Automated Assay Site Segmentation for Single-Molecule Detection
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Solution Overview
Problem
Current methods for detecting low concentrations of analyte molecules in samples are limited by the need for amplification procedures and are prone to non-specific binding, which reduces sensitivity and dynamic range, and lack automation for accurate quantification.
Innovation Solution
An automated system comprising an assay consumable handler, sealer, sample loader, imaging system, and computer-controlled components that isolate and analyze individual assay sites, allowing for precise loading and sealing of samples to detect analyte molecules with high sensitivity and accuracy.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If amplification procedures are used to increase the number of reporter molecules, then the measurable signal is improved, but the sensitivity and dynamic range are reduced
Solution Approach 1:
The invention divides the sample into multiple individual assay sites (e.g., wells, chambers, or microreaction vessels), each containing a single analyte molecule or particle. This segmentation allows direct detection of individual molecules without requiring amplification, thereby maintaining sensitivity while providing measurable signals through ensemble reading of multiple sites.
Solution Approach 2:
The invention replaces the mechanical/biochemical amplification system with an optical/enumeration-based detection system. Instead of amplifying the signal through additional reporter molecules, the system directly counts or detects the presence of analyte molecules in individual assay sites using imaging or detection apparatus, eliminating the need for amplification procedures.
2Measurement precision
If a large number of molecules are present in the ensemble, then the aggregate signal is above the detection threshold, but the lowest concentration that may be accurately detected is limited
Solution Approach 1:
The invention segments the ensemble detection into individual molecular-level assay sites, where each site can be independently evaluated for the presence or absence of an analyte molecule. This allows accurate detection of low concentrations by counting positive sites among many negative sites, maintaining statistical power while detecting single molecules.
Solution Approach 2:
The invention creates local assay sites with specific properties optimized for single-molecule detection, such as controlled volume, surface functionalization for specific binding, and isolation from neighboring sites. Each local site has the quality needed to detect individual molecules, while the ensemble of sites provides the statistical power for accurate quantification.
3Quantity of substance
If non-specific binding occurs to sites other than those expected, then the background signal increases, but the sensitivity and accuracy are reduced
Solution Approach 1:
The invention segments the assay into isolated individual sites that prevent non-specific binding between sites. Each site is independently controlled and can be sealed or isolated, ensuring that non-specific binding events are confined to individual sites and do not create cross-site interference or elevated background signals.
Solution Approach 2:
The invention extracts or removes the source of non-specific binding by isolating assay sites from each other and from non-specific binding surfaces. By confining reactions to discrete, isolated sites with controlled surfaces and volumes, the system eliminates the heterogeneous environment that promotes non-specific binding.
4Adaptability or versatility
If manual operation is used for loading and sealing assay sites, then flexibility is maintained, but automation and productivity are reduced
Solution Approach 1:
The invention employs universal assay site formats (such as standardized wells, chambers, or microreaction vessels) that can be manually or automatically loaded and sealed. The standardized geometry and interface allow the same assay format to be used across different automation levels, maintaining flexibility while enabling automation through robotic liquid handling, magnetic manipulation, or other automated systems.
Solution Approach 2:
The invention employs self-sealing or self-isolating assay sites that automatically seal or isolate after sample loading without requiring manual intervention. For example, microreaction vessels with self-sealing caps, or sites that form seals through surface tension or magnetic forces, enable automated processing while maintaining the integrity and isolation of individual assay sites.
Data Source
AI summary
Described are systems, devices, and methods which related to various aspects of assays for detecting and/or determining a measure of the concentration of analyte molecules or particles in a sample fluid. In some cases, the systems employ an assay consumable comprising a plurality of assay sites. The systems, devices, and/or methods, in some cases, are automated. In some cases, the systems, devices, and/or methods relate to inserting a plurality of beads into assay sites, sealing assay sites, imaging assay sites, or the like.