Microfluidic Nucleic Acid Isolation via Centrifugal Inhibitor Separation
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Solution Overview
Problem
Current methods for isolating nucleic acids from complex biological samples, such as blood, are not suitable for high-throughput microfluidic devices due to issues like inhibitor coprecipitation, require toxic chemicals, or are cumbersome, making them inefficient for early disease diagnosis and research applications.
Innovation Solution
A microfluidic device-based method involving a loading chamber, valved process chamber, and mixing chamber is used to separate nucleic acids from inhibitors by forming a concentrated region of nucleic acids and a less concentrated region of inhibitors, allowing for the removal of inhibitors and subsequent dilution and concentration to achieve pure nucleic acid isolation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If conventional nucleic acid isolation methods are used, then nucleic acids can be isolated from complex biological samples, but inhibitors are coprecipitated with nuclei and nucleic acid requiring additional removal steps
Solution Approach 1:
The invention extracts and removes inhibitors from the sample before nucleic acid isolation. The method involves separating inhibitors through a series of wash steps using buffers of increasing ionic strength, thereby extracting the harmful substances away from the nucleic acid-containing pellet, preventing their coprecipitation and subsequent interference with PCR reactions.
Solution Approach 2:
The invention performs preliminary removal of inhibitors before the main nucleic acid isolation process. By conducting multiple wash steps with buffers of gradually increasing ionic strength prior to final nucleic acid extraction, the method prepares the sample by eliminating harmful substances in advance, ensuring cleaner nucleic acid isolation without requiring additional post-isolation purification steps.
2Quantity of substance
If alkaline extraction with large volume water is used, then DNA can be extracted from whole blood, but the large volume makes the method unsuitable for microfluidic devices
Solution Approach 1:
The invention changes the parameters of the extraction buffers by using a series of buffers with gradually increasing ionic strength rather than large volumes of water. This parameter change allows for effective inhibitor removal and DNA extraction using small, controlled volumes of buffer that are compatible with microfluidic device constraints while maintaining extraction efficiency.
Solution Approach 2:
The invention segments the extraction process into multiple steps using different buffers with specific ionic strengths. Instead of using a single large volume of water, the method divides the wash process into sequential steps with buffers of 50 mM, 150 mM, and 500 mM ionic strength, allowing for progressive removal of inhibitors using small volumes suitable for microfluidic devices.
3Quantity of substance
If phenol chloroform extraction is used, then nucleic acids can be isolated, but toxic and corrosive chemicals are required making automation difficult
Solution Approach 1:
The invention replaces toxic phenol chloroform chemicals with disposable buffer solutions that can be easily discarded after use. The method uses a series of aqueous buffers with different ionic strengths that are non-toxic and can be simply discarded after the wash steps, eliminating the need for hazardous chemical handling and making the process suitable for automated microfluidic devices.
Solution Approach 2:
The invention substitutes the chemical extraction mechanism of phenol chloroform with a mechanical/physical separation approach using buffers of different ionic strengths. The method relies on differential solubility and precipitation based on ionic strength rather than chemical denaturation, replacing toxic chemical interactions with a safer physical separation process that is easier to automate.
4Quantity of substance
If silica solid phase extraction with alcohol wash is used, then nucleic acids can be isolated, but residual alcohol must be completely removed to prevent enzyme inhibition
Solution Approach 1:
The invention extracts and removes residual alcohol from the nucleic acid preparation through a series of wash steps using buffers that progressively dilute and remove alcohol. By washing with buffers of increasing ionic strength, the method effectively extracts residual alcohol from the nucleic acid pellet, ensuring complete removal to prevent enzyme inhibition in subsequent PCR reactions.
Solution Approach 2:
The invention performs preliminary removal of residual alcohol before the final nucleic acid elution step. By conducting multiple wash steps with buffers of different ionic strengths prior to the final elution, the method prepares the nucleic acid sample by eliminating residual alcohol in advance, ensuring that the final preparation is free from substances that would inhibit enzyme activity without requiring additional post-elution drying 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
This method enables efficient and purified nucleic acid isolation suitable for amplification processes, reducing inhibitor interference and adapting to microfluidic formats, facilitating early disease diagnosis and research applications.
Implementation Method 1
forming a concentrated region of the sample in the valved process chamber, wherein the concentrated region of the sample includes a majority of the nucleic acid-containing material and the less concentrated region includes at least a portion of the inhibitors
Data Source
AI summary
The present invention provides methods and kits for isolating nucleic acid from a sample, preferably from a biological sample, using a microfluidic device and a concentration step.