The development of sensitive nonisotopic detection systems for use in biological assays has significantly impacted many research and diagnostic areas, such as DNA sequencing, clinical diagnostic assays, and fundamental cellular and molecular biology protocols. Current nonisotopic detection methods are mainly based on organic reporter molecules that undergo enzyme-linked color changes or are fluorescent, luminescent, or electroactive (Kricka; Ed., Nonisotopic Probing, Blotting, and Sequencing, Academic Press, New York, 1995; Issac, Ed., Protocols for Nucleic Acid Analysis by Nonradioactive Probes, Humana, Totowa, N.J., 1994; and Diamandis and Christopoulos, Eds., Immunoassay, Academic Press, New York, 1996). While these nonisotopic systems solve the problems associated with radioisotopic detection, such as short half-lives of radioisotopes, health hazards and expensive removal of radioactive waste, they are not as sensitive or stable as nonisotopic detection systems that utilize luminescent semiconductor quantum dots. For example, highly luminescent semiconductor quantum dots, such as ZnS-capped CdSe quantum dots, are twenty times brighter, one hundred times more stable against photobleaching, and three times narrower in spectral line width than organic dyes, such as fluorescent rhodamine.
Over the past decade, much progress has been made in the synthesis and characterization of a wide variety of semiconductor quantum dots. Recent advances have led to large-scale preparation of relatively monodisperse quantum dots (Murray et al., J Am. Chem. Soc., 115, 8706–15 (1993); Bowen Katari et al., J Phys. Chem., 98, 4109–17 (1994); and Hines et al., J Phys. Chem., 100, 468–71 (1996)). Other advances have led to the characterization of quantum dot lattice structures (Henglein, Chem. Rev., 89, 1861–73 (1989); and Weller et al., Chem. Int. Ed. Engl. 32, 41–53(1993)) and also to the fabrication of quantum-dot arrays (Murray et al., Science, 270, 1335–38 (1995); Andres et al., Science, 273, 1690–93 (1996); Heath et al., J. Phys. Chem., 100, 3144–49 (1996); Collier et al., Science, 277, 1978–81 (1997); Mirkin et al., Nature, 382, 607–09 (1996); and Alivisatos et al., Nature, 382, 609–11 (1996)) and light-emitting diodes (Colvin et al., Nature, 370, 354–57 (1994); and Dabbousi et al., Appl. Phys. Let., 66, 1316–18 (1995)). In particular, IIB–VIB semiconductors have been the focus of much attention, leading to the development of a CdSe quantum dot that has an unprecedented degree of monodispersivity and crystalline order (Murray (1993), supra).
Further advances in luminescent quantum dot technology have resulted in a dramatic enhancement of the fluorescence efficiency and stability of the quantum dots. The remarkable luminescent properties of quantum dots arise from quantumsize confinement, which occurs when metal and semiconductor core particles are smaller than their exciton Bohr radii, about 1 to 5 nm (Alivisatos, Science, 271, 933–37 (1996); Alivisatos, J Phys. Chem., 100, 13226–39 (1996); Brus, Appl Phys., A 53, 465–74 (1991); Wilson et al., Science, 262, 1242–46 (1993); Henglein (1989), supra; and Weller (1993), supra). Recent work has shown that improved luminescence can be achieved by capping a size-tunable lower band gap core particle with a higher band gap shell. For example, CdSe quantum dots passivated with a ZnS layer are strongly luminescent (35 to 50% quantum yield) at room temperature, and their emission wavelength can be tuned from blue to red by changing the particle size. Moreover, the ZnS capping protects the core surface and leads to greater stability of the quantum dot (Hines (1996), supra; and Dabbousi et al., J Phys. Chem. B 101, 9463–75 (1997)).
Despite the remarkable advances in luminescent quantum dot technology, the capped luminescent quantum dots are not suitable for biological applications because they are not water-soluble. In addition, it has not been possible to attach a quantum dot to a biomolecule in such a manner as to preserve the biological activity of the biomolecule. However, because luminescent quantum dots offer significant advantages over currently available nonisotopic detection systems, there remains an unfulfilled desire for a luminescent quantum dot that can be used for detection purposes in biological assays. In view of this, it is an object of the present invention to provide a luminescent quantum dot that is suitable for biological applications. It is another object of the present invention to provide a biomolecular conjugate of a luminescent quantum dot that is suitable for biological applications. In particular, the present invention seeks to provide a biomolecular conjugate of a luminescent quantum dot in which the biomolecule retains its biological activity and the resultant conjugate is suitable for biological applications. Accordingly, it is yet another object of the present invention to provide a method of making such a luminescent quantum dot and a method of making a biomolecular conjugate thereof. Still yet another object of the present invention is to provide a composition comprising such a quantum dot or a biomolecular conjugate thereof. A further object of the present invention is to provide methods of using the biomolecular conjugate for ultrasensitive nonisotopic detection in vitro and in vivo. These and other objects and advantages, as well as additional inventive features, of the present invention will become apparent to one of ordinary skill in the art upon reading the detailed description provided herein.
USPTO Backgrounds
USPTO Backgrounds