Sequential Lateral Flow Device Multiplexed Assay Sensitivity
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Solution Overview
Problem
Current lateral flow immunoassays (LFIs) face challenges in maintaining sensitivity when detecting multiple target analytes, particularly due to the limited surface area of signaling particles and the conjugation capacity on their surface.
Innovation Solution
The method involves separating the binding agent from the signaling agent at the initiation of the LFI test procedure, allowing the target analytes to form sandwich complexes at the capture zone before the release of the signaling agent, thereby overcoming the limitations of conventional methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If detector antibodies are directly conjugated to signaling particles, then the assay is simpler to perform, but sensitivity deteriorates when multiple target analytes are detected
Solution Approach 1:
The assay is divided into two sequential steps: first, binding agents form sandwich complexes with target analytes without signaling particles; second, signaling particles are introduced to form detectable complexes. This segmentation allows optimization of each step independently, maintaining simplicity while improving sensitivity for multiplexed assays.
Solution Approach 2:
Sandwich complexes are formed in advance before introducing signaling particles. This preliminary formation of antigen-antibody complexes ensures that all binding events occur before the limited surface area of signaling particles becomes a constraint, thereby preserving sensitivity even when detecting multiple analytes.
2Adaptability or versatility
If more target analytes are detected on a single test strip, then the multi-analyte capability increases, but the limit of detection for each analyte deteriorates
Solution Approach 1:
By separating the binding step from the detection step, the system can accommodate multiple analytes without compromising individual detection limits. Each analyte forms its sandwich complex independently in the first step, and signaling particles are then distributed to all complexes in the second step, ensuring uniform sensitivity across all detected analytes.
Solution Approach 2:
All sandwich complex formation occurs before signaling particles are introduced. This preliminary action ensures that the full capacity of binding agents is utilized for each analyte without competition for limited signaling particle surface area, maintaining low limits of detection even when detecting forty or more analytes simultaneously.
3Measurement precision
If the surface area of signaling particles is increased to improve sensitivity, then more binding agents can be conjugated, but the device complexity increases
Solution Approach 1:
Sandwich complexes are formed before introducing signaling particles, eliminating the need to increase signaling particle surface area. The preliminary formation of complexes ensures that all binding events occur when binding agents are freely available, and standard-sized signaling particles suffice for detection without requiring complex high-capacity conjugation systems.
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 significantly improves the limits of detection (LoDs) by two-fold to as much as two log-fold in multiplexed assays compared to direct assays, enabling the detection of up to forty or more target analytes with enhanced sensitivity.
Implementation Method 1
a capillary flow bed residing in the cavity, wherein the capillary flow bed is configured to transport the sample from a proximal region of the capillary flow bed to a distal region of the capillary flow bed
Implementation Method 2
allowing the target analytes to form sandwich complexes at the capture zone before the release of the signaling agent
Data Source
AI summary
The present disclosure provides methods and lateral flow devices for detecting a plurality of target analytes in a liquid sample. In some implementations, the disclosed lateral flow device comprises a housing unit, a capillary flow bed, a sample-receiving zone, a buffer-receiving zone, and a capture zone. The device is configured to control the flow of the sample and reagent buffer in a sequential manner with minimal mixing. In some implementations, the disclosed method is capable of detecting a plurality of target analytes in an assay by applying the binding agents and the signaling agents in separate or sequential steps.