a-d show schematicsschematics for positive (
The present invention provides novel assays for use in drug discovery, bioagent detection and medical diagnostics. The present invention additionally provides assays based on cooperative self-assembly of complexes between biopolymers and fluorescent dyes. Such assays may be based on the formation, disruption and destruction of the complexes.
The unique optical properties of cyanine dyes have long held the interest of the scientific community. Cyanine dyes form several different types of aggregates characterized by spectral shifts from the emissions of the monomer. These spectral shifts may be formed depending on the molecular environment, concentration and physical state of the dye. Of the various aggregate types observered, the J-aggregates, characterized by very narrow intense absorption and narrow, only slightly red-shifted fluorescence relative to the monomer dye, have been the focus of most interest. Although J-aggregation has been observed in a variety of environments, it has previously been unclear which factors tip the balance in favor of J-aggregation for structurally similar families of cyanines.
Studies have shown that a series of polyelectrolytes with a variable number of cationic polymer repeat units constructed with cyanine chromophores pendant, but not conjugated, on a poly-L-lysine backbone, exhibit characteristic J-aggregate absorption and fluorescence in aqueous solution and when adsorbed onto anionic supports. For fluorescence of the polymers in solution, J-aggregate fluorescence became prominent for polymers having at least 33 cationic polymer repeat units. The fluorescence of the polymer is subject to very strong “super-quenching” by oppositely charged electron acceptors and energy-transfer quenchers that equals or exceeds that observed for conjugated polyelectrolytes by the same kind of quenchers. (Whitten et al., Pure Appl. Chem., Vol. 78, No. 12, pp. 2313-2323 (2006), incorporated by reference in its entirety.) Any cyanine dye capable of exhibiting J-aggregate fluorescence and absorption spectra when activated may be used in the assays of the present invention. In some embodiments, the ideal cyanine dye candidates would be ones where in aqueous solution there is no J-aggregate or only weak J-aggregate fluorescence but once complexed with a biomolecule the cyanine converts to J-aggregate and exhibits strong J-aggregate fluorescence. In exemplary embodiments, a cyanine dye of formula I, below, is used. This cyanine dye exhibits moderate water solubility and is nearly non-fluorescent in aqueous media.
According to various embodiments of the present invention, certain cyanine dyes and related chromophores, including but not limited to, cyanine dyes of Formula I, can form stable, tight complexes with chiral polymeric molecules in aqueous solutions resulting in the formation of intensely fluorescent J-aggregates. This molecular aggregation occurs via cooperative self assembly in which both the dye and the polymer undergo conformational changes to adopt a supramolecular helical structure. In some embodiments, a charged cyanine dye may associate with an uncharged or oppositely charge biopolymer. It may be desirable for chirality to be taken into account, for example, when detection methods such as induced circular dichroism are used.
Geometrics of nature have been the subject of intense interest to chemists, biologists, physicists and mathematicians. Molecular and supramolecular aggregates assemble into interesting geometries such as helices through non-convalent interactions. Chem-bio-helices play diverse roles in a variety of scientific disciplines including chemistry, biology and physics. These helices illustrate an ordered, 3-dimensional geometry present in natural and synthetic molecular entities such as deoxyribonucleic acid, ribonucleic acid and collagen. In addition, proteins and carbohydrates with helical structures participate in key biological processes such as signal transduction, cartilage formation, joint lubrication, and host-pathogen interactions. Helical structures are also prominent in chemistry, for example, the carbon nanotube.
Evidence supporting the cooperative self-assembly of cyanine dyes has been obtained from atomic force microscopy and circular dichroism spectroscopy. See, e.g. Kim, O.-K.; Je, J.; Jernigan, G.; Buckley, L.; Whitten, D. J. Amer. Chem. Soc. 128, 510-516 (2006), which is hereby incorporated by reference.
The assays formed by the methods herein may be used to detect both linear and helical biopolymers as well as non-polymeric small molecular weight analytes at a variety of concentrations, including, but not limited to, picomolar and femtomolar concentrations, and may be adapted to the format best suited for the detection of the analyte in question. In some embodiments, the assays herein use cyanine dyes to form chem-bio-helices with chem-biopolymers such as nucleic acids, proteins including helical and partially helical proteins, carbohydrates, linear and helical lipids, foldamers, linear chemical polymers and chemical helicates. (H. Gorner, A. K. Chibisov, T. D. Slavnova, J. Phys. Chem. B110:3917-3923 (2006); A. S. Tatikolov, I. G. Panova, High Energy Chem. 39: 232-236, (2005); and I. G. Panova, A. S. Tatikolov., Doklady Biol Sci. 402:183-185 (2005), each of which is incorporated by reference herein in its entirety), linear polymers (O-K. Kim, J. Je, J. W. Baldwin, S. Kooi, P. E. Pehrsson, L. J. Buckley., J. Am. Chem. Soc. 125L4426-4427 (2003); O-K Kim, J. Je, G. Jernigan, L. Buckley, D. Whitten. J. Am. Chem. Soc. 128:510-516 (2006); and D. Whitten, Ok>k Kim, K. E. Achyuthan, “Cooperative Self-Assembly of Cyanines on Carboxymethylamylose and other Anionic Scaffolds” IUPAC Mtg., Kyoto, Japan, April, 2006; each of which is incorporated by reference herein in its entirety) and carbohydrates. Exemplary biopolymers include, but are not limited to, linear polysaccharides such as cellulose, including carboxymethylamylose (CMA) (O-K Kim, J. Je, G. Jernigan, L. Buckley, D. Whitten., J. Amer. Chem. Soc. 128, 510-516 (2006), incorporated by reference herein in its entirety) and carboxymethylcellulose (CMC), and glycosaminoglycans including hyaluronic acid (G. Weindl, M. Schaller, M. Schafer-Korting, G. C. Korting. Skin Pharmacol. Physiol. 17:207-213 (2004); T. Sagawa, H. Tobata, H. Ihara. Chem. Comm. 18:2090-2091 (2004); S. Arnott, A. K. Mitra. S. Raghunathan. J. Mol. Biol. 169:861-872 (1983), each of which is incorporated by reference herein in its entirety); DNA; RNA; and proteins or structures composed of proteins such as albumin (I. G. Panova, A. S. Tatikolov., Doklady Biol. Sci. 402:183-185 (2005), incorporated by reference herein in its entirety), Bacillus collagen-like antigen (S. Rety, S. Salamitou, I. Garcia-Verdugo, D. J. S. Holmes, F. Le Hegarat, R. Chaby, A. Lewit-Bentley, J. Bio. Chem. 280:43073-43078 (2005), incorporated by reference herein in its entirety), cell membranes (M. Reers, T. W. Smith, L. B. Chen, Biochemistry 30:4480-4486 (1991), incorporated by reference herein in its entirety), phospholipids bilayers K. L. Vedvik, H. C. Eliason, R. L. Hoffman, J. R. Gibson, K. R. Kupcho, R. L. Somberg, K. W. Vogel, Assay Drug Dev. Technol. 2:193-203 (2004), incorporated by reference herein in its entirety), liposomes (H. F. Gilbert, “Basic Concepts in Biochemistry”, McGraw-Hill, Inc., New York, pp. 81-108 (1992), incorporated by reference herein in its entirety), reverse micelles S. M. Andrade, S. M. B. Costa, Chem. Eur. J. 12:1046-1057 (2006), incorporated by reference herein in its entirety), gelatin (H. Gorner, A. K. Chibisov, T. D. Slavnova, J. Phys. Chem. B110:3917-3923 (2006), incorporated by reference herein in its entirety), and patho-physiologically vital proteins including, but not limited to, potassium channel displaying extensive helices (D. A. Doyle, J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, R. MacKinnon., Science 280:69-77 (1998), incorporated by reference herein in its entirety) or the Streptococcal phage-encoded virulence factor (N. L. Smity, E. J. Taylor, A-M Lindsay, S. J. Charnock, J. P. Turkenburg, E. J. Dodson, G. J. Davies, G. W. Black, Proc. Natl. Acad. Sci. USA 102:17652-17657 (2005), incorporated by reference herein in its entirety). The creation, destruction, and/or disruption of such biopolymer:cyanine dye helices may be used to determine the presence, absence and amount of the biopolymer or enzyme of interest.
Changes associated with the formation of J-aggregation and changes in fluorescence and emission spectra of cyanine dye complexes may be read by means well known to those of skill in the art. There are a number of commercially available UV and fluorescence readers capable of viewing and quantifying such changes. In some embodiments, ratiometric analyses of the changes will be used. Such analyses may increase the specificity of the assay.
Assays of the present invention utilizing both the absorption and emissive properties for the light and spectral changes associated with the formation of J-aggregates of cyanine dyes may take a variety of forms. Such assays may be of any type generally used by those skilled in the art including, but not limited to, direct assays; indirect assays such as sandwich assays; competitive assays; non-competitive assays; vertical (columnar) format assays; and lateral flow assays such as, but not limited to, lateral flow enzymatic reaction assays, lateral flow immunoassays, displacement assays, hybrid lateral flow enzymatic/immunoreaction assays, and lateral flow displacement assays. In some embodiments, the assays of the present invention may be used in high throughput screenings.
In some embodiments, assays may be conducted using solid phase supports. Such solid phase supports may include any solid phase or semi-porous surface known to those of skill in the art including, but not limited to, glass, polystyrene, polypropylene, nitrocellulose, latex, cellulose, agarose, clay, silica, dextran or other materials. Suitable forms of the solid phase supports include beads, microparticles, tubes, fabrics, plates, latex particles, magnetic particles, paper, dipsticks, nano-particles, coupons, or tickets, formed from or coated with these materials as well as alternative flow-based formats such as channels and multiplexed assays on patterned substrates and the like (D. M. Olive., Expert Rev. Proteomics 1:327-341 (2004); W. Miaomiao, G. L. Silva, B. A. Armitage, J. Am. Chem. Soc. 122:9977-9986 (2000); K. M. Sovenhazy, J. A. Bordelon, J. T. Petty, Nucleic Acids Res. 31;2561-2569 (2003); Robert M. Jones, Liangde Lu, Roger Helgeson, Troy S. Bergstedt, Duncan W. McBranch, and David G. Whitten, Proc. Natl. Acad. Sci. USA 98:14769-14772 (2001); R. Jones, T. Bergstedt, C. Buscher, D. McBranch, D. Whitten, Langmuir 17:2568 (2001) and L. Lu, R. M. Jones, D. McBranch, D. Whitten, Langmuir 18:7706-7713 (2002), each of which is incorporated by reference herein in its entirety). In some embodiments, the assay may test for a single analyte. In other embodiments, the assay may test for multiple analytes including 2, 3, 4, or more analytes In some embodiments, as many as 100 or more analytes may be used. For example, those of skill in the art will be familiar with assays available from Luminex Corporation (Austin, Tex.) that include dye-labeled microspheres capable of testing 100 different analytes within a single sample. A similar system could be used incorporating the systems and methods described herein.
In some embodiments, the analytes may be labeled by means other than or in addition to bonding with a cyanine dye. Such labeling may include any means of generating a detectable signal. Illustrative labeling includes, but is not limited to, chromogens; catalysts, both enzymatic and non-enzymatic; molecules having an enzymatically labile bond which upon enzymatic cleavage provides a compound that can be detected either directly or indirectly; labeling with magnetic particles and isotopic labeling. In other embodiments, FRET labeled reagents that either sensitize or quench cyanine aggregate fluorescence may be utilized. In still other embodiments, the analytes may be unlabeled removing the need to chemically modify either the substrate or the enzyme. The ability of the assays of the present invention to use substrates in their natural conformational states provides a greater degree of confidence regarding the validity of the data to mimic in vivo conditions.
The assays of the present invention may function in a biosynthetic or metabolic fashion increasing or decreasing fluorescence as well as shifting the absorption spectrum emitted by the cyanine dyes. In some embodiments, the assays of the present invention may function in a biosynthetic fashion such as scaffold formation of a cyanine dye:biopolymer helix. In other embodiments, the assays may be based on metabolic processes such as scaffold destruction or disruption of a cyanine dye:biopolymer helix. A schematic representation of a scaffold formation/scaffold destruction assay based on complex formation between a biopolymer and a dye is shown in
Assays of the present invention may indicate the presence or absence of an analyte as well as the amount of the analyte of interest based on changes in the fluorescence and/or absorption spectra or lack thereof emitted by a biopolymer:dye complex. For example, a biopolymer may form a chem-bio-helix with cyanine dye, resulting in a change in the fluorescence of the cyanine dye in comparison to the monomer and/or a shift in the absorption spectrum.
According to various embodiments, a wide variety of enzyme/biopolymer pairs may be used or detected by the assays of the present invention. Exemplary enzymes which may be used in or may be detected by the assays of the present invention may include, but are not limited to, amylases, hyaluronidases, DNases, RNases, helicases, gyrases, cellulases, lipases including phospholipases, various synthases (i.e. biosynthetic enzymes such as DNA polymerases) especially those that lead to biochemical synthesis of helices such as DNA, RNA, cellulose, amylose, or collagenases.
In one embodiment, cyanine dye may be combined with a carbohydrate such as, but not limited to, carboxymethylamylose (CMA). Such a combination results in the formation of a J-aggregate and thus a change of the absorption spectrum of the dye (
Scaffold destruction assays may also involve helicases. For example, in some embodiments cyanine dye may assemble on a DNA duplex. The addition of a sample containing a helicase may destroy the DNA scaffolding as shown in
In another embodiment, instead of a scaffold destruction assay, the assay may be a scaffold disruption assay as shown in
Assays of the present invention may be conducted by any means applicable, for example using a solid phase support. In some embodiments, one or more of the reagents may be pre-loaded on a solid phase support. For example, the solid phase support may contain cyanine dye, biopolymers, enzymes, cyanine dye: biopolymer complexes, or any combination thereof. In another embodiment, the solid phase support may contain one or more capture zones capable of binding cyanine dye, biopolymers, enzymes, biopolymer fragments, cyanine dye: biopolymers, analytes, or any combination thereof. Such capture zones may or may not contain additional labels capable of emitting a signal when a reaction is completed. In some embodiments, capture zones may include, but are not limited to, one or more reaction zones containing one or more bound antibody-enzyme conjugates or other recognition molecules bound to an enzyme that disrupts biopolymer:cyanine dye complexes, biopolymer:dye complexes, molecules specific for the biopolymer under investigation; one or more recognition zone containing molecules specific for biopolymers under investigation; one or more detection zones containing biopolymer:cyanine dye complexes; one or more test zones containing antibodies or other affinity ligands for intact biopolymer and/or biopolymer fragments, including but not limited to DNA strands, additional biopolymers capable of trapping cyanine dye; one or more control zones containing molecules capable of forming chem-bio helices with cyanine dye and/or producing the spectral changes associated with J-aggregate formation such as, but not limited to, clay nanoparticles, DNA or RNA duplexes, or biopolymers such as, but not limited to, linear polysaccharides such as cellulose, including carboxymethylamylose (CMA) and carboxymethylcellulose (CMC), and glycosaminoglycans; and proteins or structures composed of proteins such as albumin, Bacillus collagen-like antigen, cell membranes, phospholipids bilayers, liposomes, reverse micelles and gelatin, or any other appropriate molecules; as well as any other applicable capture zones. Antibodies, affinity ligands, biopolymers, cyanine dye or recognition molecules in the capture zones may be adsorbed, covalently linked, conjugated via ligand protein association or other non-covalent linkage, trapped or otherwise attached to or associated with the solid phase support. It is known to functionalize cyanine, accordingly it should be possible to biotinylate, cyanine. Biotinylated cyanine may be immobilized via streptavidin-biotin interactions. For example, in some embodiments, the molecules may be isolated in a capture zone using a size permeation filter. In another embodiment, an antibody may be adsorbed to the solid phase supports. In other embodiments, the antibodies, antibody-enzyme conjugates, affinity ligands, recognition molecules, or recognition molecule-enzyme conjugates may be mobile. Signals including, but not limited to, shifts in J-aggregate emissions, may be measured at some or all of the capture zones.
In one embodiment, each reagent may be introduced individually to the solid support. In a further embodiment, combinations of reagents may be introduced to the solid support, for example a sample may be combined with cyanine dye. Alternatively, a sample may be combined with a cyanine dye:biopolymer complex before being introduced to the solid support.
In one embodiment, the capture zone on a solid phase support may be a reaction zone. The reaction zone may contain antibodies or other recognition molecules specific for the analyte to be detected. As seen in
In various embodiments, sandwich-based assays such as that shown schematically in
In another embodiment, the assay may be a lateral flow enzymatic reaction (LFER) assay. An exemplary embodiment of a LFER assay is shown in
In one embodiment, once the enzyme/polymer reaction mixture has been introduced to the solid phase support, cyanine dye may be added and allowed to travel along the solid phase support, binding to the biopolymer in the capture zones. The difference in the light absorption and emission intensities between the capture zones is an index of the enzymatic activity in the sample. In some embodiments, the difference in the light absorption and emission intensities may be used to determine the presence or absence of the analyte of interest. In other embodiments, the difference in the light emission intensities may be used to determine the amount present of the analyte of interest.
In one embodiment, the LFER assay may additionally include a capture zone such as a reaction zone containing antibodies or other affinity ligands to biopolymer:cyanine dye complexes. An embodiment of this assay is shown in
In some embodiments, the LFER assay may include multiple test zones that contain antibodies or recognition elements to bind either or both the biopolymer fragments and the liberated cyanine dye. In one embodiment, the test zone may contain a second biopolymer that serves to capture a portion of the liberated cyanine. The remaining liberated cyanine may then bind to other cyanine binding entities in the control zone. A positive test result may be indicated by the formation of J-aggregates in the test and control zones. In a further embodiment, the test and control zones may contain recognition elements that bind to the biopolymer fragments. Such elements may contain one or more labels that are activated when the biopolymer fragments are bound. The activation of such markers in the test and control zone would indicate a positive test result. In additional embodiments, the test zone and/or the control zone may contain antibodies or affinity ligands to either biopolymers or cyanine dye. In yet another embodiment, the LFER assay may contain a reaction zone and either a test zone or a control zone.
Assays may additionally comprise hybrid lateral flow enzymatic/immunoreaction (LFEIR) assays. An example of such an assay is shown in
In one embodiment, as shown in
In some embodiments, the assays may be based on biosynthetic processes or scaffold formation. In scaffold formation assays, cyanine dyes are prevented from forming cyanine dye:biopolymer complexes that create J-aggregates. The addition of a compound that allows the cyanine dye:biopolymer complexes to form J-aggregates, creating a signal that can be measured. Such signals may reveal the presence and quantity of analytes of interest.
For example, the cyanine may be prevented from forming chem-bio-helices with a product by tethering the cyanine via covalent bonds to a suitable substrate as shown in
Appropriate tethering compounds include any compound that forms a bond with cyanine that may be cleaved by hydrolytic enzymes such as, but not limited to, lipases, glycosidases, phosphatases, sialidases, caspases, influenza viral neuraminidases, proteases or peptidases. Tethering compounds may include, but are not limited to, lipases, proteins, DEVD, glycoside, sialic acid, or 4,7-Di-O-Me-N-Acetyl-Neuraminic Acid. Such compounds may form tethered substrates such as DEVD-cyanine, protein-cyanine, glycoside-O-cyanine, sialic acid-o-cyanine and 4,7-Di-O-Me-N-Acetyl-Neuraminic Acid-O-cyanine
In some embodiments, the tethered biopolymer-dye complex, and enzyme (analyte) may be pre-loaded on a solid phase surface. In another embodiment, the tethered biopolymer-dye complex may be pre-loaded on a solid phase surface. In a further embodiment, the analyte may be preloaded on a solid phase surface.
In another embodiment, the cyanine dye 90 may be encapsulated as shown at 92 in
For example, cyanine may be incorporated in vesicles composed of a phospholipid such as 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). Cyanine dye will assemble on the bilayer walls of the vesicles composed of DPPC and result in fluorescence. Disruption of the vesicle by the action of an appropriate phospholipase or a detergent such as Triton X-100 will result in disaggregation and optical activity being switched off.
In another example, cyanine dyes may be incorporated inside hydrogels that may be proteolytically degraded resulting in the liberation of the “free” cyanine which then assembles upon a suitable chem-bio-polymer resulting in strong fluorescence emission signaling the proteolytic event. An example of a patho-physiologically important protease family is the matrix metalloproteinases (MMPs) such as collagenases, stromelysins, matrilysins and gelatinases. The MMPs play an important role in tissue remodeling associated with various physiological and pathological processes such as morphogenesis, angiogenesis, tissue repair, cirrhosis, arthritis and metastasis. MMP-2 and MMP-9 are thought to be important in metastasis. MMP-1 is thought to be important in rheumatoid and osteo-arthritis.
A third example might be the encapsulation of the cyanine dye inside enzymically-degradable dendrimers. Dendrimers assembled with certain polyesters may be degraded by enzymes such as poly(3-hydroxybutyrate)-depolymerase or a catalytic antibody such as 38C2 catalyzing a retro-aldol process upon dendritic modified aliphatic polyesters. The degraded dendrimer then releases the trapped cyanine which can then assemble upon a suitable chem-bio-polymer/helical scaffold resulting in fluorescence turning “on” signaling the catalytic event of the enzyme or the antibody. Even non-catalytic events may be detected via the release of trapped or encapsulated cyanine. An example is the release of cyanine trapped within a photocleavable liposome following exposure to ultra-violet light.
Another example of a non-catalytic degradation of encapsulated cyanines might be the lability of vesicles under the influence of a pH change. For example, vesicles might be synthesized that are stable at low pH (such as those encountered in the gastric juice of the digestive system) but then disintegrate when the pH is raised to neutral. Such pH shifts might release the trapped/encapsulated cyanine dye which then can assemble upon chem-bio scaffolds resulting in an optical switch being turned “on” that signals the pH shift event. Biocompatible polymeric nanoparticles and molecular bio-capsules may also be prepared that encapsulate the cyanine and are released under a physiological or pahtological trigger such as apoptosis.
The released cyanine 92 may then assemble into helices with the appropriate biopolymers 94, resulting in the formation of J-aggregates 96, an increase in fluorescence and a shift in the absorption spectra.
In a further embodiment, scaffold formation may take place using polymerase as shown in
Scaffold formation assays may take any form applicable. An exemplary assay using tethered or otherwise isolated cyanine dye is shown in
In another embodiment, the scaffold formation assay may be a Lateral Flow Immunoassay (LFIA) as shown in
In some embodiments, the LFIA may be conducted as a sandwich assay in which a second reagent is added to the solid phase support after the test sample is introduced. In the sandwich assay, there may be one or more capture zones including at least one test zone containing antibodies or recognition molecules specific for the analyte of interest and one or more control zones containing molecules capable of forming chem-bio helices with cyanine dye and/or producing the spectral changes associated with J-aggregate formation.
The sample containing the analyte of interest is introduced to the solid phase support and binds to the test zone. A second reagent containing a biopolymer capable of binding to cyanine dye and forming J-aggregates and capable of binding to the analyte of interest or coupled to another molecule capable of binding the analyte of interest is then introduced to the solid phase support. Once the second biopolymer has bound to the captured biopolymer of interest, cyanine dye may be introduced. If the analyte of interest is present, the cyanine dye will bind to both the biopolymer attached to the analyte of interest and the biopolymer in the control zone yielding a positive result. This permits the detection of both helical biopolymers and non-helical or other small molecular mass analytes that do not bind to cyanine or cause J-aggregation.
In another embodiment, the LFIA assay may be a displacement assay. In a standard displacement assay, a sample flows through a membrane having binding elements with binding sites saturated with a labeled form of the analyte. The analyte in the sample displaces, under non-equilibrium conditions, the labeled form of the analyte from the membrane. The displaced labeled form of the analyte may then be detected. In the present invention, it may not be necessary to use a labeled form of the analyte though labeled forms may also be used. For example, a biopolymer that reacts with cyanine dye may be weakly bound to a solid phase support in an additional capture zone such as a binding zone. A sample potentially containing a biopolymer of interest may then be added to the solid phase support. The biopolymer of interest displaces the weakly bound biopolymer, washing it downstream to a second capture zone where it may be immobilized by an antibody or other recognition molecule. The addition of cyanine dye to the solid phase support may generate J-aggregation fluorescence in the binding zone, the second capture zone or a control zone. Emissions in the second capture zone and control zone indicate a positive test for the analyte of interest. Emissions in only the control zone would indicate a negative test result.
Assays may further comprise lateral flow displacement assays (LFDA). An exemplary LFDA is shown in
If the biopolymer of interest is present in the sample, it will displace the biopolymer or biopolymer:cyanine dye complex from the recognition zone. The displaced biopolymer or biopolymer:cyanine dye complex then travels downstream to a second capture zone, the test zone. Cyanine dye may then be added to the solid support. If the original antibody captured molecule already has a cyanine dye, the strip 118 will be colored before the test and the color will be depleted as the cyanine-biopolymer-complex is “displaced” by the analyte. If there is no dye in the system to begin with, dye may be captured at all three sites (118, 120 and 122) depending on the affinity of the bound analyte complex for the cyanine. Accordingly, the recognition zone might produce characteristic emissions but it also may be tuned so that it does not.
In a further embodiment, the assay may be a lateral flow without PCR (LFWP) assay. An exemplary LFWP is shown in
If the sample contains the target DNA, it hybridizes with the immobilized DNA strands. Addition of cyanine dye to the solid phase support will result in the cyanine dye intercalcating with the duplex DNA in the test zone to produce emission and absorption spectral changes. In the absence of the DNA of interest, only the control zone will produce the expected emission and absorption changes.
In some embodiments, biotinylated oligonucleotides may be added to the solid phase support prior to the addition of the cyanine dye. These oligonucleotides have short DNA sequences that are complementary to regions of the target DNA. After the oligonucleotides have bound to the captured DNA, streptavidin or other biotin binding fragment conjugated biopolymer is added to the solid phase support. The addition of cyanine dye to the complementary-DNA-target-DNA-oligonucleotide-biotin-streptavidin-biopolymer complex will produce the characteristic absorption and fluorescence changes indicative of the formation of J-aggregates in the test zone indicating the presence of the DNA of interest.
Changes in fluorescence and emission spectra in the assays described above may be read by any appropriate commercially available reader or scanner as well as specifically designed readers or scanners. In one embodiment, such units may be bench-top or portable field instruments with an optical source, lamp and other electronics to facilitate measurements of light absorption and fluorescence emission. Such readers or scanners may be powered by any means applicable including direct current, wall-plug or batteries. In one embodiment, the reader or scanner used may be capable of exciting radiation from ultra-violet (UV) through the entire visible light spectra. For example, such a reader will be capable of exciting radiation in a range of about 220 nm to 750 nm, more specifically from about 220 nm to 700 nm, 300 to 600 nm, or from about 350 to 540 nm. In another embodiment, the reader or scanner used may be capable of detecting fluorescence or luminescence emission light wavelengths over the ultraviolet through visible light spectrum. For example, such a reader will be capable of detecting fluorescence in a range of about 220 nm to 750 nm, more specifically from about 220 nm to 700 nm, 300 to 600 nm, or from about 350 to 540 nm.
The electronic reader or scanner may be able to read a variety of assay platforms including, but not limited to, beads, microparticles, tubes, fabrics, plates, latex particles, magnetic particles, paper, dipsticks, nano-particles, coupons, or tickets, formed from or coated with these materials as well as alternative flow-based formats such as channels and multiplexed assays on patterned substrates and the like and will have slots of the appropriate shape and size to accommodate such platforms.
In some embodiments, the electronic reader or scanner may be capable of measuring changes intensities of light absorption or fluorescence emission as well as wavelengths (spectral) shifts. Such measurements may be outputted as raw data, or relative comparisons such as current strengths of light signals, relative fluorescence units, or counts per second, or any other appropriate output units.
In other embodiments, the electronic reader or scanner may be integrated either through hardwiring or wirelessly to remote stations where the data may be downloaded and analyzed. Appropriate software may be used to process the data and/or to facilitate minimal intervention with the instrument on the part of the processor. In some embodiments, the electronic reader or scanner may have charting or graphing capabilities as well as visual and audible indications of test results.
The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims.
As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “a channel” includes a plurality (for example, a culture or population) of such channels, and so forth.
The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also forms part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.
The examples below provide evidence of novel label-free screening assays using cyanine dyes in order to identify enzyme activity and specific biopolymers. Such assays may be used in either a “turn on” or “turn off” mode increasing or decreasing the emissions spectra of the cyanine dyes.
Human saliva was collected from a volunteer and then centrifuged at 14000 rpm (16000×g) for one minute at ambient temperature (25° C.), using a Galaxy 16D microcentrifuge (VWR International, West Chester, Pa.). The resulting supernatant was collected and re-centrifuged as described above. The supernatant from the second centrifugation was centrifuged again as described above and the supernatant from the third centrifugation was used as the source of amylase.
75 μM of carboxymethylamylose (CMA) was mixed with 5 μL of saliva in a total volume of 60 μL water and incubated at ambient temperature (25° C.). At 0, 10, 20, 30, 40, 50, and 60 minutes, one half of the reaction volume was removed and added to 270 μL of a 7.5 μM solution of cyanine of Formula I dissolved in 20% methanol-80% water mixture and dispensed into the wells of a 96 well microplate (Optiplate™-96, Perkin Elmer Life Sciences, Boston, Mass.). Fluorescence from the resulting J-aggregate was then immediately measured using a Mithras LB 940 microplate spectrofluorometer (Berthold Instruments, Oak Ridge, Tenn.). As can be seen in
75 μM of carboxymethylamylose (CMA) was mixed with 0, 1, 2, 3, 4, 5 and 6 μL of saliva in a total volume of 60 μL water and incubated at ambient temperature (˜25° C.). After 60 minutes, one half of the reaction volume for each respective combination was removed and added to 270 μL of 7.5 μM cyanine dye of Formula I dissolved in 20% methanol-80% water mixture dispensed into the wells of a 96 well microplate (Optiplate™-96, Perkin Elmer Life Sciences, Boston, Mass.). Fluorescence from the formation of J-aggregates in the sample was then immediately measured using a Mithras LB 940 microplate spectrofluorometer (Berthold Instruments, Oak Ridge, Tenn.). As can be seen in
Carboxymethylcellulose (CMC) in 0, 20, 40, 60, 80 and 100 μM concentrations was added to the wells of a 96-well microplate containing 10 μM of cyanine dissolved in 20% methanol-80% water mixture and was dispensed into the wells of a 96 well microplate (Optiplate™-96, Perkin Elmer Life Sciences, Boston, Mass.) to a total volume of 275 μL. Fluorescence was immediately measured using a Mithras LB 940 microplate spectrofluorometer (Berthold Instruments, Oak Ridge, Tenn.). As can be seen in
Cyanine dye of Formula I dissolved in 20% methanol-80% water was added to wells of a 96-well microplate (Berthold Instruments, Oak Ridge, Tenn.) in concentrations of 0, 20, 40, 60, 80 and 100 μM. Then, either 10, 20, or 40 μM of carboxymethylcellulose was added to the various concentrations of cyanine dye to reach a total volume in each microwell of 275 μL. Fluorescence emission was measured immediately after the addition of the carbodymethylcellulose (CMC) using a Mithras LB 940 microplate spectrofluorometer (Berthold Instruments, Oak Ridge, Tenn.). As can be seen in
Hyaluronic acid in concentrations of 1, 10, 100, 1000 or 10,000 ng was added to 20 μM concentrations of cyanine dye of Formula I dissolved in a 20% methanol-80% water mixture and dispensed into the wells of a 96 well microplate (Optiplate™-96, Perkin Elmer Life Sciences, Boston, Mass.) for a total volume of 300 μL. Fluorescence emission was measured after 60 minutes of incubation at 25° C. using a Mithras LB 940 microplate spectrofluorometer (Berthold Instruments, Oak Ridge, Tenn.). As can be seen in
Concentrations of 0, 20, 40, 80 and 100 μM cyanine dye cyanine dissolved in 20% methanol-80% water mixture was dispensed into the wells of a 96 well microplate (Optiplate™-96, Perkin Elmer Life Sciences, Boston, Mass.). Then, 1.0 μg of hyaluronic acid was added to each well. The total volume inside each microwell was 275 μL. Fluorescence was measured 60 minutes after the addition of hyaluronic acid using a Mithras LB 940 microplate spectrofluorometer (Berthold Instruments, Oak Ridge, Tenn.). As can be seen in
Hyaluronidase enzyme (11.3 U of E.C. 3.2.1.35) was mixed with 8 μg of hyaluronic acid in a total volume of 500 μL at 25° C. After 0, 20, 40, 60, 80, 100, 120 and 140 minutes, a portion of the reaction mixture was diluted into 20 μM concentrations of cyanine dye of Formula I dissolved in 20% methanol-80% water mixture dispensed into the wells of a 96 well microplate (Optiplate™-96, Perkin Elmer Life Sciences, Boston, Mass.) to a total volume of 275 μL. Sixty minutes after adding the reaction mixture to the cyanine solution, the fluorescence was measured using a Mithras LB 940 microplate spectrofluorometer (Berthold Instruments, Oak Ridge, Tenn.).
The enzymatic reaction volume that was added to the cyanine dye solution represented approximately 364 ng of intact hyaluronic acid that might be present in the complete absence of enzymatic hydrolysis. This amount of hyaluronic was equivalent to the mid-point in the rise of the hyaluronic titration curve in
Varying concentrations of hyaluronidase enzyme of 2, 3, 4, 5, 6, 7, 8, 9, and 10 U respectively were mixed with 10 μg of hyaluronic acid in a total reaction volume of 500 μl. After 180 minutes at 25° C., a portion of the reaction mixture was diluted into 20 μM concentrations of cyanine dye of Formula I dissolved in 20% methanol-80% water mixture and was dispensed into the wells of a 96 well microplate (Optiplate™-96, Perkin Elmer Life Sciences, Boston, Mass.) to a total volume of 275 μL.
Fluorescence was measured 60 minutes after the addition of the enzymatic reaction mixture to the cyanine dye solution using a Mithras LB 940 microplate spectrofluorometer (Berthold Instruments, Oak Ridge, Tenn.). As can be seen in
Hyaluronic acid in amounts of 0, 200, 400, 600, 800 and 1000 ng respectively were mixed with 1 U of hyaluronidase in a total volume of 100 μL. After allowing the enzymatic reaction to proceed for 240 minutes at 25° C., a portion of the reaction mixture equivalent to approximately 364 ng of intact HA that might be present in the complete absence of enzymatic hydrolysis was added to the wells of a 96 well microplate (Optiplate™-96, Perkin Elmer Life Sciences, Boston, Mass.) containing 20 μM concentrations of cyanine dye of Formula I dissolved in a 20% methanol-80% water mixture to a total volume of 275 μL.
The dose response behavior of the hyaluronidase activity to increasing concentration of the hyaluronic acid substrate is shown in
Although the foregoing invention has been described in detail by way of example for purposes of clarity of understanding, it will be apparent to the artisan that certain changes and modifications may be practiced within the scope of the appended claims which are presented by way of illustration not limitation. In this context it will be understood that this invention is not limited to the particular formulations, process steps, and materials disclosed herein as such formulations, process steps, and materials may vary somewhat. It will also be understood that the terminology employed herein is used for the purpose of describing particular embodiments only, and is not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof. It is further noted that various publications and other reference information have been cited within the foregoing disclosure and listed in the reference section below for economy of description. Each of these references are incorporated herein by reference in its entirety for all purposes. It is noted, however, that the various publications discussed herein are incorporated solely for their disclosure prior to the filing date of the present application, and the inventors reserve the right to antedate such disclosure by virtue of prior invention.
The following application claims benefit of U.S. Provisional Application No. 60/839,001 filed Aug. 21, 2006 and U.S. Provisional Application No. 60/843,171 filed Sep. 8, 2006, each of which is hereby incorporated by reference in its entirety.
Aspects of this work were supported by grant no. CT 503323315 from the National Science Foundation. The United States Government has certain rights in the subject matter.
Number | Date | Country | |
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60839001 | Aug 2006 | US | |
60843171 | Sep 2006 | US |