Single-Cell Target Molecule Detection with Autofluorescence Correction
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Solution Overview
Problem
Existing flow cytometry methods suffer from high background signals and autofluorescence, leading to measurement errors and reduced sensitivity in detecting target molecules within cells, particularly due to unspecific binding of markers and variations in cell populations.
Innovation Solution
A method involving immobilizing cells on a solid substrate, determining their position, measuring autofluorescence, and then contacting them with a detection reagent to calibrate fluorescence signals individually, allowing for cell-by-cell correction of background noise and autofluorescence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Difficulty of detecting and measuring
If flow cytometry is used to detect target molecules in cells, then cell analysis capability is improved, but measurement precision deteriorates due to high background signals and autofluorescence
Solution Approach 1:
The patent segments the cell population into individual single cells, analyzing each cell separately rather than as a bulk population. This segmentation allows for individual background signal measurement and subtraction, thereby improving measurement precision while maintaining cell analysis capability
Solution Approach 2:
The patent performs preliminary measurement of the background signal and autofluorescence for each individual cell before adding the detection reagent. This preliminary action enables subsequent subtraction of the background from the total signal, improving detection sensitivity by eliminating the confounding effect of high background signals
2Measurement precision
If control samples are used to eliminate background signals, then measurement accuracy is improved, but reliability deteriorates due to population variations
Solution Approach 1:
The patent enables each cell to serve its own control function by measuring the background signal and autofluorescence of that specific cell before detection reagent addition. This self-service approach eliminates the need for separate control samples and ensures that each cell's background is corrected using its own baseline measurement, improving both precision and reliability
3Productivity
If average control sample values are used for calibration, then processing efficiency is improved, but measurement precision deteriorates due to individual cell variations
Solution Approach 1:
The patent divides the calibration process into individual cell-level operations rather than applying a single average calibration value to all cells. Each cell receives its own background-corrected measurement, improving detection accuracy while the automated single-cell analysis workflow maintains processing efficiency
Solution Approach 2:
The patent applies local quality correction by measuring and subtracting the background signal specific to each individual cell's location and characteristics, rather than applying a global average correction. This local approach improves measurement precision for cells with varying autofluorescence levels while maintaining overall workflow efficiency
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 reduces measurement errors by calibrating each cell's fluorescence against its own background signal, enhancing sensitivity and reliability in detecting target molecules.
Implementation Method 1
a detection reagent that binds to a given target molecule
Implementation Method 2
fluorescence signals from given target molecules
Data Source
AI summary
Methods for single cell analysis by determining the presence and/or amount of one or more target molecules in a plurality of cells may include: (i) immobilizing said plurality of cells on a solid substrate, wherein the cells are immobilized in form of a monolayer; (ii) determining the position of the individual immobilized cells on the solid substrate; (iii) measuring the auto-fluorescence of the individual immobilized cells; (iv) contacting the immobilized cells with a first detection reagent comprising (a) a moiety that specifically recognizes and binds a first target molecule and (b) a fluorescent label under conditions that allow binding of the detection reagent to the first target molecule; (v) measuring the fluorescence of the fluorescent label of the detection reagent bound to the first target molecule for the individual immobilized cells; (vi) determining the presence and/or amount of the first target molecule in the individual immobilized cells by comparing the fluorescence measured in step (v) with the fluorescence measured in step (iii) on a cell-by-cell basis.