Highly Sensitive Flow Cytometry Biosensor with a High Signal-to-Background Ratio for FEN1 Analysis via Solid-Phase...

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Flap endonuclease 1 (FEN1) is vital for DNA replication and repair.
• FEN1 overexpression correlates with tumor progression and drug resistance.
• Sensitive analytical methods for FEN1 detection are lacking.

Purpose of the Study:

• To develop a highly sensitive flow cytometry biosensor for FEN1 analysis.
• To improve upon existing primer exchange reaction amplification methods.
• To enable sensitive detection of FEN1 in biological samples and for inhibitor screening.

Main Methods:

• Development of a flow cytometry biosensor incorporating solid-phase interface-mediated primer exchange reaction amplification (s-PER).
• Comparison of s-PER with homogeneous PER amplification (h-PER).
• Assay validation using FEN1 standards, cell samples, and serum samples.

Main Results:

• Solid-phase PER amplification (s-PER) significantly suppressed background noise, achieving a signal-to-background ratio over 46-fold higher than h-PER.
• The developed FCsperA biosensor demonstrated high sensitivity with a limit of detection (LOD) of 2.53 × 10^-6 U/μL for FEN1.
• FCsperA showed excellent selectivity, anti-interference ability, and was capable of detecting FEN1 in approximately 10 cancer cells and serum samples.

Conclusions:

• FCsperA provides a highly sensitive and selective method for FEN1 detection.
• The biosensor is applicable for analyzing FEN1 in complex biological matrices like cells and serum.
• FCsperA holds promise for clinical diagnostics and the screening of FEN1 inhibitors for cancer therapy.