Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1999-05-20
2003-04-08
Chin, Christopher L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C436S002000, C436S004000, C436S005000, C436S518000, C436S523000, C436S524000, C436S164000, C436S805000, C436S823000, C435S007100, C435S174000
Reexamination Certificate
active
06544732
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to compositions and methods for encoding, decoding and using microsphere array sensors utilizing nanocrystals (also referred to in the art as quantum dots).
BACKGROUND OF THE INVENTION
There are a number of assays and sensors for the detection of the presence and/or concentration of specific substances in fluids and gases. Many of these rely on specific ligand/antiligand reactions as the mechanism of detection. That is, pairs of substances (i.e. the binding pairs or ligand/antiligands) are known to bind to each other, while binding little or not at all to other substances. This has been the focus of a number of techniques that utilize these binding pairs for the detection of the complexes. These generally are done by labeling one component of the complex in some way, so as to make the entire complex detectable, using, for example, radioisotopes, fluorescent and other optically active molecules, enzymes, etc.
Of particular use in these sensors are detection mechanisms utilizing luminescence. Recently, the use of optical fibers and optical fiber strands in combination with light absorbing dyes for chemical analytical determinations has undergone rapid development, particularly within the last decade. The use of optical fibers for such purposes and techniques is described by Milanovich et al., “Novel Optical Fiber Techniques For Medical Application”, Proceedings of the SPIE 28th Annual International Technical Symposium On Optics and Electro-Optics, Volume 494, 1980; Seitz, W. R., “Chemical Sensors Based On Immobilized Indicators and Fiber Optics” in
C.R.C Critical Reviews In Analytical Chemistry
, Vol. 19, 1988, pp. 135-173; Wolfbeis, O. S., “Fiber Optical Fluorosensors In Analytical Chemistry” in
Molecular Luminescence Spectroscopy, Methods and Applications
(S. G. Schulman, editor), Wiley & Sons, New York (1988); Angel, S. M.,
Spectroscopy
2 (4):38 (1987); Walt, et al., “Chemical Sensors and Microinstrumentation”,
ACS Symposium Series
, Vol. 403, 1989, p. 252, and Wolfbeis, O. S.,
Fiber Optic Chemical Sensors
, Ed. CRC Press, Boca Raton, Fla., 1991, 2nd Volume.
When using an optical fiber in an in vitro/in vivo sensor, one or more light absorbing dyes are located near its distal end. Typically, light from an appropriate source is used to illuminate the dyes through the fiber's proximal end. The light propagates along the length of the optical fiber; and a portion of this propagated light exits the distal end and is absorbed by the dyes. The light absorbing dye may or may not be immobilized; may or may not be directly attached to the optical fiber itself; may or may not be suspended in a fluid sample containing one or more analytes of interest; and may or may not be retainable for subsequent use in a second optical determination.
Once the light has been absorbed by the dye, some light of varying wavelength and intensity returns, conveyed through either the same fiber or collection fiber(s) to a detection system where it is observed and measured. The interactions between the light conveyed by the optical fiber and the properties of the light absorbing dye provide an optical basis for both qualitative and quantitative determinations.
Many of the recent improvements employing optical fiber sensors in both qualitative and quantitative analytical determinations concern the desirability of depositing and/or immobilizing various light absorbing dyes at the distal end of the optical fiber. In this manner, a variety of different optical fiber chemical sensors and methods have been reported for specific analytical determinations and applications such as pH measurement, oxygen detection, and carbon dioxide analyses. These developments are exemplified by the following publications: Freeman, et al.,
Anal Chem
. 53:98 (1983); Lippitsch et al.,
Anal. Chem. Acta
. 205:1, (1988); Wolfbeis et al.,
Anal. Chem
. 60:2028 (1988); Jordan, et al.,
Anal. Chem
. 59:437 (1987); Lubbers et al.,
Sens. Actuators
1983; Munkholm et al.,
Talanta
35:109 (1988); Munkholm et al.,
Anal. Chem
. 58:1427 (1986); Seitz, W. R.,
Anal. Chem
. 56:16A-34A (1984); Peterson, et al.,
Anal. Chem
. 52:864 (1980): Saari, et al.,
Anal. Chem
. 54:821 (1982); Saari, et al.,
Anal. Chem
. 55:667 (1983); Zhujun et al.,
Anal. Chem. Acta
. 160:47 (1984); Schwab, et al.,
Anal. Chem
. 56:2199 (1984); Wolfbeis, O. S., “Fiber Optic Chemical Sensors”,
Ed. CRC Press
, Boca Raton, Fla., 1991, 2nd Volume; and Pantano, P., Walt, D. R.,
Anal. Chem
., 481A-487A, Vol. 67, (1995).
More recently, fiber optic sensors have been constructed that permit the use of multiple dyes with a single, discrete fiber optic bundle. U.S. Pat. Nos. 5,244,636 and 5,250,264 to Walt, et al. disclose systems for affixing multiple, different dyes on the distal end of the bundle, the teachings of each of these patents being incorporated herein by this reference. The disclosed configurations enable separate optical fibers of the bundle to optically access individual dyes. This avoids the problem of deconvolving the separate signals in the returning light from each dye, which arises when the signals from two or more dyes are combined, each dye being sensitive to a different analyte, and there is significant overlap in the dyes' emission spectra.
U.S. Ser. Nos. 08/818,199 and 09/151,877 describe array compositions that utilize microspheres or beads on a surface of a substrate, for example on a terminal end of a fiber optic bundle, with each individual fiber comprising a bead containing an optical signature. Since the beads go down randomly, a unique optical signature is needed to “decode” the array; i.e. after the array is made, a correlation of the location of an individual site on the array with the bead or bioactive agent at that particular site can be made. This means that the beads may be randomly distributed on the array, a fast and inexpensive process as compared to either the in situ synthesis or spotting techniques of the prior art. Once the array is loaded with the beads, the array can be decoded, or can be used, with full or partial decoding occurring after testing, as is more fully outlined below.
Unfortunately, the above systems all suffer from the disadvantages of working with conventional detection labels, typically organic dyes such as rhodamine. Conventional dye molecules impose stringent requirements on the optical systems used to make these measurements; their narrow excitation spectrum makes simultaneous excitation difficult in most cases, and their broad emission spectrum with a long tail at red wavelengths introduces spectral cross talk between different detection channels, making quantitation of the relative amounts of different probes difficult.
Therefore, it is desirable to provide assay components and methods which utilize detectable labels which emit spectrally resolvable energies and have narrow, symmetric emission spectrums, and wherein whole groups of detectable labels can be excited at a single wavelength.
SUMMARY OF THE INVENTION
In accordance with the above objects, the present invention provides compositions comprising a substrate with a surface comprising discrete sites, and a population of microspheres distributed on the sites. At least one of the microspheres comprises a nanocrystal. The nanocrystal can be embedded in the microsphere, for example using the sol-gel polymerization process, or it can be attached to the microsphere. The microspheres optionally comprise bioactive agents and/or identifier binding ligands.
In an additional aspect, the population of microspheres comprises at least a first and a second subpopulation comprising a first and a second bioactive agent, respectively, and a first and a second optical signature, respectively, capable of identifying each bioactive agent. At least one of the optical signatures comprises a nanocrystal.
In a further aspect, the invention provides methods of making a composition comprising forming a surface comprising individual sites on a substrate and distributing microspheres on the surface such that the individual
Barnard Steven M.
Chee Mark S.
Zhao Chanfeng
Chin Christopher L.
Do Pensee
Dorsey & Whitney LLP
Foster David C.
Illumina, Inc.
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