pH-Responsive 1,8-Naphthalimide Probe for a One-Step Loop-Mediated Isothermal Amplification Assay

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Loop-mediated isothermal amplification (LAMP) is a rapid nucleic acid detection method suitable for point-of-care applications.
• Current LAMP assays face challenges including insufficient sensitivity, poor probe stability, and subjective result interpretation.
• These limitations hinder the widespread adoption and acceptance of LAMP technology in clinical and field settings.

Purpose of the Study:

• To develop a novel fluorescent probe, FC-NAPH-PZ, to overcome the limitations of existing LAMP assays.
• To enhance the sensitivity, stability, and objectivity of LAMP-based nucleic acid detection.
• To enable reliable and accurate detection of target nucleic acids in a one-step format.

Main Methods:

• Development and characterization of a novel fluorescent probe (FC-NAPH-PZ) with high Stokes shift and pH reversibility.
• Application of the FC-NAPH-PZ probe in closed-tube loop-mediated isothermal amplification (LAMP) assays.
• Detection of lambda DNA and *Escherichia coli* O157:H7 using visual inspection and quantitative real-time fluorescence monitoring.

Main Results:

• The FC-NAPH-PZ probe generated a large, stable, and pH-reversible 'turn-on' fluorescence signal.
• Achieved highly sensitive (single-copy level) and ultrahigh selective detection capabilities in LAMP assays.
• Demonstrated successful one-step detection of lambda DNA and *E. coli* O157:H7 with high specificity and sensitivity.

Conclusions:

• The novel FC-NAPH-PZ fluorescent probe significantly improves LAMP assay performance.
• This probe addresses key challenges of sensitivity, stability, and subjectivity in LAMP detection.
• FC-NAPH-PZ enables robust and accurate nucleic acid testing for various applications, including pathogen detection.