Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications

Area of Science:

• Biotechnology
• Molecular Biology
• Biosensing

Background:

• Strand-displacement amplification (SDA) is a powerful isothermal nucleic acid amplification technique.
• Real-time monitoring of SDA reactions is crucial for advancing biosensing applications.
• Current methods often lack real-time capabilities or require labels.

Purpose of the Study:

• To develop a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions.
• To promote the application of SDA techniques in biosensing platforms.
• To demonstrate the utility of the strategy for detecting specific enzyme activities.

Main Methods:

• Introduced G-quadruplex structures into SDA products.
• Utilized the fluorogenic dye thioflavin T for specific G-quadruplex recognition.
• Performed real-time monitoring on a commercial real-time PCR instrument.
• Designed biosensors for uracil-DNA glycosylase (UDG) and EcoRI activity.

Main Results:

• Successfully achieved real-time monitoring of both linear and exponential cross-triggered SDA.
• Demonstrated high sensitivity and specificity for UDG (6×10⁻⁵ U/mL) and EcoRI (0.016 U/mL) detection.
• Validated detection in complex biological matrices like cell lysates and human serum.
• Showcased suitability for commercial real-time PCR instruments.

Conclusions:

• The developed label-free, real-time SDA strategy is effective for biosensing.
• This approach offers advantages over traditional end-point detection, including speed, sensitivity, and reduced contamination risk.
• The strategy has the potential to significantly advance SDA-based biosensor design and application.