Fluorescent Microparticle Barcoding with Cleavable Linkers
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Solution Overview
Problem
Current bead encoding techniques for high-throughput applications are limited by the low encoding power of fluorophore-labeled beads, requiring complex sequential hybridization and sequencing steps for code identification, which are time-consuming and inefficient.
Innovation Solution
The use of fluorophores and cleavable linkers to generate millions of code combinations, allowing for simple fluorescent imaging to quickly and efficiently identify bead codes by selectively cleaving or activating fluorophores, thereby simplifying the identification process.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If genetic codes are used to encode beads at high encoding power, then the encoding capacity increases to millions of combinations, but the complexity of code identification increases requiring sequential hybridization and sequencing steps
Solution Approach 1:
The patent replaces complex biochemical identification processes (hybridization and sequencing) with a simpler optical detection system. Fluorophores are attached to beads through cleavable linkers, allowing codes to be read by fluorescent imaging instead of sequential hybridization and sequencing steps. This substitution dramatically simplifies the identification process while maintaining high encoding capacity.
Solution Approach 2:
The patent changes the detection parameter from biochemical signals (requiring hybridization and sequencing) to optical signals (fluorescence). By using fluorophores with distinct emission wavelengths and cleavable linkers that can be selectively removed, the system enables high-capacity encoding to be read through simple fluorescent imaging, transforming the detection mechanism to achieve both high encoding power and simplified identification.
2Measurement precision
If sequential hybridization and sequencing steps are used to identify bead codes, then genetic codes can be read, but the time required for code identification increases significantly
Solution Approach 1:
The patent replaces time-consuming sequential biochemical steps (hybridization and sequencing) with a rapid optical imaging process. Fluorophores attached via cleavable linkers allow multiple code bits to be simultaneously visualized through fluorescent microscopy, reducing identification time from hours or days to minutes or seconds while preserving accurate code reading.
Solution Approach 2:
The patent performs preliminary attachment of fluorophores to beads during bead manufacturing, with cleavable linkers already in place. This pre-preparation allows codes to be directly visualized when needed, eliminating the need for time-consuming sequential processing steps during actual code identification, thus significantly reducing identification time while maintaining accuracy.
3Ease of operation
If fluorophore-labeled beads are used for encoding, then fluorescent readouts are standard for image-based screening, but the encoding power is limited to only hundreds of combinations
Solution Approach 1:
The patent segments the encoding system into multiple independent components: multiple fluorophore types (each with distinct emission wavelengths) and multiple cleavable linker types (each selectively removable by specific agents). By combining these segmented elements, the system achieves millions of unique code combinations while maintaining the simplicity of fluorescent imaging for detection.
Solution Approach 2:
The patent creates composite bead structures containing multiple fluorophores attached through different cleavable linkers. This composite approach combines the simplicity of fluorescent detection with high encoding capacity, as each bead can carry a unique combination of fluorophore-linker pairs that can be systematically decoded through selective cleavage and imaging.
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 the creation of millions of unique bead-specific barcodes, improving data quality in single cell sequencing, antibody discovery, T-cell receptor identification, CRISPR screening, and drug resistance mechanism analysis by eliminating the need for hybridization and sequencing steps, enabling high-throughput and efficient optical readouts.
Implementation Method 1
Fluorescent readouts are standard for image-based screening applications
Implementation Method 2
the cleavable linkers are sequentially treated with an agent that either selectively cleaves the cleavable linkers to release the fluorophores, or activates the fluorophores that are attached to the cleavable linkers
Data Source
AI summary
The invention provides a method of barcoding microparticles with fluorescent moieties. The fluorescent moieties are linked to the microparticles by variable cleavable linkers. The combination of fluorescent moieties and variable cleavable linkers provides microparticles having an exponentially expandable number of fluorescent barcodes that can be manufactured and decoded in a sequential manner. The fluorescent barcodes can be used to identify microwells, cells, chemicals and oligonucleotides in wide variety of high throughput assay applications.