring of drug levels is a fertile area of application of the present invention. Patients typically exhibit a wide range of metabolic levels and liver activity. This is particularly the case for those who are hospitalized. Because the levels of the drug in the blood can not be easily determined, the clinician is often forced to medicate with a smaller amount a patient who could benefit from higher levels of administration. Unfortunately, the doctor must go to the side of caution in order to avoid the possibility of reaching toxic levels. The present invention allows a more accurate titration of drug administration, which leads to better pain relief and other benefits of the drug.

The present invention has important application in the abuse of drugs. When a patient suffers from a possible overdose, the actual levels in the blood of the drug and also its identity, can be analyzed very quickly by the doctor making the treatment using the present invention. This information prevents potentially harmful treatment for overdoses by drugs that exhibit the same symptoms, such as those of the actual overdose substance. Additionally, less draconian detoxification measures can be taken if lower levels of drug are detected than those that were suspected, using the present invention. Conversely, toxic levels can be detected, even when the patient does not present symptoms that alert the clinician to the level of real danger.

The present invention is also used in a wide variety of industrial applications. For example, industrial enzymes can be monitored as well as their binding strength and presence in a medium. Its loss can be monitored in an effluent and its proper dispersion can be monitored in food materials and media in general.

The invention is very useful for determining the optimal conditions of enzymatic activity of any particular substrate. Additionally, the enzyme can be easily designed to be optimal, including adjustment for specific uses or work environments. This is done in an analogous way to design the evaluation of the drug, as explained elsewhere. Therefore, it can be developed for industrial enzymes and other active materials, tolerance for environments with extreme pH, concentrated, cold and hot feed materials, additional interference materials and other desirable tolerances.

The ability of the present inventive films to detect small molecules using RTMs as described in the previous drug development section also has excellent use in environmental and industrial applications. It should be noted that among the RTMs that are used for this purpose are the olfactory RTMs. These can bind small odorous molecules and have important applications as an environmental detector, among others.

The need for chemical detectors to measure analyte concentrations for industrial process control applications, for warning and safety systems, in environmental analysis, etc., is great. Classical clinical analyzes, such as mass spectrometry and gas chromatography, do not lead to on-site field analysis due to analytical time, high cost and impractical equipment for use in the field and because of the need for technically experienced personnel. The detector that would be useful in field work analysis, therefore requires material that is chemically sensitive and that can specifically bind the analyte in question and a simple method that is carried out without difficulty, "to detect when the union has occurred. of the analyte Monitoring can be developed in the place of public water supplies, (for example, swimming pools, drinking water, waste water streams, etc.) for contaminants.

EXAMPLE 1 Preparation of polymer film for the detection of influenza virus.

This example illustrates the process used for the preparation of a polymer film suitable for the detection of influenza virus.

A two-layer polymerized assembly, shown in Figure 1, was prepared, comprised of a self-assembled monolayer of octadecyl trichlorosilane (OTS) and a monolayer of functionalized polydiacetylene.

The films were prepared by a technique Modified LB in which, the carbohydrate ligand detection group is presented on the surface of the bilayer. Mixtures of 2% 5% glycolipid monomer (13) seen in Figure 3 and matrix lipid monomer (11) were sprayed onto the water surface of a common LB tundish.

The matrix lipid uniformly dispersed the lipid to the sialolate, which allowed the optimal union of the virus. 1% to 5% of the lipid of the sial side, gave a maximum union of the virus to polymerized liposomes. The ideal mixture of the two components was determined by analyzing the Langmuir isotherms. Several ratios of the monomers (11) and (13) gave isotherms whose boundary areas and collapse pressures change in direct proportion to the mole fraction of (2) as expected by miscibility. The mixed monolayer was compressed and polymerized on the surface of the water.

The floating polymerized film was raised by horizontal touch method on a slide previously covered with an OTS monolayer to another assembly. The resulting bilayer assembly presents an arrangement of carbohydrate ligands on the surface. The spacer of tetraethylene glycol in the lipid (13). Sialolated serves to extend the carbohydrate ligand beyond the parent lipid carboxylic acid (11) header groups.

Films prepared in this way, exhibited a high degree of order at a macroscopic range (50 to 150 microns) as evidenced by optical microscopy with the use of intertwined polarizers as shown in figure 2. The films were also characterized by ellipsometry and X-ray photoelectron spectroscopy (XPS) resolved by angle. The resulting XPS indicates that the nitrogen atoms of the amide and the carbon atoms of the carbonyl of the heading groups, are located on the surface relative to the methylene carbons of the lipid chains, which shows that the sialolate detection group is present on the surface of the film. The ellipometric analysis of the monolayer of polydiacetylene coated on silicon treated with HF indicated a film thickness of approximately 40 A, according to the expected value based on the molecular model. The bilayer assembly has a maximum visible absorption of 620 nm and appeared as a blue film.

EXAMPLE 2 Qualitative detection of influenza virus unions.

This example illustrates the detection of the binding of influenza viruses to the polymer film of example 1.

When the film obtained in Example 1 was incubated with influenza A virus X31, in a phosphate buffered saline buffer (PBS), at a pH of 7.41, the binding of the viral hemagglutinin to the residues of sialic acid on the surface , resulted in a color transition from blue to red.

Figure (2) B shows the colorimetric response of the film, supported on a microscope glass slide, easily visible to the single eye for qualitative evaluation of the presence of the virus. The film on the left (blue) has been exposed to a white solution of PBS. The film on the right (red) has been exposed to 100 HAUs of virus (CR = 77%) a colorimetric response of approximately 15% was visually observed. No color change was observed when the blue film was incubated with a white PBS buffer solution.

This result demonstrates a color transition of the polydiacetylene that appears from the affinity bond (affinity chromism) instead of thermal annealing (thermal chromism).

Previous studies have shown that, the LB films composed of lipid (1), reaction scheme 1, undergo a color change from blue to red when heated to 70CC, which corresponds to the endothermic transition to chain fusion. lipid The disorder and entanglement of the lipid chain decreases the effective conjugation length of the polydiacetylene skeleton. Similarly, the infrared of the Fourier transform, and the Raman resonance spectroscopy, as well as the X-ray data, demonstrate that the lipid chain package of the red form of the polymer is different from that of the form blue. Therefore, conformational changes in the lipid chains affect the optical properties of the polymer backbone. The binding of the viral hemagglutinin to the assembly of two layers to the sialolate appears to affect the lipid chain conformations in a manner analogous to thermal annealing.

EXAMPLE 3.- Quantitative detection of influenza virus binding.

This example describes the quantitative detection of the binding of influenza viruses to the polymeric film of the present invention.

In addition to qualitative evaluation by visual inspection, the degree of color change is easily quantified by normal visible absorption spectroscopy. The visible absorption spectrum of a two-layer assembly before a (continuous line) and after (interrupted line) viral incubation, are shown in figure 4. The two-layer assembly was inserted into a quartz cuvette containing SPB buffer (pH 7.4) and the absorption spectrum was obtained. The addition of influenza virus in the SPB buffer (pH 7.4) resulted in a chromatic transition after an incubation period of 30 min. Although the color of the film began to change in seconds after exposure to the virus, it was found that 30 min. is the average time required for the RC (colorimetric response) to reach a silver value in a solution without shaking. These spectra represent an RC of 50%.

The blue color film (solid line) before exposure to the virus had a maximum absorption mark at 620 nm and a weaker absorption at 550 nm. After incubation with influenza virus (interrupted line, a dramatic change in the visible absorption spectrum occurred.) The maximum at 550 nm increased with a concurrent decrease in the maximum at 620 nm, resulting in a colored film Red.

In order to quantify the response of a film to a given amount of virus, the visible spectrum of the film was analyzed before exposure to the virus, according to equation (1): B0 = IQZO / (I530 + I620) where B0 is defined as the absorption intensity at 620 nm divided by the sum of the absorption intensities at 550 nm and 620 nm. After. In exposure to influenza, the equation was (2): where Bv represents the new ratio of absorption intensities after incubation with the virus. The colorimetric response of a film is defined as the Change in% in B under exposure to the virus (3): CR = [(B0 - Bv) / B0)) 100% The colorimetric response was directly proportional to the amount of influenza virus, measured in haemagglutination units (HAUs), where an HAU is defined as the highest dilution of virus in existence that completely agglutinates a common erythrocyte suspension.

Figure 5 shows a graph of the colorimetric response of a two-layer assembly to the sialolate against successive additions of influenza viruses. A blue film containing 2% lipid 13 (figure 3), the sialolate, and 98% lipid 11 (figure 3) matrix, was previously incubated in a SPB buffer for 30 min., After which, they were added successive aliquots of influenza A X31 virus. The film was incubated for 30 min. after each virus addition and the visible absorption spectrum was recorded. The CR was calculated according to equation 3. The linear regression analysis of the first six data points gives a slope of 0.93% (r = 0.985).

Saturation of the colorimetric response occurs at approximately 80 HAUs. Incubation of the red film with a white buffer (without virus) for one hour did not result in a return to the blue color. Therefore, the structural changes that result in the change of color seem to be irreversible under these conditions.

EXAMPLE 4.- Competitive inhibition tests for specific detection of influenza virus.

This example illustrates the utility of the invention in competitive inhibition tests.

The specific nature of the interaction between the influenza virus and the surface of the film to the sialolate, was confirmed by competitive inhibition tests. Figure 6 shows that the RC of the film can be inhibited by. compounds that bind to viral hemagglutinin. The incubation of a two-layer assembly to the sialolate with 32 HAUs of influenza virus produced a color response of 22.6%.

However, the same concentration of virus in the presence of a concentration of 17.3 mM of comp or t-17 (K = a 2 mM) (figure 6, column 4) completely suppresses the RC to less than 0.5% due to competitive inhibition.

The CR did not decrease in the presence of a concentration of 17. 3 mM of compound 19 (K> 50 mM) or in the compote " (Fig. 6, col 6), which is not competent for binding to viral hemagglutinin. The known inhibitor of influenza haemagglutination, compound 17, which is observed in figure 3, has a constant of dis c? = Tc? - :. Kd of 2 mM, as determined by a common hemagglutination inhibition test (H7AI). L ?. inciib i n the assembly of two layers of the sialolado, with influenza virus in the presence of the known binding inhibitor 17, - l? as a result no RC (RC <0.5%) and the film remained blue. This result demonstrates that, the inhibitor effectively competed with the surface of the sialolate for binding to the virus. When the blue film was exposed to the same amount of influenza in the presence of a non-inhibitor (figure 3, compound 19, Kd> 50 mM, or glucose, compound 21), the color change was identical to an exposed film to influenza alone.

In order to test the ability of the film to predict the Kd value for an inhibitor, the RC was measured for a series of inhibitory concentrations. The RC was increased in a linear manner (r2 = 0.995) with decreasing concentrations of inhibitor 17. Extrapolation of this graph to RC = 0% gives the concentration of inhibitor that completely prevents the binding of virus to the surface. This value represents the concentration and minimum inhibitor required to effectively compete with the surface on the sial side. The value obtained,? .5 0.93 mM for 4 HAUs of virus, is in accordance with the value of 2 x 1.1-m-xs obtained by a common HAI test and 2.8 plus or minus 0.30 mM as obtained by resonance spectroscopy. nuclear magnetic The inhibition test of the invention described herein is carried out directly and easily. This solution avoids the need for red cell cells, which are used in the normal HAI test. In addition, the subjectivity of reading microliter plates in the common HAI test is replaced by a quantitative, electro-spectrophotometric method. This methodology can be applied to examine new drug candidates or binding ligands.

EXAMPLE 5 Non-specific adhesion.

This example illustrates the non-existence of non-specific adhesion that interferes with the proof of the invention.

In order to appreciate the CR due to non-specific adhesion, two experiments were performed. In the first experiment, films that incorporate the lipid (15) of lactose were incubated with the influenza virus. { figure 3). Lactose was not a ligand for the hemagglutinin lectin. Incubation with 100 HA c £ -1 -rirsz, ~ .z z r. concentration corresponding to a maximum response in the sialolated films show small effect (RC of 2% to 4%) In the second experiment, films containing liquid 13 to the sialolate, were exposed to concentrated solutions of bovine serum albumin. Again, the same small RC was observed. These results indicate that the non-specific adhesion of virus or protein to the surface of the film does not produce the dramatic color change observed from the specific binding of receptor-ligand.

EXAMPLE 6.- Development of the drug.

This example illustrates the utility of the invention for the development of the drug.

We selected a receptor and its reciprocal binding partner (receptor-binding molecule) that are known to be involved in the physiological regulation of interest. The binding partner was incorporated into the inventive film according to example 1.

In the case of development of the neurological drug and -z-ricirotransmission, for example, a dopamine receptor was used. In the case of the development of the drug towards pathogens such as influenza, for example, hemagglutinin receptor viruses were used. The binding partner incorporated in the film was dopamine, or an analogue of dopamine or sialic acid or a sialic acid analog, respectively. The goal of the test was to select a drug that interacts with the binding site in a way that performs the physiological functions.

When the described receiver was present, it caused a color change when it was allowed to join the inventive membrane that incorporates the binding partner.

A candidate drug was then introduced into the system. If the drug binds to the receptor or modifies the binding capacity of the binding partner, there is a concomitant decrease in the color change observed in the present inventive membrane due to competitive inhibition. The ability of the candidate drug to influence the binding was quantified, observing the degree of decrease in the signal compared to the control.

Different variations of the previous solution were used to satisfy different systems. In some cases, it is necessary to organize the receivers into large assemblies such as incorporation into polymers, liposomes or membranes. This arrangement amplifies the changes of the film when the receivers are joined. However, when a candidate drug that binds to the receptor is introduced as before, there is a concomitant decrease in the observed color change.

Another variation that is applicable to this invention requires that the portion of the receiver be attached to the film. A known receiver is covalently linked to the film at one or more points. This can be done by adhering the receptor to the monomer before the polymerization of the film (as in the previous example for binding partners) or after the film is polymerized by means of the modification of the surface of the film. Then the binding partners interact with the immobilized receptors distorting the film, resulting in the color change. In this way, test candidates can be directly examined with receivers that give a positive film response, instead of no color change as in the previous variation.

EXAMPLE 7. Generalized solution.

This example illustrates the general utility of the invention.

A generic film is made in which the binding partner remains constant and a remainder or intermediate link half is varied to accommodate a new auxiliary binding partner.

A film that has biotin on its surface binds protein streptavidin. This protein is tretavalent, therefore, in its bound state, it still has one or more sites available for biotin binding. New binding partners are derived with biotin to bind themselves to the surface of the film via the streptavidin protein. Only a single biotinylated film is prepared and formed in a sandwich with streptavidin, the biotinylated test binding partner. The exposure of this assembly to the test receiver gives the desired color change. As in the previous example, competitive tests are carried out to identify new drug candidates.

EXAMPLE 8 Entrapment and detection of small organic molecules.

This example shows the development of a new class of functional materials that specifically trap small organic compounds and report the entrapment event by means of a colorimetric change that can be detected visually. These materials act as simple color-based detector devices that detect the presence of compounds such as solvents or other toxic contaminants in streams of water or in the air.

The first stage comprises the synthesis of analogs of the one and two diacetyl lipid compounds as seen in reaction scheme 1, by the preparation of the secondary methyl group in the targeting group. The enantiometrically pure ester of pentacosadinoic acid 3 (APD) is hydroxylated through the oxidation of molybdenum peroxide to alcohol. The diastereomers are separated and the ester is hydrolysed to chiral lactate analogs 5 and 6. The ethyl esters are formed and treated with Grignard reagents to give the desired chiral lipid analogs 7 and 8. Variations in the R groups result in a wide variety of new materials in which specific entrapment capabilities are reviewed.

The monomer-lipid clathrate is sorted and compensated on the surface of the water using a Langmuir-Blodgett film apparatus. Polymerization of the monolayer, by ultraviolet radiation, produces blue colored material as previously described. This is lifted on a hydrolyzed microscope slide. The ability of the 7 and 8 films to trap dioxane and 1-butanol was tested and to experience the expected color transition. Because the technique can be generalized, the appropriate derivatives of 1 and 2 are determined and selected and adapted to the chemistry to specifically detect a particular small molecule. To date, little is known about why materials 1 and 2 are highly selective for dioxane and 1-butanol, respectively. Examining a series of compounds, a variety of solvents were displayed, using the colorimetric detection technique to determine which solvent forms the most suitable host compound. Using computer models, cavities of the specific size and shape were designed for the union of analyte molecules. The non-complex film and the complex film were completely characterized with a variety of common surface techniques. These include ellipsometries of XPS, Auger, Leed, Raman spectroscopy and STM. All these allow the determination of the structural requirements of the clathrates in order to rationally design new materials with specific clamping properties.

EXAMPLE 9.- Detection of malaria merozoites, This example describes the films and conditions used for the detection of malaria merozoites.

The films contained sialic acid and were prepared identically to those described in Example 1. The films were exposed to solutions containing erythrocytes of malaria merozoites. After exposure to pathogens overnight the films turned pink. The color response (RC) in each case was almost 100%.

TABLE 1.- PATHOGENO MOLECULA RECEPTOR.

HIV D414; intestinal peptide7 active vessel, peptide T, acid Vaccines Factor1 of epidemic development Rabies Receptor2 of acetylcholine Epstein Barr Receptor3'4 complement Rheo Receptor5 Beta-adrenergic rhinovirus ICAM-16 '10' "; N-CAM, Mab13 glycoprotein with associated myelin Polio virus Receptor9 of poliovirus. Influenza Sialic acid15 Cytomegalovirus Glycoprotein (not sialic acid) 16 '17, 18.

Corona virus Sialic acid 9-OAC & sialic acid Encephalomyelitis Sialic acid 9-OAC Rubella virus Measles virus Glycoprotein (not sialic acid) 20, 21 22, 23.

Herpes Oligosaccharide glycoportein 2 25 26 Chlamydia Sialic acid 27 '28' 29 '30 Rhino virus Proteins31' 32 licosylated Rotavirus Sialic acid 9 -OAC. Polioms virus Sialic acid. Reo virus Sialic acid Streptococcus Suis Sialic acid to 2 - 3 poly-N-acetyl lactosamine- Salmonella Sialic acid. Tifimurium Paramyxovirus Sendivirus Sialic acid Mumps Sialic acid Ne castle Sialic acid Viral disease Mixovirus Sialic acid Escherichia Coli Oligomannose, galactose at 1 - »4 galactose, sialic acid at 2 - 3 galactose Encephalon-myocarditis virus Sialic acid Cholera toxin Ca (A ganglosial of sialic acid, galactose, glucose, N-acetyl-galactose Meningitis Sialic acid.) BIBLIOGRAPHY.

I. Natura, 318: 663 (1985) 2.- Science, 215: 182 (1982) 3.- Proc. Nati Acad. Sci; USA, 81: 4510 (1984) 4. - J. Of Biol. Chem., 265: 12293 (1990) 5. Proc. Nati Acad. Sci. USA, 82: 1494 (1985) 6. - Nature, 344: 70 (1990) 7. J. of Neuroscience Research: 18: 102-107 (1987) 8. FEBS Letters, 211: 17-22 (1987) 9. Cell, 56: 855-865 (1989) 10. Cell, 56: 839-842 (1989) II. Cell, 56: 849-853 (1989) 12. Nature, 333: 426-431 (1988) 13. Proc. Nati, Acad, Science, USA, 85: 7743-47 (1988) 14. Nature, 312: 763-770 (1985) 15. Cell, 56: 725-728 (1989) 16. J. Virol, 63: 3991 ( 1989) 17. Inas, 86: 10100 (1989) 18. Virol, 176: 337 (1990) 19. Med. Microbe. Imm., 179: 105 (1990) 20. Infect. Imm. , 24: 65 (1979) 21. Proc Soc. Exp.Bio Med, 162: 299 (1979) i 22. Virol, 172: 386 (1989) 23. J. Clin. Inv., 86: 2569 (1990) 24. J. Virol, 64: 2569 (1990) . Science, 248: 1410 (1990) 26. Febs Lett., 277: 253 (1990) 27. Infect. Im., 57: 2378 (1989) 28. Microb. Lett., 57: 65 (1989) 29. Infect. I m. , 40: 1060 (1990) . Infect. Imm. , 25: 940 (1983) 31. Med. Virol, 8: 213 (1989) 32. J. Virol, 64: 2585 (1990) REACTION SCHEME 1 1: R = H 2: R = CMe3 (R1 »CH3 (CH2) U- = - = ~ (CH2) 7- ° > -xD b:

Claims (38)

1. R E I V I N D I C A C I O N S 1. - A polymerized two-layer film for direct detection of the presence of analytes, comprising: (a) a ligand comprising a detection group that has direct affinity for the analyte or functions as a competitive linker to the analyte; (b) a linear structural linker having two terminal ends, wherein the linker is attached at its first terminal end to the ligand; (c) a conjugated polymer backbone to which the structural linker is attached at its second terminal end; (d) targeting groups that are bonded to the surface of the conjugated polymer backbone in positions not occupied by the structural linker; and (e) a support structure; wherein the film is subjected to a detectable spectral modification when it binds to an objective analyte for the ligand. 2. - The film of claim 1, wherein the analytes are biomedical materials, pathogens, drugs or industrial materials. 3. - The film of claim 2, wherein the biomedical materials are selected from the group comprising pathogens and cells infected therewith, drugs (pharmaceuticals), hormones, blood components, disease indicators, cellular components, antibodies, lectivas, enzymes, genetic material and their metabolic derivatives. 4. - The film of claim 2, wherein the pathogens are selected from the group comprising viruses, bacteria, parasites and other pathogens. 5. - The film of claim 4, wherein the virus is selected from the group comprising influenza, coryza, rubella, varicella, hepatitis A, hepatitis B, herpes simplex, polio, smallpox, plague, HIV, vaccines, rabies, Epstein Barr, reoviruses, rhinoviruses and mutations, strains and / or recognizable parts of ligands thereof. 6. - The film of claim 4, wherein the bacteria are selected from the group comprising E. coli, tuberculosis, salmonella, streptococci and mutations, strains and degraded parts thereof. 1 . - The film of claim 4, wherein the parasites and other pathogens are selected from the group consisting of malaria, sleeping sickness (lethargic encephalitis), river blindness and toxoplasmosis. 8. - The film of claim 1, wherein the ligand is provided for the detection of a pathogen analyte. 9. - The film of claim 8, wherein the analyte is a virus. 10. - The film of claim 8, wherein the ligand is selected from the group consisting of an epidermal growth factor for vaccine analyte, an acetylcholine receptor for rabies analyte, a complement receptor for Epstein Barr analyte, a receptor beta-adrenergic for reovirus analyte, ICAM-1 for rhinovirus analyte, a poliovirus receptor for poliovirus analytes, a trisaccharide analyte for cholera toxin analyte, tetrasaccharide for neurotrophin analyte and derivatives and analogs thereof capable of association with analyte. 11. - The film of claim 8 wherein the ligand is sialic acid and its derivatives and analogs that bind to corona virus, influenza virus, encephalomyelitis virus, chlamydia, sendi virus, mumps virus, disease pathogen of the ne castle, myxovirus, encephalon-myocarditis virus, meningitis virus or malaria virus. 12. The film of claim 8, wherein the analyte ligand pairs are tetrasaccharides and neutrophils, adhesion peptides of cells and target cells, trisaccharides and bacterial toxins, or transmembrane receptors and hormones. 13. - The film of claim 8, wherein the ligand provided for detecting HIV analytes is selected from the group comprising CD4, sCD4, CD26, vasoactive intestinal peptide, T peptide and sialic acid and derivatives and analogs thereof capable of associating with HIV 14. - The film of claim 1, wherein the polymer comprises polymerizable lipid monomers. 15. - The film of claim 14, wherein the monomer is selected from the group consisting of acetylenes, diacetylenes, alkenes, thiophenes, imides, acrylamides, methacrylates, vinyl ether,. malic anhydride, urethanes, allylamines, siloxanes, anilines, pyrroles and vinylpyridinium. 16. - The film of claim 15, wherein the backbone of the polymer consists of diacetylene monomers. 17. - The film of claim 1, wherein the orientation heading groups are hydrophilic groups with the ability to mutually form a hydrogen bond. 18. - The film of claim 17, wherein the targeting header groups are selected from the group consisting of -CH2OH, -CH2OCONHPh, -CH2OCONHEt, -CH2CH (Et) OCONHPh, - (CH2) 9OH, -CH20C0Ph, CH20C0NHMe, -CH20Ts, -CH (0H) Me; -CH20C0R2, wherein R2 is n-C5Hn, n-C7H? 5, n-C9H? 9, n-CuH23, n-C? 3H27, n-C? 5H3? , nC? 7H35, Ph, PhO, or o- (H02C) C6H4, -OS02R2, where R2 is Ph, p-MeC6H4, p-FC6H4, p-CIC6H4, p-BrC6H4, p-MeOC6H4, m-CF3C6H4, 2-C? 0H7 or Me; -C02M, wherein M is K, HNA or Ba / 2; or -CH2OCONHR2, or -CH2CONHR2, wherein R2 is Et, n-Bu, n-C6Hi3, n-C8H? , nC? 2H25, cyclic? -C6Hn, Ph, p-MeC6H4, m-MeC6H4, o-ClC6H4, m-ClC6H4, p-Cl C6H4, o-MeOC6H4, 3-thienyl, Me, Et, Ph, I-C10H7 Et, Ph, EtOCOCH2, BuOCOCH2, Me, Et, i-Pr, n-C6H13 / EtOCOCH2, BuOCH0CH2, Ph, 2, 4 (N02) 2C6H3OCH2, CH2CH2OH. 19. - The film of claim 17, wherein the targeting heading group is a carboxylic acid. twenty . - The film of claim 1, wherein the monomer targeting group is selected from the group consisting of CH3-, CH30-, neo-C5HnO-, cyclo-C6HnO-, PhCH20-, p-AcC6H40-, p- BzC6H40-, p-BrC6H4COCH20-, p- (PhCH = CHCO) C6H40-, p- (PhCOCH = CH) C6H40-, or-BZC6H4NH-, p-BZ C6H4NH-, MeOCH2CH2H-, n-C6H? NH-, or EtO-. 21. - The film of claim 20, wherein the terminal group is a methyl group. 22. - The film of claim 1, wherein the film is additionally provided with a support. 23. - The film of claim 22, wherein the support is selected from the group consisting of plastic, mica, metal, ceramic, glass and other polymer surfaces and hydrophobic derivatives thereof. 24. - The film of claim 23, wherein the support is a hydrophobized microscope slide. 25. - A test set comprising a container that incorporates the film of claim 1. 26.- The test set of the claim 25, wherein a solid structure, provided with holes, is mounted on the surface of the film to provide structures with wells that are impervious to fluids and analytes. 27. - The test set of claim 25, wherein the set container also incorporates instructions for implementing the test procedure. 28. - An analytical device that incorporates the film of claim 1. 29. - The analytical device of claim 28, wherein said device is provided with a component for automatic chromatographic reading. 30. - The analytical device of claim 29, wherein the device is provided with a component for automatic handling of the sample to allow continuous readings on a flow stream or a series of samples. 31. - A method for making the test film with two polymerized layers of claim 1, comprising the steps of: a) .- binding a receptor binding ligand to a first terminal end of a linear structural unit; b) - attaching a monomer to a second terminal end of the linear structural unit to produce a moiety or ligand moiety of a monomer-linear structural unit; c) .- join a heading group of orientation to the monomers, producing a monomer residue of heading group of orientation; d). - mixing half the unit - structural ligand of linear monomer with a plurality of halves or residues of targeting monomer groups of heading; e) .- extending the mixture of stage (d) on a surface; and f) .- polymerize the extended mixture. 32. - The method of claim 31, comprising the step of transferring the polymerized mixture to a support. 33. - The method of claim 31, wherein after step (e) of the process, the extended mixture is compressed. 34. - The method of claim 31, wherein after step (d) the mixture is spread on a water surface. 35. A method for the direct detection of an analyte, characterized in that it comprises the steps of: a) contacting the test sample film of two polymerized layers of claim 1, with a sample containing an analyte; and b) .- inspect the film for a change in its optical properties. 36. - The method of claim 35, wherein the observed optical property is a color change. 37. - The method of claim 36, wherein the color change is from blue to red. 38. - The method of claim 37 wherein the color change is detectable at a wavelength of 620 nm to 550 nm. SUMMARY . A polymerized film, a test sample and method for the direct detection of analytes using observable spectral changes in monomolecular films that occur under the selective binding of analytes to the film.