Direct detection of single molecules on microparticles
Inventors
Ruan, Qiaoqiao • MacDonald, Patrick J. • Swift, Kerry M. • Tetin, Sergey Y. • Calfin, Brenda • Lin, Zhen • Haack, Richard • Pope, Mark R. • Prostko, John • Qiu, Xiaoxing • Bogdan, M. Felicia
Assignees
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Publication Number
US-12498376-B2
Publication Date
2025-12-16
Expiration Date
Abstract
The disclosure provides methods of analyzing an analyte of interest in a biological sample using fluorescent agents and macroconjugates which comprise a core containing a cross-linked polymer or protein, tags, specific binding members or fragments thereof, and optionally carrier proteins. Also provided are methods of analyzing two or more analytes of interest in a biological sample in a single assay using microparticles and detection conjugates comprising different fluorophore labels, acquiring transmitted light and fluorescent images of the microparticles, and using a customized image analysis process to analyze the acquired images.
Core Innovation
The invention relates to methods of analyzing two or more analytes of interest in a single assay by capturing different analytes on different microparticles and visualizing binding on the microparticles. A first analyte is captured on a surface of a first microparticle comprising a plurality of specific binding members immobilized on the surface, and the first microparticle is labeled with a first fluorophore. A second analyte is captured on a surface of a second microparticle comprising a plurality of specific binding members immobilized on the surface, with the first analyte different from the second analyte.
The captured analytes are then reacted with conjugates that include specific binding members labeled with different fluorophores for binding to the respective analytes. In the method, transmitted light images of the microparticles are obtained together with separate fluorescent images corresponding to different fluorophores, and image analysis is performed on the obtained transmitted and fluorescent images to determine the presence and amount of each analyte.
The invention further describes analyzing the images in a way that isolates fluorophore-specific signals using image masking and subtraction, and calculating emitted signals to detect a first analyte and a second analyte. The method can be extended to more than two analytes by capturing additional analytes on distinct microparticles, reacting them with additional conjugates, obtaining transmitted and separate fluorescence images corresponding to additional fluorophores, and analyzing the images.
Claims Coverage
The document chunk explicitly provides one independent claim directed to a single-assay method for analyzing two or more analytes using fluorophore-labeled microparticles, fluorophore-labeled conjugates, transmitted light imaging, separate fluorescent imaging, and image analysis.
Single-assay multiplex capture with distinct microparticle fluorophores
capturing a first analyte of interest on a surface of a first microparticle with a plurality of specific binding members immobilized on the surface and labeled with a first fluorophore, and capturing a second analyte of interest on a surface of a second microparticle with a plurality of specific binding members immobilized on the surface, the first analyte different from the second analyte
Conjugate reaction with different fluorophore-labeled specific binding members
reacting the captured first analyte with a first conjugate comprising a specific binding member labeled with a second fluorophore and binding to the first analyte, and reacting the captured second analyte with a second conjugate comprising a specific binding member labeled with a third fluorophore and binding to the second analyte, wherein the first, second, and third fluorophores are different
Transmitted light plus separate multi-fluorophore fluorescence imaging
obtaining a transmitted light image of the first and second microparticles and obtaining separate fluorescent images of the first and second microparticles corresponding to the first, second, and third fluorophores, respectively
Image analysis for multi-analyte detection
analyzing the images to determine information from the obtained transmitted and fluorescent images
The independent claim covers a multiplex imaging-based immunoassay workflow in a single assay, combining distinct fluorophore-labeled microparticles, fluorophore-labeled conjugates, transmitted light imaging with separate fluorescence channels for different fluorophores, and image analysis.
Stated Advantages
Not explicitly described in patent.
Documented Applications
Not explicitly described in patent.
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