Picoliter Well Array Chip-Based Digital Recombinase Polymerase Amplification for Absolute Quantification of Nucleic...

Area of Science:

• Biotechnology
• Molecular Biology
• Analytical Chemistry

Background:

• Accurate nucleic acid quantification is crucial for research and diagnostics.
• Digital polymerase chain reaction (dPCR) offers precision but requires thermal cycling, limiting its use in resource-limited settings.
• Isothermal methods like recombinase polymerase amplification (RPA) are attractive alternatives.

Purpose of the Study:

• To develop and validate a novel picoliter well array (PWA) chip for digital RPA (dRPA) enabling isothermal DNA quantification.
• To assess the performance of dRPA on the PWA chip for sensitivity, accuracy, and speed.
• To demonstrate the potential of this method for applications in resource-limited environments.

Main Methods:

• Development of a PWA chip with 27,000 picoliter reaction wells.
• Utilized a scraping liquid blade for simple and rapid sample loading (<20 μL).
• Employed methoxy-PEG-silane surface passivation to prevent cross-contamination and a novel optical design for in situ imaging.

Main Results:

• Achieved consistent picoliter reaction volumes (314 pL) and effective isothermal DNA quantification at 39°C.
• Quantified serial dilutions of Listeria monocytogenes gDNA with an average error <11%.
• Demonstrated a total processing time of <30 minutes, a 4-fold improvement over dPCR.

Conclusions:

• dRPA on the PWA chip provides a simple, sensitive, and rapid method for nucleic acid quantification without thermal cycling.
• The developed system is suitable for fast field analysis and critical clinical diagnostics in resource-limited settings.
• This technology overcomes limitations of traditional dPCR, enhancing accessibility and speed in molecular diagnostics.