IDF Solution Permeabilization for Fixed Cell Detection
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Solution Overview
Problem
Current methods for permeabilizing cell membranes after fixation are not effective for all specimens, are too rigorous, or require expensive equipment, limiting the detection of larger sized detection molecules and the accuracy of diagnostic methods for cellular targets and pathogens.
Innovation Solution
The use of an IDF solution comprising chaotropic salts, ionic and non-ionic detergents, and other reagents to create channels in cell walls and membranes, allowing probes to penetrate and detect targets such as nucleic acids, peptides, and pathogens within fixed cells, using a method that includes fixation, treatment with the IDF solution, hybridization, and counterstaining for visualization.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If fixation is performed to preserve cellular structure, then cellular molecules are set in place and degradation is prevented, but cell permeability to foreign particles is reduced and detection of internal components is hampered
Solution Approach 1:
The patent applies preliminary action by performing permeabilization treatment on fixed cells before probe hybridization. The IDF solution is applied to create channels in the cell membrane and walls after fixation, enabling subsequent probe penetration without compromising the fixed cellular structure. This sequential approach allows both preservation and detection to be achieved.
Solution Approach 2:
The patent uses an intermediary substance - the IDF solution comprising chaotropic salts, ionic and non-ionic detergents - to mediate between the fixed cellular structure and the probe. This intermediary creates permeabilizing channels that allow probe penetration while maintaining the integrity of the fixed cellular components, thus resolving the contradiction between preservation and detection.
2Difficulty of detecting and measuring
If conventional permeabilization methods are used after fixation, then cell membrane permeability is improved, but the methods are either not effective for all specimens, too rigorous destroying structures, or require expensive equipment
Solution Approach 1:
The patent applies parameter changes by formulating a specific chemical composition (IDF solution) with defined parameters - chaotropic salts at specific concentrations, ionic and non-ionic detergents in particular ratios - that optimizes permeabilization effectiveness across different specimen types while maintaining cellular structure integrity. This standardized chemical approach replaces variable physical methods.
Solution Approach 2:
The patent substitutes mechanical permeabilization methods (sonication, electroporation) with a chemical approach using the IDF solution. This replacement eliminates the need for expensive equipment while achieving effective permeabilization through chemical action of chaotropic salts and detergents that create channels in cell membranes and walls.
3Difficulty of detecting and measuring
If mechanical methods such as sonication or electroporation are used for permeabilization, then cell membrane permeability is improved, but these methods require expensive equipment and usually only work on unfixed samples
Solution Approach 1:
The patent replaces mechanical permeabilization systems (sonication apparatus, electroporation equipment) with a simple chemical treatment using the IDF solution. This substitution eliminates complex equipment requirements while enabling permeabilization of fixed samples through chemical action of chaotropic salts and detergents that dissolve cell membrane and wall structures.
Solution Approach 2:
The patent changes the permeabilization mechanism from physical parameters (mechanical vibration, electrical field) to chemical parameters (chaotropic salt concentration, detergent type and amount). This parameter change enables the method to work on fixed samples and eliminates the need for expensive mechanical or electrical equipment.
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 enhances the detection of cellular targets and pathogens by improving cell permeability, allowing for more accurate and sensitive identification of nucleic acids and other cellular components, even in fixed specimens, using a cost-effective and equipment-efficient process.
Implementation Method 1
The IDF solution can comprise of any combination of the following reagents: chaotropic salts (e.g., guanidine thiosulphate or hydrochloride), ionic detergents (e.g., SDS) and/or non-ionic detergents (e.g., IPGEL, deoxycholate, cholate or bile salts)
Implementation Method 2
ionic detergents (e.g., SDS) and/or non-ionic detergents (e.g., IPGEL, deoxycholate, cholate or bile salts)
Implementation Method 3
The probe complex (comprising binding agents specific for the target) is contacted with the target under conditions appropriate for hybridization or binding
Implementation Method 4
Most agents that are used as fixatives (e.g., alcohols such as ethanol and aldehydes such as paraformaldehyde) work by crosslinking cellular molecules, especially proteins
Data Source
AI summary
The present invention provides a method for allowing foreign particles to penetrate, very efficiently, the cell wall, cell membrane, organelle membrane and/or nuclear membrane of a cell and hybridizing or binding to the complimentary target in the cell. The cells may be from a culture or from specimens obtained from a patient. The foreign particle can be a probe consisting of, for example, either individually or in any combination of two or more of the following: DNA, RNA, peptide nucleic acids (PNA), glycopeptides, lipopeptides, glycolipids or prions. The target is a cell, a cell component or, preferably, a pathogen or pathogen component. The pathogen can be, for example, bacteria, fungi, yeast or viruses.