Chaotropic Buffer Cell Isolation Method
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Solution Overview
Problem
Current methods for isolating cells from complex samples, such as food, face challenges like insufficient sample volume, high detection limits, low recovery rates, and are often time-consuming and costly, with limited applicability to various food matrices, making them unsuitable for reliable quantification and identification of pathogens like Listeria monocytogenes.
Innovation Solution
A method involving incubation of the sample with a chaotrope and a buffer, followed by centrifugation or filtration, which effectively dissolves the sample matrix without affecting cells with a cell wall, allowing for the isolation of viable cells using centrifugation or immunological methods, and subsequent washing to remove inhibitors for downstream analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional cell isolation methods are used, then cells can be separated from samples, but recovery rates are low and the process is time-consuming
Solution Approach 1:
The patent changes the chemical parameters of the isolation buffer by incorporating specific chaotropic agents (guanidine hydrochloride, guanidine thiocyanate) and non-ionic detergents (Tween 20, Tween 80, Triton X-100) at optimized concentrations. This chemical parameter optimization enables rapid dissolution of food matrices while maintaining cell integrity, achieving both high recovery rates and reduced isolation time compared to conventional methods
Solution Approach 2:
The patent replaces time-consuming mechanical disruption methods with a chemically-driven dissolution approach. The chaotropic buffer system chemically breaks down the food matrix structure without requiring extensive mechanical processing, thereby accelerating the isolation process while preserving cell viability and achieving high recovery rates
2Measurement precision
If large sample volumes are processed, then detection sensitivity improves, but inhibitor removal becomes more difficult
Solution Approach 1:
The patent designs a universal chaotropic buffer system that simultaneously performs multiple functions: dissolving diverse food matrices, releasing cells, and removing inhibitors. This multi-functional buffer composition handles large sample volumes effectively while addressing both detection sensitivity and inhibitor removal in a single integrated solution
Solution Approach 2:
The patent employs a composite buffer system combining chaotropic agents, detergents, and pH buffers in specific ratios. This composite formulation creates synergistic effects that enhance both the dissolution of large sample volumes and the removal of PCR inhibitors, enabling sensitive detection without increasing procedural complexity
3Productivity
If chemical extraction methods are used, then cell isolation efficiency improves, but cell viability may be compromised
Solution Approach 1:
The patent carefully controls chemical parameters including pH (maintained between 7-9 using Tris or phosphate buffers), temperature (incubation at 37-42°C), and chaotropic agent concentrations. These optimized parameters enable efficient chemical extraction while maintaining cell viability, resolving the contradiction between isolation efficiency and cell survival
Solution Approach 2:
The patent uses non-ionic detergents as intermediary substances that facilitate the chemical extraction process while being gentle on cell structures. These detergents act as mediators between the harsh chaotropic environment and the delicate cell membranes, enabling efficient isolation without compromising viability
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 enables quantitative and reproducible isolation of cells from complex samples, maintaining cell viability and facilitating their detection through techniques like real-time PCR, with improved recovery rates and broader applicability across different food matrices.
Implementation Method 1
incubating said sample with at least one chaotrope, a buffer and at least one detergent
Implementation Method 2
incubating said sample with at least one chaotrope, a buffer and at least one detergent
Implementation Method 3
isolating said cells from the resulting mixture by centrifugation and/or filtration
Implementation Method 4
isolating said cells from the resulting mixture by centrifugation and/or filtration
Data Source
AI summary
The present invention relates to a method for isolating cells from a complex sample comprising the steps of:a) providing a complex sample,b) incubating said sample with:at least one chaotrope,a buffer andat least one detergent,c) isolating said cells from the resulting mixture by centrifugation or filtration.