Cell Isolation Using MgCl2 and Ionic Liquid Extraction
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Solution Overview
Problem
Current methods for isolating cells from complex samples, such as food and blood, face challenges including insufficient sample volume, high detection limits, low recovery rates, and inability to distinguish between viable and non-viable cells, leading to inefficient PCR analysis and contamination issues.
Innovation Solution
A method using an extraction solution containing magnesium chloride (MgCl2) and/or an ionic liquid to effectively isolate viable cells from complex matrices through mild extraction conditions, allowing for centrifugation, affinity binding, or filtration, while maintaining cell viability and reducing matrix interference.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional extraction methods using chaotropic agents and detergents are used, then cells can be isolated from complex samples, but cell viability is reduced and matrix interference remains
Solution Approach 1:
The invention changes the chemical parameters of the extraction buffer by using MgCl2 (0.5-3 M) and ionic liquids instead of conventional chaotropic agents and detergents. This parameter change maintains cell viability while effectively disrupting the food matrix, resolving the contradiction between cell integrity and matrix disruption.
Solution Approach 2:
The extraction buffer combines MgCl2 and ionic liquids in a composite formulation that leverages the matrix-disrupting properties of ionic liquids while MgCl2 maintains cell viability. This composite approach allows simultaneous achievement of matrix disruption and cell preservation.
2Productivity
If strong lysis conditions are applied to break down complex food matrices, then cell isolation is improved, but cell viability is compromised
Solution Approach 1:
The invention uses mild extraction parameters (MgCl2 concentration 0.5-3 M, specific ionic liquids) that are sufficient to disrupt food matrices but gentle enough to preserve cell viability, avoiding the need for strong lysis conditions that would compromise cell integrity.
Solution Approach 2:
The ionic liquid acts as an intermediary substance that mediates between the food matrix and the cells. It disrupts the complex matrix structure while MgCl2 provides a protective environment for the cells, enabling efficient isolation without direct harsh treatment of the cells.
3Measurement precision
If conventional PCR methods are used on complex samples, then DNA amplification can occur, but inhibitory effects from sample components reduce detection sensitivity
Solution Approach 1:
The invention extracts and removes inhibitory matrix components from the sample using the MgCl2 and ionic liquid-based buffer, separating them from the cellular DNA. This purification step eliminates PCR inhibitors while retaining the genetic material, thereby improving detection sensitivity.
Solution Approach 2:
The extraction buffer acts as an intermediary that selectively removes inhibitory substances from the complex matrix while preserving cellular integrity and DNA quality. This intermediary step prepares the sample for sensitive PCR detection by eliminating harmful components.
4Reliability
If existing isolation methods are applied to distinguish viable cells, then some viability assessment is possible, but the methods are time-consuming and have high costs
Solution Approach 1:
The extraction buffer with MgCl2 and ionic liquids performs preliminary selection by selectively preserving viable cells during the extraction process itself. This preliminary action inherent in the extraction step reduces the need for additional time-consuming viability assessment steps.
Solution Approach 2:
The extraction buffer serves multiple functions simultaneously: it disrupts the food matrix, isolates cells, and selectively preserves viable cells. This multi-functionality consolidates several steps into one, reducing overall isolation time while maintaining viability discrimination capability.
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 method achieves high recovery rates of viable cells, enabling efficient downstream analysis, including real-time PCR, with improved reproducibility and reduced contamination, particularly in complex food samples like cheese.
Implementation Method 1
an extraction solution that comprises at least MgCl2 and/or an ionic liquid
Implementation Method 2
incubating said sample with an extraction solution that comprises MgCl2 in concentrations between 0.5 and 3 M
Implementation Method 3
isolating said cells from the mixture of step b) by centrifugation
Data Source
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AI summary
The present invention relates to a method and kit for the isolation of cells from a sample. The sample is treated with an extraction solution that comprises at least MgCl2 and/or an ionic liquid resulting in the isolation of preferably viable cells.