Influence of segmenting fluids on efficiency, crossing point and fluorescence level in real time quantitative PCR

Area of Science:

• Biotechnology and Analytical Chemistry
• Microfluidics and Biosensing

Background:

• Two-phase segmented flow in microdevices offers advantages for biological sample analysis.
• Compatibility of segmenting fluids with biological samples and reactions is poorly understood.
• Previous studies reported reduced yield and enzyme inhibition with certain segmenting fluids.

Purpose of the Study:

• To investigate the compatibility of various segmenting fluids with real-time quantitative PCR (qPCR).
• To understand the physicochemical requirements for qPCR in segmented flow systems.
• To identify factors influencing reaction efficiency in microfluidic devices.

Main Methods:

• Utilized real-time quantitative PCR (qPCR) assays.
• Tested compatibility with diverse segmenting fluids at a static interface with the PCR mix.
• Analyzed reaction efficiency, crossing threshold (Ct), and end fluorescence.

Main Results:

• Static interfaces between segmenting fluids and PCR mix showed negligible impact on qPCR efficiency.
• Key qPCR parameters (Ct, end fluorescence) remained unaffected across various segmenting fluids.
• Inhibitory effects previously observed are likely due to dynamic fluid motion, not static contact.

Conclusions:

• Static segmenting fluid/PCR mix interfaces are compatible with qPCR, validating their use.
• Dynamic interfaces in continuously flowing systems are the probable cause of previously reported inhibition.
• This research is a foundational step toward optimizing segmented flow methodology for broader applications.