Isotachophoresis Nucleic Acid Extraction for FFPE Sample Purification
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Solution Overview
Problem
Existing methods for nucleic acid extraction and purification from formalin-fixed paraffin-embedded (FFPE) samples are labor-intensive, difficult to automate, and result in low yield and quality issues due to cross-linked proteins binding DNA and RNA, leading to failed downstream assays.
Innovation Solution
The use of isotachophoresis (ITP) for sample preparation, including extraction, purification, and enrichment, which selectively focuses nucleic acids using a discontinuous buffer system to achieve high yield and quality nucleic acid samples from FFPE and other biological samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Extent of automation
If traditional manual methods are used for nucleic acid extraction and purification from FFPE samples, then the process can be completed with existing equipment, but the procedure becomes labor-intensive and difficult to automate
Solution Approach 1:
The sample preparation process is divided into distinct functional zones within the microfluidic device: a first region for deparaffinization and a second region for nucleic acid extraction and purification. This segmentation allows each region to be optimized for its specific function while enabling automated operation through integrated fluid control.
Solution Approach 2:
The patent introduces an intermediary microfluidic device that acts as a bridge between sample input and downstream analysis. This device incorporates automated fluid handling, temperature control, and separation mechanisms, transforming a manual multi-step process into an automated integrated system.
2Object-affected harmful factors
If traditional deparaffinization methods using xylenes are employed, then paraffin removal can be achieved, but the process becomes hazardous due to flammability and requires additional safety measures
Solution Approach 1:
The patent replaces the hazardous xylene-based deparaffinization process with a water-based or alcohol-based system that uses elevated temperature and enzymatic treatment. This converts a harmful chemical process into a safer thermal-biological process that achieves the same deparaffinization goal without flammability hazards.
Solution Approach 2:
The deparaffinization method changes the physical parameters of the process by using elevated temperatures (e.g., 60-100°C) and alternative solvents (water or alcohol) instead of room temperature xylene. This parameter change eliminates the flammability hazard while maintaining effective paraffin removal through enhanced solubility and enzymatic activity at higher temperatures.
3Quantity of substance
If cross-link reduction and enzyme treatment are performed to release bound nucleic acids, then nucleic acid availability improves, but the process requires additional time and effort
Solution Approach 1:
The microfluidic device performs preliminary deparaffinization and enzymatic treatment in the first region before nucleic acid extraction in the second region. By pre-processing the sample to remove paraffin and activate enzymes, the device reduces the time required for subsequent nucleic acid release and improves overall yield without adding significant time to the total process.
Solution Approach 2:
The patent implements continuous processing where deparaffinization, enzymatic treatment, and nucleic acid extraction occur in sequence without interrupting the sample flow. The microfluidic system maintains continuous fluid movement and reagent delivery, eliminating idle time between steps and keeping the useful action continuous throughout the process.
4Reliability
If non-crosslinked nucleic acids are not removed after digestion, then the process remains simple, but downstream assays may fail due to contamination with non-crosslinked material
Solution Approach 1:
The patent extracts and removes non-crosslinked nucleic acids from the sample in the second region of the microfluidic device. This selective removal of contaminating non-crosslinked material improves the reliability of downstream assays by ensuring that only crosslinked nucleic acid-protein complexes proceed to analysis, while eliminating the need for complex additional purification steps.
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
ITP methods and devices enable faster, less manually intensive sample preparation with higher yield and quality, suitable for both small and large starting amounts of tissue, and improve the success rate of downstream analyses.
Implementation Method 1
Isotachophoresis (ITP) is an electrophoretic technique which can use a discontinuous buffer containing a leading electrolyte (LE) with a higher effective mobility magnitude and a trailing electrolyte (TE) with a lower effective mobility magnitude
Implementation Method 2
a first loading reservoir in fluid communication with said first channel via a first aperture in said first channel, wherein said first aperture is positioned between said first and second capillary barriers
Data Source
AI summary
The present disclosure relates to fluidic systems and devices for processing, extracting, or purifying one or more analytes. These systems and devices can be used for processing samples and extracting nucleic acids, for example by isotachophoresis. In particular, the systems and related methods can allow for extraction of nucleic acids, including non-crosslinked nucleic acids, from samples such as tissue or cells. The systems and devices can also be used for multiplex parallel sample processing.