Reversible Bis-Carbamate Fixation for Rapid Partition Assay Unfixing
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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, as existing fixation methods do not allow for rapid and efficient un-fixing to perform assays before degradation occurs.
Innovation Solution
The use of bis-imidazole-carboxylate compounds to form reversible bis-carbamate crosslinks in biomolecules, allowing for the fixation and subsequent un-fixing of biological samples in discrete partitions, using a fixing reagent composition comprising compounds of formula (I) and an unfixing agent 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 preserve biological samples, then sample stability is improved, but the ability to rapidly and efficiently un-fix the sample for subsequent assays deteriorates
Solution Approach 1:
The patent employs reversible chemistry where the fixation state can be toggled by changing chemical parameters. The bis-imidazole-carboxylate compounds form carbamate crosslinks that can be reversed by adding specific reagents (like DTT or hydroxylamine), allowing the sample to transition between fixed and un-fixed states without permanent damage. This enables rapid un-fixing compared to irreversible methods.
Solution Approach 2:
The patent introduces an intermediary chemical system (carbamate crosslinks formed by bis-imidazole-carboxylate compounds) that acts as a reversible mediator between the fixed and native states of biological samples. These intermediates can be easily broken down by specific reagents, facilitating rapid un-fixing while maintaining sample integrity during storage.
2Stability of the object's composition
If existing chemical fixing agents that create covalent crosslinks are used, then sample preservation is improved, but sample degradation occurs before assays can be performed
Solution Approach 1:
The patent uses reversible carbamate crosslinking chemistry that can be toggled on and off. During storage, the fixed state preserves sample integrity. Before assays, adding reversing reagents (DTT, hydroxylamine) breaks the crosslinks and restores the native state, ensuring assay accuracy. This dynamic parameter control solves the contradiction between preservation and reliability.
Solution Approach 2:
The fixation system transitions from static/irreversible to dynamic/reversible. The carbamate crosslinks can be formed and broken on demand, allowing the sample to adapt between preserved (fixed) and functional (un-fixed) states. This dynamic property ensures both long-term stability and assay reliability.
3Ease of operation
If biological samples are removed from their viable niche, then sample processing for assays is enabled, but physical decomposition begins immediately
Solution Approach 1:
The patent applies preliminary fixation using bis-imidazole-carboxylate compounds immediately after sample collection and before processing. This pre-protection step stabilizes the sample during storage and transport, preventing decomposition. The reversible nature ensures the sample remains assay-ready when processing occurs.
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 the preservation of biological samples for extended periods while allowing for their subsequent un-fixing and analysis in partition-based assays, maintaining the stability of nucleic acids and proteins for accurate and reproducible results.
Implementation Method 1
bis-imidazole-carboxylate compounds to form reversible bis-carbamate crosslinks in biomolecules
Data Source
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.