Multiplexed Assay Sample Segmentation for Analyte Concentration
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Solution Overview
Problem
Conducting multiplexed assays for analytes with widely different abundances poses challenges as it is difficult to find a single set of conditions, such as sample dilution or binding reaction time, that brings all analytes within the assay's dynamic range, leading to either saturation or insensitivity issues.
Innovation Solution
The method involves processing different portions of the sample differently, such as diluting to different extents or using different detection reagents, to optimize the measurement of each analyte, allowing for simultaneous measurement in a single volume or chamber, even when analytes have different affinities or require incompatible detection reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If a single set of conditions (dilution or binding reaction time) is used for multiplexed assays, then the assay procedure is simple, but analytes with widely different abundances cannot all be brought within the assay's dynamic range
Solution Approach 1:
The sample is divided into multiple portions, each processed with different dilution factors or binding reaction times. This segmentation allows each analyte to be measured under optimized conditions while maintaining a single overall assay procedure, resolving the contradiction between operational simplicity and measurement precision.
Solution Approach 2:
The assay conditions (dilution factor, binding reaction time) are made dynamic and adjustable for different sample portions rather than fixed for the entire assay. This dynamic approach enables optimization for each analyte's specific abundance level while keeping the overall procedure straightforward.
2Object-affected harmful factors
If sample dilution is increased to prevent saturation of high abundance analytes, then saturation is avoided, but low abundance analytes fall below the assay's detection threshold
Solution Approach 1:
The sample is segmented into portions with different dilution factors. High abundance analytes are measured in more dilute portions to prevent saturation, while low abundance analytes are measured in less dilute portions to maintain detection sensitivity. This resolves the contradiction between avoiding saturation and maintaining sensitivity.
Solution Approach 2:
Different dilution factors are applied locally to different sample portions based on the specific analyte being measured. This local optimization allows each analyte to be measured under conditions appropriate to its abundance level, preventing both saturation and loss of sensitivity.
3Reliability
If binding reaction time is extended to improve detection of low abundance analytes, then sensitivity increases, but high abundance analytes saturate binding sites
Solution Approach 1:
The assay is segmented into multiple experiments with different binding reaction times. Low abundance analytes are measured with extended binding times to improve sensitivity, while high abundance analytes are measured with shorter binding times to prevent saturation. This resolves the contradiction between sensitivity and saturation.
Solution Approach 2:
The binding reaction time is made dynamic and adjustable for different analytes rather than fixed for the entire multiplexed assay. This allows optimization of sensitivity for low abundance analytes without causing saturation of high abundance analytes.
4Measurement precision
If multiple dilutions are made and individually tested to ensure one is within dynamic range, then measurement accuracy improves, but sample volume and handling complexity increase
Solution Approach 1:
Multiple dilutions that would normally be prepared and tested separately are merged into a single multiplexed assay plate. Different sample portions with different dilution factors are measured simultaneously in the same assay, reducing handling complexity while maintaining measurement accuracy.
Solution Approach 2:
A single multiplexed assay protocol is designed to handle multiple dilutions and measure multiple analytes simultaneously. This universal approach eliminates the need for separate assay procedures for different dilutions, reducing handling complexity while preserving measurement precision.
5Measurement precision
If multiple experiments with different binding reaction times are conducted to capture analytes within dynamic range, then measurement accuracy improves, but throughput decreases
Solution Approach 1:
Multiple experiments with different binding reaction times are merged into a single multiplexed assay. Different sample portions are measured simultaneously in the same plate, achieving the same measurement accuracy as separate experiments but with significantly improved throughput.
Solution Approach 2:
The assay design allows dynamic selection of binding reaction times for different analytes within a single experiment. This enables optimization of measurement precision for each analyte while maintaining high throughput through simultaneous measurement.
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 enables accurate measurement of multiple analytes with varying concentrations and properties by optimizing processing conditions for each analyte, ensuring they fall within the assay's dynamic range, thereby improving assay sensitivity and throughput.
Implementation Method 1
contacting a first portion of the sample with one or more binding surfaces; contacting the binding surface(s) with a first detection reagent that binds to the first analyte
Implementation Method 2
contacting the binding surface(s) with a first detection reagent that binds to the first analyte; contacting the binding surface(s) with a second detection reagent that binds to the second analyte
Data Source
AI summary
The present invention is directed to methods for conducting multiplexed assays. The methods are particularly well suited for measuring a plurality of analytes that may be present in very different abundances. The invention also relates to systems, devices, equipment, kits and reagents for use in such methods.