Reversible Fixation Reagents for Single-Cell Sample Preservation
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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 of biomolecules, optionally combined with paraformaldehyde for additional stabilization, and subsequent unfixing with agents like DETA or DTT to reverse the fixation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If standard fixation methods (cryopreservation, dehydration, high-salt storage, or chemical crosslinking agents) are used to stabilize biological samples, then sample stability is improved, but the ability to rapidly un-fix the sample for subsequent assays is worsened
Solution Approach 1:
The patent applies preliminary action by pre-installing cleavable crosslinks using bis-imidazole-carboxylate compounds during the fixation step. These crosslinks are designed to be stable during storage but can be rapidly cleaved by specific reagents (like DTT or hydrazine) when un-fixing is needed, thus preparing the sample in advance for rapid recovery without requiring lengthy reversal procedures.
Solution Approach 2:
The patent employs parameter changes by using fixing reagents with specific chemical properties (bis-imidazole-carboxylate compounds that form cleavable crosslinks) that can be switched between stable and unstable states. The crosslink stability can be controlled by adjusting reagent concentration, incubation time, and the specific chemical structure of the bis-imidazole-carboxylate compound, allowing optimization between stability and reversibility.
2Stability of the object's composition
If strong chemical crosslinking agents (e.g., paraformaldehyde or DSP) are used to fix biological samples, then sample stability is improved, but sample degradation still occurs over time limiting assay accuracy
Solution Approach 1:
The patent uses parameter changes by employing bis-imidazole-carboxylate compounds with specific molecular structures (varying the R groups and linker lengths) to create crosslinks with controlled stability. This allows optimization of the fixation strength while maintaining reversibility, improving both sample stability and subsequent assay accuracy by preventing irreversible degradation.
Solution Approach 2:
The patent applies composite materials by combining bis-imidazole-carboxylate compounds with complementary fixing agents like paraformaldehyde or using them in combination with specific unfixing reagents (DTT, hydrazine). This composite approach creates a synergistic effect where the bis-imidazole-carboxylate provides reversible crosslinking while other components enhance stability or facilitate controlled reversal, overall improving sample reliability.
3Reliability
If biological samples are processed without fixation to maintain native state, then assay accuracy is improved, but sample degradation occurs immediately limiting reproducibility
Solution Approach 1:
The patent introduces an intermediary substance (bis-imidazole-carboxylate compound) that mediates between the need for sample stability and the need to maintain native structure. This intermediary forms temporary, reversible crosslinks that preserve the sample during storage and transport, but can be easily removed before assays to restore the native state, thus ensuring both stability and assay accuracy.
Solution Approach 2:
The patent applies preliminary action by performing reversible fixation before storage and transport, then rapidly removing the fixation before assays. This preliminary stabilization prevents degradation during handling, while the rapid un-fixing ensures the sample is in its native state during the actual assay, improving both reproducibility and accuracy.
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
The method allows for the preservation of biological samples, enabling their use in partition-based assays by maintaining sample integrity for extended periods before un-fixing and analysis, thus facilitating accurate and reproducible assays.
Implementation Method 1
forming bis-carbamate crosslinks with amine-bearing moieties of biomolecules
Implementation Method 2
subsequent unfixing with agents like DETA or DTT to reverse the fixation
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.