1. Field of the Invention
The present invention concerns dendritic polymer chemiluminescent substrate conjugates; synthesis of dendritic polymer chemiluminescent substrate conjugates; compositions containing dendritic polymer chemiluminescent substrate conjugates; and methods of using dendritic polymer chemiluminescent substrate conjugates. The invention also concerns the use of enhancer substances in combination with the dendritic polymer chemiluminescent substrate conjugates. The dendritic polymer chemiluminescent substrate conjugates and enhancer dendritic polymer chemiluminescent substrate conjugates, which amplify the detectable chemiluminescent signal by coupling multiple (i.e., from 3 to 3072) enzyme activable chemiluminescent substrates, are useful in detecting the presence or determining the concentration of chemical or biological substances in immunoassays, chemical assays and nucleic acid probe assays, and in chemical/physical probe procedures for studying the microstructures of macromolecules, including synthetic polymers, proteins, nucleic acids and the like.
2. Background of the Technology
Dendritic polymers (otherwise known as “dendrimers”) are uniform polymers, variously referred to in the literature as hyperbranched dendrimers, arborols, fractal polymers and starburst dendrimers, having a central core, an interior dendritic (hyperbranched) structure and an exterior surface with end groups. These polymers differ from the classical linear polymers both in form and function. Linear polymer chemistry results from chaotic, uncontrolled processes that yield a distribution of product species from microscopic to macroscopic size. The molecular linearity of the polymers produces a heavily entangled macromolecular population that defines macroscopic behavior such as sharply increasing viscosity with increased molecular weight, mediocre chemical reactivity due to hidden sites in random coils and variable or mediocre solubility. In contrast, dendrimer chemistry constructs macromolecules with tight control of size, shape topology, flexibility and surface groups. In what is known as divergent synthesis, these macromolecules start by reacting an initiator core in high-yield iterative reaction sequences to build symmetrical branches radiating from the core with well-defined surface groups. Alternatively, in what is known as convergent synthesis, dendritic wedges are constructed from the periphery inwards towards a focal point and then several dendritic wedges are coupled at the focal points with a polyfunctional core. Dendritic syntheses form concentric layers, known as generations, with each generation doubling the molecular mass and the number of reactive groups at the branch ends so that the end generation dendrimer is a highly pure, uniform monodisperse macromolecule that solubilizes readily over a range of conditions. For the reasons discussed below, dendrimer molecular weights range from 300 to 700,000 daltons and the number of surface groups (e.g., reactive sites for coupling) range from 3 to 3072.
As dendrimers grow with each generation, the steric constraints from congestion of the branches force the polymer shape to change from a starfish-shaped molecule to a globular molecule. For example, with starburst polyamidoamine (“PAMAM”) dendrimers, generations 0-3 are dome-shaped, generation 4 is a transition generation with an oblate spheriod shape, and generations 5 and greater are symmetrically spherical with a hollow interior and a surface skin. This change of shape, from domes to spheres, with increasing size (caused by increasing surface congestion at the branch ends) is a general feature of dendritic polymers.
Dendritic growth, shape and topology are controlled by the core, the interior branch structure and the surface groups. Dendrimers expand symmetrically in a way that maintains a constant terminal surface group area. For example, with starburst dendrimers the surface group —CO2Me requires 93 A2/group whereas the surface group —NH2 requires 150 A2/group. Terminal groups with greater steric bulk, such as NH2, promote greater core hollowness by surface steric interaction as observed with the PAMAMs. In contrast, polyether starburst dendrimers become congested within 3 generations with very little internal cavity. In general, dendritic growth becomes self-limiting as steric congestion of the surface reactive sites precludes further chemical modification; for PAMAM starburst dendrimers this occurs at generations 9-10.
As discussed herein, dendritic surfaces can have from 3 to 3072 end groups available for surface chemistry; the number of end groups depending on the type of dendrimer structure (which defines steric congestion) and the dendrimer generation. Amino (NH2) terminated dendrimers react with, e.g., Michael acceptors (CH2═CHCO2R), α-haloesters, epoxides, aziridines, activated carboxylic acids, acid chlorides, benzyl halides, carbonates and aldehydes. Hydroxyl (OH) terminated dendrimers react with, e.g., halosulfonic esters, activated carboxylic acids and acid chlorides. Ester and acid (CO2R, CO2H) terminated dendrimers react with, e.g., amines and halide terminated dendrimers react with, e.g., amines and alkoxide and thioalkoxide anions. Other reactive surface groups include carboxyhalide, imino, imido, alkylamino, dialkylamino, alkylarylamino, cyano, sulfonic esters, dithiopyridyl and sulfhydryl, among others. Chemical reactions on the dendrimer surface usually occur as readily as with single organic molecules, with high specificity and high yields, as long as there is no steric congestion on the surface. Structures of commercially available dendrimers, which include the Starburst PAMAM dendrimers (NH2-terminated, OH-terminated and CO2R-terminated) and Astramol PEI dendrimers (NH2-terminated), which are shown in FIGS. 1A to 1D are available from Aldrich Chemical Co., Dendritech Inc. and DSM Fine Chemicals. In addition, many other dendritic polymers with reactive surface groups have been synthesized and reported in the literature (for a review, see, for example, “Dendritic Molecules: Concepts, Syntheses, Perspectives,” G. R. Newkome, C. N. Moorefield, F. Vogtle, VCH Publishers, Inc. New York (1996) and references cited at the end of this application).
In recent years, it has been found that the size, shape and properties of dendritic polymers can be molecularly tailored to meet specialized end uses. Dendritic polymers thus have significant advantages which can provide a means for the delivery of high concentrations of carried material per unit of polymer, controlled delivery, targeted delivery and/or multiple species delivery or use.
U.S. Pat. Nos. 5,338,532; 5,527,524 and 5,714,166 disclose dense star polymers or starburst polymers associated with a variety of materials, including drugs, toxins, metal ions, radionuclides, signal generators, signal reflectors, chelated metal, signal absorbers, antibodies, hormones, biological response modifiers, diagnostic opacifiers, fluorescent moieties and scavenging agents; processes for preparing the conjugates; compositions containing the conjugates; and methods of using the conjugates.
U.S. Pat. No. 5,482,698 discloses methods for detecting or treating lesions that includes a polymer comprising multiple avidin or biotin binding sites; adding a detection or therapeutic agent to the avidin or biotin; and detecting or treating the lesion.
U.S. Pat. Nos. 5,443,953 and 5,635,603 disclose a soluble immunoconjugate that includes a glycosylated antibody fragment, and an intermediate conjugate comprising a polymer carrier having at least one free amine group and a detectable label molecule covalently bound to the polymer carrier.
None of the references cited above disclose the association of dendritic polymers with chemiluminescent substrates.
1,2-dioxetane enzyme substrates have been well established as highly efficient chemiluminescent reporter molecules for use in enzyme assays of a wide variety of types. These assays provide a preferred alternative to conventional assays that rely on radioisotopes, fluorophores, complicated color shifting, secondary reactions and the like. Dioxetanes developed for this purpose include those disclosed in U.S. Pat. Nos. 4,978,614; 5,112,960; 5,538,847 and 5,582,980, as well as U.S. application Ser. No. 09/362,047 (pending). U.S. Pat. No. 4,978,614 discloses, among others, 3-(2′-spiroadamantane)4-methoxy-4-(3″-phosphoryloxy)phenyl-1,2-dioxetane, which is commercially available from Applied Biosystems under the trade name AMPPD®, which is a registered trademark of PE Corporation (NY). U.S. Pat. Nos. 5,112,960; 5,538,847 and 5,582,980 disclose similar compounds, wherein the adamantyl stabilizing ring is substituted, at either bridgehead position, with a variety of substituents, including hydroxy, halogen, and the like, which convert the otherwise static or passive adamantyl stabilizing group into an active group involved in the kinetics of decomposition of the dioxetane ring. Compounds of this type give a faster and stronger signal than AMPPD® in many applications. CSPD®, which is a registered trademark of PE Corporation (NY), is a second-generation dioxetane with a chlorine substituent on the adamantyl group. This material is also available from Applied Biosystems. CSPD® gives improved light intensity and detection sensitivity. U.S. application Ser. No. 09/362,047 discloses enzymatically cleavable chemiluminescent 1,2-dioxetanes that emit in wavelengths close to the red or green end of the visible spectrum. Each of the patents and applications cited in this paragraph are incorporated herein by reference in their entirety.
Reactions that produce chemiluminescence exemplify yet another instance in which the medium, although not the message, can determine the intensity of the message transmitted. Chemiluminescent compounds that, upon decomposition in substances such as moderately polar or polar aprotic organic solvents, e.g., n-butanol, acetonitrile, dimethylsulfoxide or dimethylformamide, produce fluorophores that in turn emit light of adequate intensity for easy detection and quantitation, will produce light of considerably lessened intensity when decomposed in a polar protic environment, and especially in aqueous media. But since all biological systems are aqueous—indeed, humans are approximately 97% water—the need to enhance the intensity of light produced by chemiluminescent labels or substrates in immunoassays, nucleic acid probe assays, chemical/physical probe techniques and other bioassays is obvious. One way to provide such enhancement is to use expensive optical or electronic equipment: single photon counters, luminometers, scintillation counters, etc.
Although dioxetanes have been particularly developed for enhanced sensitivity in assays for the presence of analytes in concentrations as low as 10−12 M, in certain applications, dioxetanes are used in conjunction with enhancers to detect analytes in concentrations of 10−12 M or lower. These enhancement agents, which include natural and synthetic water-soluble macromolecules, are disclosed in detail in U.S. Pat. No. 5,145,772. Preferred enhancement agents include water-soluble polymeric quaternary ammonium, phosphonium or sulfonium salts, and copolymers and/or mixtures thereof, such as poly(vinylbenzyltrimethylammonium chloride) (TMQ), poly(vinylbenzyltributylammonium chloride) (TBQ) and poly(vinylbenzyldimethylbenzylammonium chloride) (BDMQ).
These enhancement agents improve the chemiluminescent signal of the dioxetane reporter molecules, apparently by providing a hydrophobic environment in which the dioxetane is sequestered. Water, unavoidable in most assays due to the use of body fluids, is a natural “quencher” of the dioxetane chemiluminescence. The enhancement molecules apparently exclude water from the microenvironment in which the dioxetane molecules, or at least the excited state emitter species reside, resulting in enhanced chemiluminescence. Other effects associated with the enhancer-dioxetane interaction could also contribute to the chemiluminescence enhancement.
In addition to dioxetane, luminol derivatives, acridinium esters, acridinium sulfonylamides and luciferin have been employed as chemiluminescent labels in bioassays (Schroeder et al., Anal. Chem., 50, 1114 (1978); Arakawa et al., Anal. Biochem., 79, 248 (1979); and Arakawa et al., Clin. Chem., 31, 430 (1985). For reviews, see: Kricka et al., Clinical and Biochemical Luminescence, L. J. Kricka and T. J. N. Carter (Eds.), Chapter 8, Marcel Dekker, Inc., New York (1982); Kricka, Ligand-Binder Assays, Chapter 7, Marcel Dekker, Inc., New York, (1985); McCapra et al., Bioluminescence and Chemiluminescence: Instruments and Applications, Vol. I, K. Van Dyke (Ed.), CRC Press, Inc., Boca Raton, Fla. (1985), Chapter 2 (note, in particular, the section on dioxetanes, page 13); and Barnard et al., Ibid, Ch. 7). The enzyme labels have been detected by color or fluorescence development techniques. More recently, chemiluminescent enzyme immunoassays have been based on peroxidase conjugates assayed with luminol/hydrogen peroxide, pyrogallol/hydrogen peroxide, Pholas dactylus luciferin, or luminol under alkaline conditions (Kricka et al., Clinical and Biochemical Luminescence, Chapter 8, L. J. Kricka and T. J. N. Carter (Eds.), Marcel Dekker, Inc., New York (1982)).
By way of background, the enzymatically-activated chemiluminescent substrates are used as reporter molecules by acting as substrates for enzymes which cleave an enzyme-labile group thereon. Thus, the enzyme (e.g., alkaline phosphatase) can be covalently linked or otherwise complexed with either an antigen or antibody, in conventional antigen/antibody ligand binding assays, or a nucleic acid probe in nucleic acid assays. The enzyme-bearing antigen or antibody, or nucleic acid probe, is then admixed with the analyte suspected of containing the target antigen, or nucleic acid sequence, under conditions which permit complexing or hybridization between the antigen/antibody or probe/nucleic acid sequence. After washing away or separating off all noncomplexed or nonhybridized material, the chemiluminescent substrate is added. If the suspected analyte is present, the enzyme will cleave the enzyme-labile group chemiluminescent substrate, yielding an intermediate that decomposes. The decomposition event is the light-releasing event.
To detect analytes in extremely low concentrations (below, e.g., about 10−12 M) it is desirable to improve the intensity of the signal of the chemiluminescent substrate reporter molecule, and it is simultaneously desirable to avoid increasing the background noise due to nonenzymatically-induced light release, so as to improve the overall sensitivity of the assay. Thus, further improvements in the use of chemiluminescent substrates are sought.
All patents and literature that are cited in this disclosure are incorporated herein by reference.