A rapidly-prototyped microfluidic device for size-based nucleic acid fractionation using isotachophoresis

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Biology

Background:

• Nucleic acid (NA) size fractionation is crucial for various molecular biology applications.
• Existing commercial kits for NA size selection can be expensive and time-consuming.
• There is a need for rapid, cost-effective, and efficient NA fractionation methods.

Purpose of the Study:

• To develop and demonstrate a novel microfluidic device for size-based nucleic acid fractionation.
• To utilize isotachophoresis (ITP) and an ionic spacer for efficient NA separation.
• To provide a rapid, label-free, and cost-effective alternative to commercial NA size selection kits.

Main Methods:

• A rapid-prototyped, laser-cut plastic microfluidic device with modifiable channel dimensions was designed.
• Isotachophoresis (ITP) was employed for NA preconcentration.
• An ionic spacer and sieving matrix were used for size-based segregation of NAs.
• Custom buffering reservoirs were engineered using 1 mL pipette tips for high capacity and bubble prevention.
• The device was tested with both synthetic DNA and RNA molecular weight standards.

Main Results:

• The microfluidic device successfully achieved size-based fractionation of nucleic acids.
• Two distinct fractions were generated: small (<50 nt) NAs and NAs of all sizes.
• Demonstrated fractionation of both DNA and RNA samples.
• The entire process, including preconcentration and fractionation, was completed in under 10 minutes.
• The device demonstrated a label-free and minimally-disruptive sample collection.

Conclusions:

• The developed microfluidic device offers a simple, rapid, and cost-effective solution for nucleic acid size fractionation.
• This technology presents a promising alternative to current commercially available size-selection kits.
• Further improvements in device design can enhance performance and expand utility for various NA applications.