Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form
Reexamination Certificate
1999-01-07
2001-01-23
Kulkosky, Peter F. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
C428S407000, C424S001530
Reexamination Certificate
active
06177088
ABSTRACT:
BACKGROUND
This invention relates to nanospheres with acid-functionalized polymeric outer shells and surfactant-surrounded probe ion cores, and to a method for making the nanospheres.
Immunoassay, used for detecting and quantitating organic compounds, or analytes, is a well-known analytical technique. In an immunoassay procedure, an excess quantity of an antibody known to complement a particular analyte, is exposed to a test sample containing the analyte. The analyte binds to the antibody, forming an antibody-analyte conjugate. The conjugate is isolated and measured spectroscopically or electrochemically, and the results are extrapolated to determine the level of analyte in the material tested. In a conventional immunoassay procedure, the antibody or the analyte—the host—is labeled with a dye or with enzymes, which react with other reagents to produce colored products. The dye or colored product make it easier to detect and measure the amount of conjugate formed.
Although immunoassay is a highly sensitive means for detecting organic compounds in complex mixtures, such as those found in medical, biochemical, and environmental applications, the immunoassay technique has some serious shortcomings. For example, the sensitivity is often less than desired, meaning analytes present at very low concentrations may not be detected; and the coefficient of variability between replicates is relatively high, so it can be difficult to accurately determine the concentration of the analyte present in a particular sample. The dye-labeled antibodies or analytes, in particular, suffer from low sensitivity, because only a limited number of dye molecules can be coordinated with a host without inactivating the host. With enzyme-labeled antibodies or analytes, the sensitivity is increased because a single enzyme associated with a host can essentially produce an unlimited number of colored product molecules, but the reproducibility suffers because the rate of reaction between the enzyme and the other reagents, to produce the colored product molecules, is highly dependent on reaction time and temperature.
The immunoassay technique is also not very effective for analyzing solid samples, such as soils, because the analytes typically must be extracted from the sample before the immunoassay is performed, a time-consuming and waste-generating procedure. The extraction procedure also causes the analyte to be diluted, making detection more difficult. Further, the analyte must be known a priori, limiting the procedure to one, pre-selected contaminant.
The sensitivity is increased, and the reproducibility is improved, when the signal generated from the antibody-analyte conjugate is amplified, such as with the fluorescent rare earth ions, terbium(III) and europium(III), referred to as probe ions. Single probe ions are linked to antibodies using molecules of bifunctional chelating reagents, such as 1-(p-benzenediazonium)-EDTA or 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid. However, the number of molecules of chelating reagents that can coordinate to a particular antibody is limited, with the average ratio of chelating reagent molecules linked to antibodies being less than 10. At this ratio, signal amplification from the probe ions is not strong enough to significantly alter the sensitivity of the procedure.
The patentee has previously shown that it is possible to concentrate terbium(III) ions by encapsulating them within a polystyrene, or latex, shell to form a bead having a diameter as small as 50 nm, and then to couple antibodies to the bead. Beads having diameters in the nanometer-scale range are generically referred to as nanospheres, and nanospheres with diameters of about 50 nm can contain up to about 40,000 terbium(III) ions. This high concentration of probe ions can significantly improve the sensitivity and reproducibility for immunoassay. However, coupling antibodies to the probe-containing nanospheres can be problematic. For example, bead surfaces have been functionalized with methacrylic acid groups only, and with amine plus methacrylic acid groups, in an attempt to facilitate the nanosphere-antibody coupling reaction. (See “Functionalized, Probe-Containing, Latex Nanospheres”,
Anialytical Biochemistry
, 207, 241 (1992), and “The Synthesis of Surface-Functionalized Probe-Containing Nanospheres for Bioanalysis”, C.-Y. Guo Dissertation, University of Missouri-Columbia (April, 1991), both articles incorporated herein by reference.) Antibodies coupled with the methacrylic acid functionalized nanospheres very poorly: either the antibodies did not couple with the nanospheres at all, or so many antibodies became attached to a single nanosphere that the antibodies became stressed and essentially inactive. Nanospheres functionalized with amine and methacrylic acid groups tended to conjugate more with each other than with antibodies, resulting in very low yields of nanosphere-antibody conjugates.
In addition, a nanosphere having a diameter of about 50 nm is relatively large as compared to an antibody, so when the nanosphere is conjugated to the antibody, the mobility of the conjugate is reduced as compared to an unconjugated antibody, increasing the time necessary for the nanosphere-antibody conjugate to contact and react with an analyte. However, producing nanospheres smaller than about 50 nm is extremely difficult because so many factors affect the nanosphere size, including how the probe ions are prepared prior to encapsulation within the polymer shell, and how the polymer shell is formed.
SUMMARY OF THE INVENTION
The present invention relates to a series of nanospheres, having diameters of less than about 50 nm, and having probe ion cores surrounded by layers of surfactant and by a polymer shell. The surface of the shell is functionalized with acid compounds, at least one of which includes a spacer between the nanosphere surface and the acid functionality.
The surface-functionalized, probe-containing nanospheres can be coupled to antibodies, analytes, or other compounds of immunological significance and can be used, for example, in immunoassay analyses. The probe ion core improves the sensitivity and reproducibility of the immunoassay procedure. The acid compounds on the surface minimize self-agglomeration of the nanospheres, improving the degree of coupling with antibodies or other proteins. In addition, the spacer separates the acid functionality from the nanosphere surface, significantly improving the degree of coupling between the nanospheres and antibodies, providing greater flexibility to the antibody coupled to the nanosphere, and reducing the steric stress on the antibody when the antibody is conjugated to a single nanosphere at several places.
In some alternative embodiments, the surfaces are also functionalized with long-chain ester and alcohol groups. The ester and alcohol groups enhance the aqueous suspension characteristics of the nanospheres, thereby reducing the rate of precipitation of the nanospheres. Nanospheres functionalized with acids, esters, and alcohols have a longer shelf life, and produce nanosphere-antibody conjugates in higher yields, than nanospheres functionalized with acids alone.
DETAILED DESCRIPTION OF THE INVENTION
Structure of the Nanosphere
The nanosphere of the present invention is a nanometer-scale bead having a surfactant-surrounded probe ion core and an acid-functionalized polymer shell. The beads are essentially spherical, and have relatively uniform size distribution, ranging in diameter from about 10 nm to about 50 nm. Preferably, the beads have a nominal diameter of about 30±10 nm.
The probe ion core can be any nanometer-scale complex that can be detected spectroscopically with or without an imaging agent, and can be formed from an ionic salt that can be crystallized as a nanometer-scale crystal, or precipitated as a nanometer-scale particle, in an organic solvent. The ionic salt can be any water soluble inorganic anion or cation having a labile counterion. Some example ionic salts include dysprosium(III) chloride, europium(III) ch
Guo Congyuan
Thomas Rhys N.
Fayette Environmental Services, Inc.
Kulkosky Peter F.
Simunic Joan L.
Stites & Harbison
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