Reversible Fixation Reagents for Partition-Based Single-Cell Assays
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Solution Overview
Problem
Biological samples are unstable and prone to degradation, which limits their use in partition-based assays such as droplet-based or well-based single cell assays, necessitating a need for reversible fixation methods compatible with these assays.
Innovation Solution
The use of bis-imidazole-carboxylate compounds for reversible fixation of biological samples, forming bis-carbamate crosslinks with amine-bearing moieties, followed by un-fixing with agents like DETA or DTT to maintain sample integrity for analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If standard fixation methods (cryopreservation, dehydration, high-salt storage, or chemical fixing agents) are used to stabilize biological samples, then sample stability is improved, but the ability to rapidly and efficiently un-fix the sample for subsequent assays is worsened
Solution Approach 1:
The patent employs fixation reagents that can be reversed by changing chemical parameters (pH, redox state) to enable efficient un-fixing. Specifically, the use of reversible crosslinking chemistry allows the fixed sample to be returned to its native state through controlled chemical parameter adjustments, resolving the contradiction between stability and ease of un-fixing
Solution Approach 2:
The invention uses temporary fixation chemistry that is designed to be reversible and easily removed, analogous to using disposable or temporary elements that serve their purpose and then are discarded or reversed. The fixation is intentionally made transient rather than permanent, allowing efficient recovery of the sample for downstream applications
2Productivity
If biological samples are removed from their viable niche, then they can be processed for detection and analysis, but physical decomposition begins immediately
Solution Approach 1:
The patent applies preliminary fixation action immediately upon sample collection to prevent decomposition before processing begins. The reversible fixation reagent is applied as a first step to stabilize the sample, allowing subsequent processing steps to be performed on a stabilized sample without immediate degradation
Solution Approach 2:
The invention changes the chemical parameters of the sample environment through reversible fixation, transforming the sample from an unstable state to a stable fixed state that can withstand processing. The reversibility ensures that the original biological state can be restored after processing, maintaining reliability throughout the workflow
3Measurement precision
If partition-based assays are used to process biological samples, then accurate control of respective environments is achieved, but sample degradation limits accurate and reproducible analysis
Solution Approach 1:
The patent applies preliminary reversible fixation before partitioning to ensure sample stability during the partitioning process and storage in discrete partitions. This allows samples to be divided into multiple partitions for high-throughput analysis while maintaining their integrity throughout the process
Solution Approach 2:
The reversible fixation reagent acts as an intermediary that temporarily stabilizes the sample during partitioning and storage, then can be removed to allow the sample to return to its native state for accurate measurement in the partition-based assay
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 stable preservation of biological samples for extended periods, allowing accurate and reproducible analysis in partition-based assays by reversing the fixation process to maintain sample integrity.
Implementation Method 1
The compounds of the present invention are defined in claim 1... contacting the sample with a first fixing reagent composition comprising a compound of formula (I)... forming bis-carbamate crosslinks with amine-bearing moieties
Implementation Method 2
followed by un-fixing with agents like DETA or DTT to maintain sample integrity for analysis
Data Source
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AI summary
The present disclosure relates to compositions and methods for reversible fixation of biological samples using fixation reagents that form bis-carbamate crosslinks between amine-bearing moieties in biomolecules.