Dual-domain microchip-based process for volume reduction solid phase extraction of nucleic acids from dilute, large...

Area of Science:

• Forensic Science
• Biotechnology
• Analytical Chemistry

Background:

• Forensic genetic analysis often requires processing large volumes of dilute biological samples.
• Traditional nucleic acid extraction methods can be inefficient and struggle with inhibitor removal, impacting downstream analyses like PCR.
• Existing microfluidic solid-phase extraction (muSPE) methods may not adequately address volume reduction or inhibitor removal.

Purpose of the Study:

• To develop and validate a novel dual-phase microfluidic device for integrated nucleic acid purification and volume reduction.
• To demonstrate the device's capability in removing common PCR inhibitors found in forensic samples.
• To assess the device's performance for both DNA and RNA purification from various challenging sample types.

Main Methods:

• Development of a dual-phase microfluidic device integrating silica-based and chitosan-based solid-phase extraction (SPE).
• Utilized flow-mediated mixing with a herringbone structure to interface the two SPE phases.
• Tested the device with dilute whole blood, semen, semen stains, and indigo dye-inhibited blood samples for DNA purification.
• Evaluated RNA purification from a dilute semen sample, followed by reverse transcription PCR (RT-PCR).

Main Results:

• The dual-phase device achieved substantial volume reduction (50-fold for DNA, 14-fold for RNA) and effective removal of PCR inhibitors (guanidine, isopropanol, indigo dye).
• Purified DNA and RNA were successfully amplified using PCR and RT-PCR, respectively, outperforming traditional muSPE methods.
• The device demonstrated superior performance compared to traditional muSPE, particularly in inhibitor removal and achieving PCR-amplifiable results from challenging samples.
• The system operated as a closed system, reducing reagent consumption by 2.6-fold and minimizing contamination risks.

Conclusions:

• The developed dual-phase microfluidic device offers an efficient, integrated solution for nucleic acid purification and volume reduction from dilute forensic samples.
• This technology enhances the reliability of downstream genetic analyses by effectively removing PCR inhibitors.
• The device represents a significant advancement for forensic genetic analysis, improving sample processing efficiency and data accuracy.