Capillary flow velocity-based length identification of PCR and RPA products on paper microfluidic chips.

Area of Science:

• Biotechnology
• Analytical Chemistry
• Microfluidics

Background:

• Accurate identification of nucleic acid amplification product lengths is crucial for molecular diagnostics.
• Existing methods often require fluorescence detection or complex instrumentation.
• There is a need for rapid, cost-effective, and field-deployable assays.

Purpose of the Study:

• To develop a novel, non-fluorescence method for determining the length of polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA) products.
• To utilize capillary flow velocities on paper microfluidic chips for product length identification.
• To establish a smartphone-based assay for rapid nucleic acid analysis.

Main Methods:

• Employed paper microfluidic chips with aminated microspheres to differentiate flow velocities of PCR and RPA products.
• Utilized smartphone imaging to analyze initial capillary flow velocities under ambient lighting.
• Established a linear relationship between initial flow velocities and the square root of product lengths.

Main Results:

• Achieved high correlation coefficients (R²=0.981 for PCR, R²=0.993 for RPA) between flow velocities and product lengths.
• Demonstrated assay robustness with minimal dependency on cycle numbers or initial target concentrations.
• Validated the use of initial time frames for accurate velocity analysis through error analysis.

Conclusions:

• The developed method offers a rapid, non-fluorescence approach for nucleic acid product length identification.
• This smartphone-based paper microfluidic assay is potentially suitable for various nucleic acid amplification tests.
• The technology holds promise for developing handheld, point-of-care diagnostic tools.