US 2003/0028981 A1 · Precision fluorescently dyed particles and methods of making and using same

Abstract

An improved method of making a series of bead or microsphere or particle populations characterized by subtle variation in a proportion or ratio of at least two fluorescent dyes distributed within a single bead of each population is provided. These beads, when excited by a single excitation light source are capable of giving off several fluorescent signals simultaneously. A set containing as many as 64 distinct populations of multicolored, fluorescent beads is provided and when combined with analytical reagents bound to the surface of such beads is extremely useful for multiplexed analysis of a plurality of analytes in a single sample. Thus, methods of staining polymeric particles, the particles themselves, and methods of using such particles are claimed.

Claims40

Dependent claims

A method of staining polymeric microspheres with two or more fluorescent dyes comprising:

(a) combining at least two fluorescent dyes in a solvent mixture comprising at least one organic solvent in which the at least two fluorescent dyes are soluble and at least one alcoholic solvent in which the at least two fluorescent dyes are less soluble, to provide a solution of mixed dyes which is further characterized as having the capacity to swell at least partially but not dissolve a plurality of polymeric microspheres, which is brought into contact with said solution;
(b) contacting a plurality of polymeric microspheres with said solution for a period of time sufficient to provide uniform staining of substantially all of the members of said plurality of polymeric microspheres with the at least two fluorescent dyes,
said at least two fluorescent dyes being selected such that on isolation and excitation of said dyed plurality of polymeric microspheres, a distinct fluorescence signal is emitted from each dye, the intensity of which emitted signal is proportional to the amount of said dye in said dyed plurality of polymeric microspheres.

A method of detecting a plurality of analytes in a sample, each of said analytes being recognized by a corresponding analytical reactant, comprising:

(a) contacting said sample with a plurality of populations of uniformly stained microspheres, said microspheres having at least two fluorescent dyes uniformly mixed at a specific ratio within each microsphere of each said population, each population of said microspheres having a distinct analytical reactant bound to its surface, wherein, said reactant on each population of microspheres specifically interacts with one of said analytes in said sample;
(b) providing a label reagent that specifically binds to the analyte and analyzing the microspheres to detect said label indicating binding of the analyte to the analytical reactant;
and
(c) determining the populations of microspheres having said fluorescent dyes mixed at the specific ratio within microspheres of each population to which the reactant is bound.
1 TABLE 1 Sample FL2 (CV) FL3 (CV) Events Old Method A 24.2 25.9 10000 B 27.7 25.5 10000 C 27.7 23.2 10000 Improved Method A 7.3 6.8 10000 B 7.3 6.7 10000 C 7.2 6.7 10000
2 TABLE 2 Region 47 series FL3 Red Dye (mM) FL2 Orange Dye (mM) 470/250 0.0495 0.00441 470/340 0.104 0.0041 470/415 0.0685 0.004 470/470 0.388 0.00576 470/525 0.458 0.0039 470/580 0.981 0.00662 470/635 0.132 0.00557 470/690 1.831 0.00637 470/745 3.84 0.01138 470/810 11.29 0.0278 Region 58-series FL 3 Red Dye (mM) FL2 Orange Dye (mM) 580/250 0.0201 0.00994 580/340 0.0582 0.00979 580/415 0.0763 0.00161 580/470 1.38 0.0256 580/525 0.42 0.0114 580/580 0.613 0.0162 580/635 1.15 0.0132 580/690 2.71 0.0267 580/745 0.643 0.0248 580/810 7.5 0.0489 580/870 9.71 0.008 Region 69-series FL3 Red Dye (mM) FL3 Orange Dye (mM) 690/360 0.048 0.024 690/415 0.129 0.024 690/470 0.226 0.023 690/525 0.45 0.029 690/580 0.89 0.0459 690/635 1.11 0.0322 690/690 2.45 0.07 690/745 2.88 0.0624 690/810 5.54 0.185 690/870 5.68 0.145 750/940 10.52 1.07 Region 80-series FL 3 Red Dye (mM) FL2 Orange Dye (mM) 800/415 0.0465 0.15 800/470 0.123 0.131 800/525 0.226 0.061 800/580 0.504 0.0835 800/635 0.62 0.0805 800/690 1.18 0.118 800/745 1.7 0.113 800/800 3.06 0.218 800/870 5.7 0.31 800/940 7.54 0.806 950/940 7.93 0.736 Region 90-series FL 3 Red Dye (mM) FL2 Orange Dye (mM) 900/510 0.084 0.0017 900/553 0.0666 0.0146 900/596 0.138 0.0913 900/645 1.29 0.253 900/690 0.861 0.298 900/734 0.699 0.229 900/779 0.984 0.214 900/823 1.13 0.435 900/867 2.61 0.381 900/912 3.41 0.579 900/953 4.88 0.861 Region 99-series FL 3 Red Dye (mM) FL 2 Orange Dye (mM) 990/600 — 0.274 990/640 0.125 0.21 990/680 0.21 0.268 990/720 0.472 0.216 990/760 0.712 0.275 990/800 0.82 0.278 990/840 1.12 0.446 990/880 1.87 0.431 990/920 3.24 0.477 990/980 2.93 0.763

3 more elements

Dependencies are inferred from the claim text.

Precision fluorescently dyed particles and methods of making and using same

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Precision fluorescently dyed particles and methods of making and using same

Life of this patent

Abstract

Claims40