Real-time PCR microfluidic devices with concurrent electrochemical detection

Area of Science:

• Biomedical Engineering
• Analytical Chemistry
• Molecular Biology

Background:

• Electrochemistry-based detection offers potential for portable nucleic acid analysis.
• Existing electrochemical methods for PCR amplicon analysis often require complex procedures like probe-modified electrodes or solid-phase amplification.

Purpose of the Study:

• To implement an in situ electrochemical (EC) detection method within a microfluidic flow-through EC-qPCR (FTEC-qPCR) device.
• To achieve simultaneous nucleic acid amplification and EC detection of PCR amplicons in a single device.
• To develop a simplified, sensitive, and probe-free EC detection method for nucleic acids.

Main Methods:

• Developed a microfluidic flow-through EC-qPCR (FTEC-qPCR) device.
• Utilized methylene blue (MB), an electroactive DNA intercalator, for electrochemical signal measurements.
• Investigated and addressed technical challenges including surface passivation, electrochemical measurement optimization, PCR inhibition by metal electrodes, and bubble-free PCR.
• Demonstrated solution-phase, symmetric PCR amplification and EC detection of lambda phage DNA.

Main Results:

• Successfully demonstrated simultaneous nucleic acid amplification and EC detection in the FTEC-qPCR device.
• Showcased a probe-free EC detection method that avoids the need for asymmetric PCR or solid-phase PCR.
• Observed a linear decrease in threshold cycle (C(t)) values with increasing input target quantity for both EC and fluorescence-based assays.
• Achieved sensitivity in EC-based detection comparable to optical fluorescence detection systems.

Conclusions:

• The FTEC-qPCR device enables simultaneous nucleic acid amplification and EC detection, offering a simplified approach.
• This probe-free EC method demonstrates comparable sensitivity to fluorescence-based detection, highlighting its potential for portable nucleic acid analysis instruments.
• The developed method overcomes limitations of existing EC techniques, paving the way for accessible hand-held diagnostic tools.