Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
  • G01N33/54366—Apparatus specially adapted for solid-phase testing
  • G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
  • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
  • G01N33/586—Liposomes, microcapsules or cells

Definitions

• the field of this invention is the detection of analytes using polymerized lipid layers.
• 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, chemUuminescence, 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.
• 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 describes 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. Bhattachaigee etal., 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.
• Atomic force microscopy is described in Marti et al., "Atomic Force Microscopy of an Organic Monolayer,” Science (1988) 293: 50-52.
• Methods and compositions are provided for determining the presence of analytes using a polymerized layer or material capable of undergoing a spectrophotometric or optical change, e.g. chromatic shift, in relation to a change in its condition, wherein at least one element of such change will be specific binding of analyte or an analyte mimic ("binding ligand") to a specific binding member, which may be associated with the polymerized layer or which is part of a compound which can produce a change in the environment of the polymerized layer in relation to the binding event, resulting in a chromatic shift.
• binding ligand an analyte mimic
• the chromatic shift which is measured can result from: a change in the spectrophotometric properties of the polymerized layer caused by the binding of the binding ligand to the polymerized layer; the binding of the binding ligand to the compound which produces a change in the environment of the polymerized layer in relation to the binding of the binding ligand; or the effect of the binding of the binding ligand to the polymerized layer on the triggering of an enhanced or retarded optical change, as a result of a change in the environment of the polymerized layer or the reversal of such optical change during triggering.
• Various agents may be employed to induce the triggering of the spectrophotometric shift. By comparing the results obtained with a sample to a control value, the presence of the analyte may be detected.
• FIG. 1 provides data for four parameters used to study the effect of streptavidin binding on the color shift of an ethyl morpholin PDA film.
• FIG. 2 provides data for four parameters used to study the effect of streptavidin binding on the color shift of a 5% biotin/1 ,2 propandiol pentacosadiynoic ester film.
• the methodology uses detection of a chromatic or spectrophotometric shift in light either absorbed or fluoresced, in response to a binding event between the analyte or analyte mimic ("binding ligand") and a polymerized diacetylenic (dyine material or polymer) acid, where the spectrum (absorbance or fluorescence) of the polymerized layer changes as a result of: a change in the spectrophotometric properties of the polymerized layer caused by the binding of the binding ligand to the polymerized layer; the binding of the binding ligand to the compound which produces a change in the environment of the polymerized layer in relation to the binding of the binding ligand resulting in a change in the layer's ability to respond; or the effect of the binding of the binding ligand to the polymerized layer on the triggering of an
• the polymerized layer may be an insoluble material, e.g. later, or a soluble material or in a colloidal state in an aqueous solvent.
• lipid refers to monomers having a hydrocarbon chain of at least 6 carbon atoms in length.
• the lipids of the subject invention may comprise one or more diacetylene groups and either one, e.g. polar lipophilic monomers, or two polar head groups, where the monomers which comprise two polar head groups are proximal to the termini of the linking aliphatic group, e.g. dual headed. However, either situation may be used for carrying out the analysis in accordance with this invention.
• the colloidal polymerized lipid may be prepared in a wide variety of ways, where the lipid composition may be homogenous or heterogeneous as to the nature of the lipids involved, the moieties bound to the lipids, and the like. Likewise, soluble polymer strands can be made using monomers containing two polar head groups. These compositions are referred to as soluble polymer lipids. For the lipid layers, the layers may be unilamellar or multilamellar films.
• liposomes where liposomes also fall within the definition of polymerized layer, having a conglomerated or aggregated structure, where the liposomes may then be coated onto an appropriate support, or remain in solution thereby avoiding the requirement for a solid support.
• the manner in which the polymerized layers are prepared may be varied widely, although some procedures will be preferred over others.
• the composition of the lipids may be varied widely, where again some monomers may be preferred over others.
• 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.
• Conventional Langmuir-Blodgett techniques may be employed.
• Soluble phase polymerization and subsequent solubilization can be employed.
• Methods for making vesicles, such as extrusion can 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, crystaUization time, temperature, humidity, traverse rates, will affect the nature of the polymerized film.
• Polymerization times are important for controlling the assay responsiveness of the layers, such as the films. Prolonged polymerization times (10 minutes) can lead to less responsive layers. Polymerization times between 20 seconds to 5 minutes can lead to more responsive layers dependent upon radiation intensity. Typically, times ranging from 1 second to 30 seconds give the greatest response. On the other hand, the degree of polymerization must be sufficient to provide a change in optical density which is readily determinable and allows for accurate measurement.
• 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/cm 2 to 100 mjoule cm 2 , more usually 30 mjoule/cm 2 .
• 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.
• 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 or material 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 membrane or tape.
• greater flexibility may be achieved with regards to the amount of light that is absorbed or emitted as a result of an analyte binding event.
• the polymerized groups experience a greater degree of flexibility than they do on a rigid support, thus allowing for the possibility of reversible shifts in absorbed or emitted light.
• Transferring the layer to a highly rigid support may limit the film's flexibility, and thus reversibility, of the color shif
• 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, lnterscience Publishers, I. Prigogine Editor, John Wiley and Sons (1964)].
• the polymerized material can also be transferred to the support before polymerization, and polymerized while on the support. Following transfer of the polymerized material to the appropriate support, the polymerized material is ready to be used to detect the presence of analyte.
• the various polymerized lipid compositions may be used, as appropriate, where one particular form may have advantages as compared to another.
• considerations will include economies of manufacture, reproducibility, nature of the sample, responsiveness in the assay, stability to shipping etc.
• 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.
• 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.
• Monolayer 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.
• Multilayer films can be formed by multiple film transfers to the same support surface or by modifying the film composition so that multiple layers can spontaneously form.
• Cast films or liposomes may be prepared in accordance with the procedures described in the Kuo and O'Brien references described previously.
• the initial stage in providing the cast films or liposomes is to prepare an aqueous dispersion of the lipid, where the medium will comprise from about 0.1 to 5, more usually about 0.5 to 2 mM of lipid, buffer, e.g. Tris, PBS, HEPES, etc., and other additives, such as organic, polyvalent ions, glycerol, etc.
• the aqueous lipid dispersion is then sonicated in accordance with conventional ways, generally at a temperature in the range of about 20- 60°C, for a time usually in excess of about 5 sec and not more than about 30 sec.
• the water suspension may then be irradiated with UV light in the substantial absence of oxygen for sufficient time for the polymerization process to go to at least 50% completion, preferably at least about 75% completion.
• the polymerization process may be monitored spectrophotometrically.
• the liposomes may be extruded, as described by Hope et al. , Biochimica Et. Biophysica Acta (1985) 812:55-65.
• the liposomes will be of a size in the range of about 100 nm to several microns in diameter.
• the unpolymerized or polymerized vesicles may be spread onto an appropriate support, e.g. glass, nitrocellulose membranes, nylon membranes, paper or other porous materials, and allowed to dry, with substantially complete removal of water. If unpolymerized, the film may then be irradiated and the polymerization process followed spectrophotometrically, with the previously indicated degrees of polymerization desired.
• an appropriate support e.g. glass, nitrocellulose membranes, nylon membranes, paper or other porous materials
• mono- or dioic diacetylenic compounds may be employed, particularly the dicarboxylic acids, where the carboxyl groups will be normally at the terminal positions.
• the carboxyl groups may be modified with a wide variety of hydrophilic groups, particularly uncharged or charged hydrophilic groups, such as oxy, amino, carboxy, inorganic acid groups, such as phosphorous acids, sulfur acids, etc. and the like.
• hydrophilic groups particularly uncharged or charged hydrophilic groups, such as oxy, amino, carboxy, inorganic acid groups, such as phosphorous acids, sulfur acids, etc. and the like.
• Various polyols, polyethers, polyamines, polyacids, or combinations thereof may find use.
• the carboxyl groups may be unfunctionalized or functionalized, usually at least about 50 number % of the carboxyl groups being functionalized, depending upon the length of the chain, the nature of the binding group attached to the polymer, as well as the nature of the medium in which the assay is carried out.
• soluble polymers typically the diacetylenic monomers are crystallized from an organic solvent. The crystalline monomers are then polymerized in bulk using UV or X-ray irradiation. The polymerized material is then solubilized in an aqueous or organic media, the unpolymerized monomer extracted, and then the polymeric material processed for the assay. Sonication, detergents and other emulsifiers can be used to enhance solubility. Standard biochemical methods can be used to functionalize soluble polymers once they have been formed. Functional groups can then be used for attaching various ligands or binding members.
• the surfactant or lipid molecule may have a single lipid chain, e.g. a diynoic acid or a plurality of lipid chains, e.g. diester glycendes, triester glycerides; mono- or polyesters of mono- or polycarboxylic acids, e.g. N-acyl bis (docosa-10, 12-diynil) L- glutamate; or cosa-8,10-diyn-l,20-dioic acid, and the like.
• a single lipid chain e.g. a diynoic acid or a plurality of lipid chains, e.g. diester glycendes, triester glycerides
• mono- or polyesters of mono- or polycarboxylic acids e.g. N-acyl bis (docosa-10, 12-diynil) L- glutamate
• 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.
• the functional groups may be present in the material to provide for polymerization and various optical properties, such as F ⁇ rster energy transfer.
• the functional groups will comprise diynes, although other polyunsaturated molecules may find use, such as activated monoynes, e.g., ⁇ -ketomonoynes.
• the hydrophobic portion of the polymerizable monomer will have a chain of at least 6 aliphatic carbon atoms, usually a straight chain of at least 8 carbon atoms, and generally not more than a total of about 100 carbon atoms, usually not more than about 34 carbon atoms.
• the number of carbon atoms will vary from about 6 to 32, more usually 10 to 30, and more preferably 12 to 29 carbon atoms.
• the monomers 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.
• 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
• Hydrophilic groups may include drugs, peptides, ligands, receptors, charge transfer complexes, or chromophores.
• 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 polymerizable group is typically a diacetylenic moiety, but other optical polymers may also be employed.
• 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.
• 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.
• Single or multiple polymerizable hydrophobic chains may be present per lipid unit.
• the polymerizable groups may also be incorporated between two polar head groups. These monomers may be polymerized in bulk crystals and then subsequently solubilized into soluble polymer strands. Their solubility is dictated by the polarity of the headgroups.
• the hydrocarbon tail of the surfactant may also terminate in a hydrophilic group so that the surfactant is bipolar.
• the ligand or binding molecule provides for specific binding pair member complex formation, where the amount of complex formation is related to the amount of analyte in the assay medium.
• the ligand or binding molecule may be associated with the lipid material, either directly or indirectly, being covalently bonded to the lipid material or covalently bonded to a macromolecule physically associated with the lipid material, or may be an entity whose activity is modulated as a result of complex formation, e.g. an enzyme conjugate whose activity changes upon complex formation. Therefore, the ligand or binding molecule may take many forms, since the lipid material is a sensitive detector for a broad spectrum of events, such as direct binding and changes in its rnacroenvironment.
• the complex comprising the binding molecule may involve the analyte, an analyte mimic or a molecule whose effective concentration varies with the amount of analyte, e.g. an antibody to the analyte.
• the ligand or binding molecule used in analyte detection may be bound to an independent macromolecule or to the surfactant, where the preference will depend upon the nature of the polymerized material. With the material, the preference will be for an independent macromolecule, such as a protein, peptide, sugar, or other ligand, when referring to ligand, it is intended any molecule which specifically binds to another molecule.
• the macromolecule will be at least about 0.2kD, usually 50kD, or more and may be bound covalently or non-covalently to the support for the film or bound directly to the polymerized material.
• the macromolecule may be applied to a support before or after the polymerized material is applied to the support.
• the ligand can be chemically coupled, enzymatically coupled or absorbed to the polymerized material.
• the ligand density will range from .01 % to 100 % of the surface area. If the ligand is bound to the layer, depending upon the desired density of the ligand bound to the layer, 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, carboxysaccharides, reduced saccharides, peptides, polypeptides, nucleotides, oligonucleotides, or the like.
• Specific groups include zwitterions, e.g., betaine, peptides, sugars, such as glucose, glucuronic acid, ⁇ -galacto-u-mine, sialic acid, etc., phosphatidyl esters, such as phosphatidyl glycerol serine, inositol, etc.
• the ligand or binding molecule 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, polysaccharides, 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 or binding molecules which are covalently bonded to the surfactant will normally be a member of a specific binding pair.
• the ligands may be varied widely, usually being molecules of less than about 2 kD, more usually less than about 0.5 kD.
• 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.
• the ligand bound to the surfactant may be a macromolecule, usually not exceeding about 500 kD, more usually not exceeding about 200 kD.
• proteins, nucleic acids, or other polymeric or nonpolymeric compounds of high molecular weight may also be employed.
• 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.
• 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.
• 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.
• prior treatment such as removal of cells, filtration, dilution, concentration, detergent disruption to release antigen, centrifugation, or the like.
• 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 or other 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.
• the flow cell method or a porous membrane 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.
• 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 change in the absorbed or emitted light of the layer.
• the change in chromatic signal can be as a result of the effect of binding of a specific binding member to the lipid material, the effect of specific binding of the specific binding member to the lipid material of a change in the chromatic signal due to an environmental change, or the effect of binding of a specific binding member to an entity, where the result of the binding is to produce an environmental change in the environment.
• the environmental change in the environment is normally a change in the macroenvironment of the lipid material, for example, the assay medium, irradiated light, or the like.
• the assays may involve binding of a binding ligand (analyte or analyte mimic) to the lipid material or to a different material resulting in the modulation of the change in the environment of the lipid material with a resulting difference in the chromatic shift in the presence or absence of analyte.
• a binding ligand analyte or analyte mimic
• the binding ligand may provide for binding to the lipid material as the event providing the difference in the chromatic shift, inhibiting or providing for the binding of a different entity to the lipid material as the event providing the difference in the chromatic shift, or binding to an entity other than the lipid material to modulate the change in the environment of the lipid material as the event providing the difference in the chromatic shift, or the like. Therefore, the significant factor is that the subject system allows for a chromatic shift in absorption or fluorescence in relation to a binding event related to the amount of analyte in the assay medium, which binding event directly or indirectly results in a difference in the chromatic shift in the presence and absence of analyte.
• 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, enzymes, antibodies and nucleases.
• hydrazine may be used, by itself or bound to a polymer, e.g., poly (aery lhydrazide).
• 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.
• a thiol containing molecule which may be biotin, avidin, any ligand or binding protein, sulfhydryl containing polymer, a nucleic acid, or molecular recognition analogs.
• 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.
• 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.
• sulfhydryl surfactants may be attached to sulfhydryl groups on the antibody molecules.
• the binding event can induce or modulate an absorbance or emission change in the material.
• various agents or processes can be used to enhance the optical change in the material due to the binding event.
• the optical properties of polydiacetylene films or materials can be changed due to pH, temperature, mechanical stress (e.g. atomic force methods), various solvents, ionic strength, detergents, optical induction using a specific wavelength that the polymer may respond to, inorganic mediators or the like.
• the binding event may promote or retard the polymerized material's response due to pH, temperature, mechanical stress (e.g. atomic force methods), various solvents, ionic strength, detergents, optical induction using specific wavelength that the polymer may respond to, inorganic mediators 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 environment, e.g. a change in pH.
• hydrolases e.g. phosphatases, glycosidases or esterases
• products can be produced which result in a change in pH.
• the tight 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.
• the light absorbed by a non-fluorescent blue form of polymerized film has been found to shift to a fluorescent red form as a response to a change in acidity.
• the binding ligand may compete for receptor with a binding ligand bound to an enzyme, where the enzyme conjugate comprises an enzyme which enzymatically converts a substrate to a composition which increases the pH of the medium.
• the enzyme conjugate comprises an enzyme which enzymatically converts a substrate to a composition which increases the pH of the medium.
• Groups which may augment the pH include phenols, esters, caiboxylates, 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 and can serve as substrates.
• the binding event may also be distal and affect 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 binding event may provide for the major portion of the chromatic change, one may provide for an agent which induces a major change, which major change is modulated by the binding event. For example, changes in solvent, pH, ionic strength, mechanical stress (e.g. rubbing, atomic force microscopy or other mechanical perturbation) or other change in the environment of the polymerized lipid can provide for a substantial modification of the chromatic characteristics of the polymerized lipid.
• the effect of the change in environment can be modulated by the binding event, so that the binding event in conjunction with the change in environment can result in a greater response resulting from binding to the polymerized lipid.
• the binding event can be used to retard the optical responsiveness of the polymer due to the environmental modification.
• the heating protocol one may obtain a temperature profile over a range not exceeding 90°C, usually not exceeding about 60°C, preferably being from about 30 to 50°C.
• the rate of heating will generally be from about 0.5 to 50 °C per min, more usually from about 1 to 10°C per min.
• the reading may be anywhere from every 1 °C up to about every 10°C, conveniently at 5°C intervals.
• pH As already indicated, one may use pH, whereby the effect of the change in pH can be compared in the presence and absence of the analyte.
• the pH range will vary depending on the head group charge.
• a pH range of about 1 to 4 is used to trigger positively charged head groups and a pH range of about 8 to 11 is used to trigger negatively charged head groups.
• ionic strength where the ionic strength will vary from l ⁇ m to 5 M, more usually from .1 mM to 50 mM. Ionic strength is dependent on the valence of the ions, e.g. monovalent or multivalent.
• solvents one may remove the aqueous solvent in which the binding occurred, optionally followed by washing and drying, and then followed by addition of a different solvent, particularly a non-acquiesced solvent, or one may add solvents to the original assay medium, where the solvents are dissolved in the assay medium or can displace the assay medium by contacting the polymerized lipid layer.
• solvents particularly a non-acquiesced solvent
• polar or non-polar solvents can be added directly to the aqueous media surrounding the polymerized material.
• Mechanical methods such as rubbing or atomic force microscopy or other means of mechanical stress can be used to promote a conformational change in the polymerized material.
• the conformational change results in optical changes due to perturbing the polymer's orientation.
• Atomic force microscopy can be used in a tapping or scanning mode to selectively change regions of a polymerized layer from a blue, non-fluorescing form to a red, fluorescing form.
• the polymerized layer bound with protein will respond differently to mechanical force than an unbound polymerized layer.
• an assay utilizing a mechanical optical triggering method can be employed.
• the polymerized material can be exposed to specific wavelengths to which the polymer or other substituent can respond.
• Light energy injected into the polymerized material results in a change in the material's conformational state due to interaction between the light/energy and absorbing conjugate polymer. Injecting light/energy can trigger the material to undergo an optical change from the blue form of the polymer to a red form of the polymer. This optical change can be used as an environmental change in the assay.
• the change in environment is found to provide a larger signal than the binding event, and the effect of the binding event on the change in signal resulting from the change of environment is greater than the change in signal as a result of the binding event, this triggering mechanism by changing the environment provides for a more sensitive assay.
• the binding event can inhibit the optical change in the polymer back bone due to the change in environment.
• a sensitive assay can be achieved by comparing a bound test film to an unbound control film. After triggering through environmental change, the control film will change dramatically while the test film will be partially inhibited from change in relation to the amount of analyte bound to the polymer materials.
• the method of detection focuses on the change in absorbed or emitted light of the polymerized layer which results from the presence of analyte.
• the spectral shift is the result of the change in absorbed light of the layer.
• the absorbed or emitted light is polarized or non-polarized.
• other spectroscopic methods can be used to measure the polymer's response to binding, e.g. IR spectroscopy, Raman spectroscopy, and other linear and non-linear techniques.
• the color shift will normally be from blue to red.
• the red 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.
• Reverse chromism may also find use, e.g. yellow to blue. It is found that different wave lengths can provide for more sensitive responses, in providing for greater changes in the chromatic signal. Particularly, it is found that greater sensitivity may be achieved by determining d e change in ratio of the absorbance, for example, A636/A540 as compared to A646/A540.
• 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.
• the binding event may induce or inhibit the emission spectrum of the film.
• the actual concentration of the binding events may be proportional to the degree of optical change that the film is allowed to undergo.
• the chromatic shift may result in a change in maximum absorption in the range of 565 to 850 nm to 400 to 560 rim.
• 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.
• cascade enzymes which further enhance the signal may be used.
• Various mass or size dependent labels can be used to enhance the molecule's effect on the polymer. 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.
• the enzyme can be used to catalyze the formation of an abundance of product molecule which in turn reacts with the film and propagates 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 film providing for an amplification cascade.
• 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.
• 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.
• DNA duplexing leads to signal generation.
• 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.
• 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.
• cocking may be achieved, where cocking intends 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.
• Multivalent binding can be used to enhance the effect on the material.
• large moieties including gold particles, latex spheres, red blood cells, or the like can be used as labels to have a larger impact on the effective signal difference between bound and unbound analytes.
• Two liposomes dispersed in the assay medium or used in forming a layer may have different binding moieties such that a sandwich can be formed by bridging the analyte between both liposomes. This has the added benefit when the liposome is dispersed in solution of increased specificity and potential for signal enhancement through agglutination.
• 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.
• 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 surface 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 die lipid monomer over the surface of the subphase.
• lOO ⁇ l 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/cm 2 . 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.
• a glass trough (dimensions of 6 in. x 6 in. x 12 in.) was filled with 1.0 L of 0.0° C Milli-Q water.
• a 2.0 mM ethyl morpholin pentacosadiynoic amide monomer(EMPDA)/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 ⁇ l 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).
• UVP Model UVG- 54 Mineralight The fluence of light at the subphase surface was at 30 mjoule/cm 2 .
• Morpholin PDA Film An assay for the presence of DNA in a sample was run as follows.
• 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 ⁇ l. 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 30 ⁇ l samples of 2.6 x 10"* M oligonucleotide/sterile water ( 21-mer: 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.
• a 5% biotin attached to 3 repeat Polyethylene glycol spacer/95 % ethyl morpholin PDA film was prepared by spreading the lipid mixture at the air-water interface with stirring at a sub-phase temperature of 10°C and compressed to 25 mNt m.
• the film was manually and horizontally transferred to 4/1x3" slides stuck to a holder. After being air-dried, the films were polymerized by placing the holder about three inches beneath a xenon arc lamp and turning on the lamp for 1, 5, 15, or 45 sec.
• the slides were stored in a box at room temperature. The slides were assembled in 8-well devices using double-sticky tape on glass
• Temperature triggering was done by filling each well with DI water and submerging each slide in a trough containing DI water, which was itself placed in a circulating water bath at a fixed temperature for one min. The slides were then removed from the bath, blown dry with air (at this point they were at room temperature) and then scanned. The process was then repeated at higher temperatures in the range from 20 to 80°C at 5°C increments.
• the data was analyzed by first determining that the absorbance peaks for the blue and red forms of the film occurred at 646 and 540 nm, respectively. Each scan was corrected for baseline variation by subtracting the flat portion of the scan around 750 nm. The ratio A646/A540 (R) was then computed and plotted versus temperature. The curve was fitted with a 4-parameter sigmoidal curve model using the program Tablecurve.
• Parameter a corresponds to the final R value, i.e. when the film has completely triggered ("final redness").
• Parameter b corresponds to the initial R value.
• Parameter c corresponds to die inflection point halfway between the initial and final states.
• Parameter d corresponds to the steepness of the curve, i.e. the temperature range at which R goes from to 37% to 63% of its final value; the smaller d is, the more uniform the triggering.
• biotin/diol ester PDA films were prepared by spreading the lipid mixture at an air-water interface with stirring with nitrogen at a subphase temperature 40 °C and compressed to 25mNt/m at O.Olcm/s.
• the film was manually and horizontally transferred as described in the previous experiment, where the glass was silanized. The procedure was repeated to form a bilayer. The transfer was followed by air drying and polymerizing by placing the holder of the slides about three inches beneath a xenon arc lamp and exposing the film for 1, 5, 15, or 45 sec. The slides were then stored at room temperature.
• the slides were assembled in 8-well devices as described above, except that the slides were air blown dry after the washings. Each well was scanned from 350- 800 nm ("pre-binding") and then filled in the same manner as described above. After incubating for 20 min, and rinsing 3X with TBS and 3X with DI water, the slides were then scanned again ("post-binding"). The effect of SA was determined by this procedure.
• Temperature triggering was done by filling each well with DI water and submerging each slide in a trough containing DI water, which was itself placed in a circulating water bath at a fixed temperature for one min. This lodge was then removed from the bath, blown dry with air and then scanned. The process was repeated in the range from 20 to 80°C at 5°C increments.
• FIG. 2 provides the results for the four parameter values. Theoretically a should not go below zero, but for SA and TBS at one " UV it did. For b, the biggest difference is seen in the 1" UV time. The results with b indicate that increasing UV time leads to films with lower initial values; however, regardless of the initial R value, the "direct" effect of SA binding is to convert all the films to approximately the same final R value. The results with c showed that in all cases SA binding inhibited the temperature-triggered transition relative to TBS, with the biggest difference for 1" UV.

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• 1995
  • 1995-09-12 WO PCT/US1995/011672 patent/WO1996008721A1/en not_active Application Discontinuation
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