Particle Detection via Electroporation and Flow Cytometry
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Solution Overview
Problem
Current methods for detecting biotic and abiotic particles in samples, particularly in the food and biotechnological industries, are limited by their inability to differentiate between biological and non-biological particles, require toxic dyes, and are slow, making them unsuitable for real-time monitoring and safe operation.
Innovation Solution
A method involving electroporation of liquid samples to increase cell membrane permeability, allowing non-toxic fluorescent dyes to stain biotic particles, followed by flow cytometry for rapid and sensitive detection and quantification of both biotic and abiotic particles.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If cultivation-based methods are used to detect biotic particles, then the presence of microorganisms can be determined, but the detection time is long (at least one to five days) and the method cannot detect viable but non-culturable microorganisms
Solution Approach 1:
The patent replaces the biological cultivation process with a physical electroporation method combined with flow cytometry. Instead of relying on microbial growth over days, the method uses electrical pulses to permeabilize cell membranes and immediately detects cells through fluorescent staining and flow cytometric analysis, reducing detection time from days to minutes while maintaining reliability through direct cell counting.
Solution Approach 2:
The patent changes the detection parameter from indirect cultural growth to direct physical detection of cell membranes. By using electroporation to alter membrane permeability and combining it with fluorescent dyes and flow cytometry, the method detects biotic particles based on their physical properties rather than their biological activity, enabling rapid detection of both culturable and non-culturable microorganisms.
2Productivity
If flow cytometry with fluorescent dyes is used to detect particles, then rapid detection is achieved, but toxic and mutagenic dyes are required which are not suitable for food and biotechnology industries
Solution Approach 1:
The patent changes the chemical property of the fluorescent dye from toxic/mutagenic to non-toxic. By selecting appropriate fluorescent dyes that do not compromise safety while maintaining the ability to stain cell membranes after electroporation, the method achieves rapid detection without the harmful effects associated with traditional flow cytometry dyes, making it suitable for food and biotechnology applications.
3Quantity of substance
If conventional flow cytometry methods are used, then total cell count can be determined, but differentiation between biotic and abiotic particles is not possible
Solution Approach 1:
The patent applies local quality by using electroporation specifically on biotic particles to alter their membrane permeability, while abiotic particles remain unaffected. This creates a differential response where only biotic particles take up the fluorescent dye, enabling clear differentiation between living and non-living particles based on their selective response to the electroporation-staining protocol.
Solution Approach 2:
The patent introduces electroporation as an intermediary step that mediates between the detection method and the particles. This intermediary process selectively permeabilizes biotic cell membranes, allowing the fluorescent dye to enter and stain only biotic particles. The intermediary electroporation step thus provides the discriminatory capability needed to differentiate biotic from abiotic particles while maintaining rapid detection.
4Reliability
If cultivation-based methods are used, then microbial presence can be detected, but the complexity of bacterial colonies at the culture level is not correctly assessed
Solution Approach 1:
The patent replaces the biological cultivation system with a direct physical detection system using flow cytometry. Instead of observing complex colonial growth patterns that require days of incubation, the method directly counts and characterizes individual cells based on their physical properties (size, granularity, fluorescence), providing immediate information about microbial complexity without relying on cultural development.
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 fast, safe, and sensitive discrimination between biotic and abiotic particles, reducing operational costs and avoiding thermal effects, allowing for real-time monitoring and online analysis of contaminants in food and biotechnological processes.
Implementation Method 1
subjecting a liquid sample to electroporation to obtain an electroporated liquid sample
Implementation Method 2
at least one fluorescent dye is added to the liquid sample before and/or after electroporation
Implementation Method 3
detecting and/or quantifying particles labelled with said dye in the electroporated sample and determining the presence and/or amount of abiotic particles by detecting and/or quantifying particles not labelled with said dye in the electroporated sample
Data Source
AI summary
The present invention relates to a method for determining the presence and/or amount of biotic and abiotic particles in a liquid sample.