In-Pen Assay Imaging for Accurate Microfluidic Analyte Quantification
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Solution Overview
Problem
Existing technologies face challenges in accurately determining the quantity of an analyte produced by a micro-object confined within a microfluidic chamber, particularly due to sensitivity to the position of biological micro-objects and limitations in measuring analyte concentration fluctuations.
Innovation Solution
A system and method that utilize an image acquisition unit to capture assay images, define an area of interest within sequestration pens, and analyze image areas to determine analyte quantity, incorporating an image processing unit to score the quantity of analyte in each sequestration pen, with the area of interest being most sensitive to analyte concentration fluctuations and least sensitive to micro-object position.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional imaging systems are used to measure analyte quantity, then the system can capture images of the microfluidic device, but the measurement accuracy is poor due to sensitivity to micro-object position and inability to effectively capture analyte concentration fluctuations
Solution Approach 1:
The patent divides the imaging task into segmented processing steps: (1) capturing a reference image without analyte, (2) capturing an assay image with analyte, (3) subtracting the reference image from the assay image to isolate analyte signal, and (4) analyzing only the relevant regions. This segmentation eliminates background interference and position sensitivity, directly improving measurement accuracy while reducing detection difficulty
Solution Approach 2:
The patent introduces an intermediary reference image that captures the baseline state of the microfluidic device without analyte. This reference image acts as a mediator that, when subtracted from the assay image, isolates the analyte concentration signal from position-dependent background variations, thereby improving measurement precision without increasing detection complexity
2Measurement precision
If the entire sequestration pen area is analyzed, then all potential analyte signals are captured, but the analysis complexity and processing time increase
Solution Approach 1:
The patent applies local quality by analyzing only specific regions within the sequestration pen where analyte concentration fluctuations are most likely to occur. After reference image subtraction, the system focuses analysis on areas showing signal changes, rather than processing the entire pen area. This reduces image processing complexity while maintaining complete analyte detection, as the differential imaging approach concentrates the analyte signal in specific locations
3Ease of operation
If conventional imaging without reference subtraction is used, then the imaging process is simpler, but the measurement is sensitive to background noise and position variations
Solution Approach 1:
The patent performs preliminary action by capturing a reference image before the analyte assay, which records the baseline state of the device. This reference image is then subtracted from the assay image to eliminate background noise and position sensitivity. The additional step improves measurement precision significantly while maintaining operational simplicity, as the reference image captures can be performed automatically as part of the assay protocol
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
Enables accurate quantification of analyte production by biological micro-objects, minimizing positional sensitivity and enhancing measurement precision.
Implementation Method 1
allowing a portion of the plurality of reporter molecules to diffuse into the sequestration pen and bind to the analyte secreted therein
Implementation Method 2
each reporter molecule includes: a binding component configured to bind the secreted analyte
Data Source
AI summary
Methods, systems and kits are described herein for detecting the results of an assay. In particular, the methods, systems and devices of the present disclosure rely on a difference between the diffusion rates of a reporter molecule and an analyte of interest in order to quantify an amount of analyte in a microfluidic device. The analyte may be a secreted product of a biological micro-object.