Microparticle Single-Molecule Detection Without Enzyme Amplification

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Solution Overview

Problem

Conventional immunodiagnostic assays face challenges in achieving high sensitivity and dynamic range in digital immunoassays, and multiplex immunoassays suffer from reagent interference and reduced sensitivity.

Innovation Solution

The use of macroconjugates and fluorescent agents in immunoassays allows for direct visualization and analysis of single immune complexes without enzyme amplification, combined with transmitted light and fluorescent image analysis to reduce interference and increase sensitivity in multiplex assays.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Measurement precision

If enzyme amplification is used in digital immunoassays, then sensitivity is improved, but assay complexity and time consumption increase

Engineering Contradiction:

ImprovesensitivityVSAvoidassay complexity

Core Design Contradiction:

Measurement precisionVSDevice complexity

Solution Approach 1:

The patent extracts and eliminates the enzyme amplification step from the immunoassay process. Instead of using enzyme-conjugated antibodies that require substrate conversion and amplification steps, the invention uses fluorescently labeled detection antibodies that provide direct fluorescence signal from single molecule binding events, thereby reducing assay complexity while maintaining sensitivity

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The patent substitutes the biochemical amplification mechanism (enzyme-catalyzed substrate conversion) with a direct optical detection mechanism (fluorescence labeling). This replacement eliminates the need for enzyme substrates, washing steps, and amplification time, directly reducing assay complexity and time consumption while preserving single-molecule detection sensitivity

Inventive Principle:

Principle #28Mechanics substitution (Replace mechanical system)

2Productivity

If multi-colored beads are used for multiplexing, then throughput is improved, but reagent interference and sensitivity decrease

Engineering Contradiction:

ImprovethroughputVSAvoidsensitivity

Core Design Contradiction:

ProductivityVSMeasurement precision

Solution Approach 1:

The patent applies local quality by using spatially resolved fluorescence imaging to distinguish different analytes. Instead of relying on spectrally distinct fluorophores that cause crosstalk, the invention detects fluorescent signals at different spatial locations on the microparticle surface, where each location corresponds to a specific analyte binding site, thereby eliminating reagent interference while maintaining high sensitivity

Inventive Principle:

Principle #3Local quality

Solution Approach 2:

The patent transitions from spectral multiplexing (different fluorophore colors) to spatial multiplexing (different locations on the microparticle). This dimensional change from wavelength domain to spatial domain allows multiple analytes to be detected simultaneously without fluorophore crosstalk, resolving the contradiction between throughput and sensitivity

Inventive Principle:

Principle #17Another dimension (Dimensionality change)

3Measurement precision

If single-molecule counting is implemented, then detection limit is improved, but dynamic range is reduced

Engineering Contradiction:

Improvedetection limitVSAvoiddetection dynamic range

Core Design Contradiction:

Measurement precisionVSAdaptability or versatility

Solution Approach 1:

The patent creates a universal detection system that can operate in both digital (single-molecule counting) and analog (ensemble averaging) modes. The fluorescently labeled detection antibodies and spatially resolved imaging platform serve multiple functions: they enable single-molecule detection for low concentration samples while also supporting traditional bulk detection for high concentration samples, thereby expanding the overall detection dynamic range

Inventive Principle:

Principle #6Universality (Multi-functionality)

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This approach enhances the sensitivity and reduces reagent interference in multiplex assays, enabling accurate detection of multiple analytes with improved dynamic range.

Implementation Method 1

reacting a plurality of fluorescent agents with the tags, wherein each fluorescent agent comprises a molecule which is capable of binding to the tag and a detectable label

Methodology Applied

Scientific EffectFluorescence: Fluorescence

Data Source

PatentUS12498376B2Direct detection of single molecules on microparticles

Publication Date: 2025.12.16 ABBOTT LAB INC

AI summary

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.