Microcapsule Semi-Permeable Shell for Single-Cell Lysis and Nucleic Acid Isolation
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Solution Overview
Problem
Current methods for single-cell PCR and RT-PCR face challenges such as complex microfluidic workflows, cell and nucleic acid loss, reduced sensitivity, and inhibition of nucleic acid amplification due to harsh lysis conditions.
Innovation Solution
The development of microcapsules with a semi-permeable shell and a core containing antichaotropic agents and polyhydroxy compounds, which allow for efficient cell encapsulation, lysis, and nucleic acid analysis without the need for sophisticated microfluidic systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If harsh lysis reagents (SDS, guanidinium thiocyanate) are used to efficiently lyse encapsulated cells, then cell lysis efficiency is improved, but nucleic acid amplification is inhibited
Solution Approach 1:
The reaction process is divided into two separate compartments: a first compartment for cell lysis and a second compartment for nucleic acid amplification. This segmentation allows harsh lysis reagents to be used in the first compartment without inhibiting the amplification reaction in the second compartment, as the reagents are physically separated by the semi-permeable membrane.
Solution Approach 2:
A semi-permeable membrane acts as an intermediary between the lysis compartment and the amplification compartment. This membrane allows small molecules and nucleic acids to pass through while retaining larger cellular components and lysis reagents, thereby mediating the transfer of nucleic acids from the lysis compartment to the amplification compartment while preventing inhibition.
2Reliability
If mild lysis conditions with non-ionic detergents are used to maintain compatibility with polymerase enzymes, then nucleic acid amplification compatibility is improved, but cell lysis efficiency is reduced
Solution Approach 1:
The reaction process is divided into two separate compartments: a first compartment for cell lysis and a second compartment for nucleic acid amplification. This segmentation allows harsh lysis reagents to be used in the first compartment without inhibiting the amplification reaction in the second compartment, as the reagents are physically separated by the semi-permeable membrane.
3Adaptability or versatility
If complex microfluidic workflows are used to perform multi-step reactions with reagent replacement, then reaction control is improved, but device complexity increases
Solution Approach 1:
The reaction process is divided into two separate compartments: a first compartment for cell lysis and a second compartment for nucleic acid amplification. This segmentation allows harsh lysis reagents to be used in the first compartment without inhibiting the amplification reaction in the second compartment, as the reagents are physically separated by the semi-permeable membrane.
Solution Approach 2:
The semi-permeable membrane enables automatic exchange of reagents and products between compartments based on concentration gradients and molecular size, reducing the need for complex external control mechanisms. Small molecules and nucleic acids automatically diffuse through the membrane while larger components are retained, providing self-regulating reaction control.
4Object-generated harmful factors
If droplet splitting and fusion operations are performed to dilute cell lysate, then inhibition is reduced, but workflow complexity and reagent loss increase
Solution Approach 1:
A semi-permeable membrane acts as an intermediary between the lysis compartment and the amplification compartment. This membrane allows small molecules and nucleic acids to pass through while retaining larger cellular components and lysis reagents, thereby mediating the transfer of nucleic acids from the lysis compartment to the amplification compartment while preventing inhibition.
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 microcapsules enable efficient retention of biological entities, improved cell lysis efficiency, and effective removal of inhibitors, allowing for high-sensitivity nucleic acid assays and easy handling of large numbers of cells in a massively parallel fashion.
Implementation Method 1
a semi-permeable shell surrounding the core
Implementation Method 2
the semi-permeable shell comprising a gel formed from a polyampholyte
Implementation Method 3
wherein the polyampholyte in the gel is covalently cross-linked
Implementation Method 4
the core comprising an antichaotropic agent and/or a polyhydroxy compound
Data Source
AI summary
The present invention concerns a method of performing one or more reactions on a biological entity, the method comprising: (i) isolating the biological entity in a microcapsule comprising a core and a semi-permeable shell; and (ii) performing the one or more reactions on the biological entity in the microcapsule.