Optimization of multiplexed PCR on an integrated microfluidic forensic platform for rapid DNA analysis

Area of Science:

• Biotechnology
• Forensic Science
• Microfluidics

Background:

• Current forensic DNA analysis workflows are complex and time-consuming.
• On-chip polymerase chain reaction (PCR) offers potential for miniaturization and automation.
• Integrating sample preparation, amplification, and analysis on a single device remains a challenge.

Purpose of the Study:

• To design, prototype, and develop an assay for multiplexed PCR on a plastic microfluidic device.
• To enable continuous workflow processing from sample preparation (SP) to capillary electrophoresis (CE).
• To develop a microfluidic system for high-quality forensic short tandem repeat (STR) amplification with preloaded reagents.

Main Methods:

• Development of a plastic microfluidic chip for multiplexed PCR amplification of 17 DNA loci.
• Implementation of customized control systems including Peltier assemblies, valve actuators, and software.
• Encapsulation of PCR reagents using a Solid Phase Encapsulating Assay Mix (SPEAM) bead-based hydrogel fabrication process.
• Integration of SPEAM bead technology with microfluidic metering and dispensing for on-chip reagent storage and amplification.

Main Results:

• Successful on-chip amplification of 17 DNA loci directly within the microfluidic device.
• Demonstration of a continuous workflow from sample preparation to capillary electrophoresis.
• Achieved high-quality 17-plex forensic STR amplification from a reference sample using preloaded solid-phase reagents.
• Validated the reliability and shelf-life of on-chip reagents for potential point-of-care applications.

Conclusions:

• The developed microfluidic device enables efficient and automated multiplexed PCR for forensic DNA analysis.
• The SPEAM bead technology provides a stable, on-chip reagent storage solution suitable for automation and field deployment.
• This integrated system represents a significant advancement towards portable and rapid forensic identification.