Related Experiment Video
Updated: Jul 4, 2026
Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction
Published on: October 6, 2022
Double-Strand Gated Biosensor for Ultrasensitive T4 PNK Detection via λ-Exonuclease-Driven Background Suppression and
Qi Wang1, Xiumei Chen1, Murong Fan1
1Anhui Provincial Key Laboratory of Green Carbon Chemistry, Key Laboratory of Embryo Development and Reproductive Regulation, Anhui Province Key Laboratory of Pollution Damage and Biological Control for Huaihe River Basin, School of Chemistry and Materials Engineering, Fuyang Normal University, Fuyang, Anhui236037, PR China.
A new double-strand gated biosensor accurately detects polynucleotide kinase (PNK) activity, overcoming limitations of traditional methods. This DNA repair diagnostic tool offers high sensitivity and specificity for clinical applications and inhibitor screening.
Area of Science:
- Biochemistry and Molecular Biology
- Nanotechnology and Biosensing
Background:
- Accurate monitoring of polynucleotide kinase (PNK) activity is crucial for DNA repair research and clinical diagnostics.
- Existing λ-exonuclease (λ-exo)-based biosensors face challenges with high background signals and false positives due to nonspecific hydrolysis and phosphorylation.
Purpose of the Study:
- To develop a novel biosensing platform for sensitive and specific detection of PNK activity.
- To overcome the limitations of conventional biosensors, reducing background interference and false-positive results.
Main Methods:
- Development of a double-strand gated (DSG) biosensing platform utilizing high-order dimer G-triplex (DG-3) structures as signal reporters.
- Integration of a dual-verification mechanism involving PNK phosphorylation and Klenow fragment polymerase-mediated dsDNA synthesis for λ-exo cleavage.
- Fabrication of DNA nanoflowers (DNFs) via rolling circle replication to enhance probe and enzyme concentration and stability.
Main Results:
- The DSG platform effectively eliminates false positives by requiring dual enzymatic verification.
- The DG-3 structure, upon cleavage, generates strong fluorescence signals.
- The DNF architecture improves biosensing stability and sensitivity, enabling detection of PNK activity from 10⁻⁴-1 U/mL with a detection limit of 3.15 × 10⁻⁵ U/mL.
Conclusions:
- The developed DSG biosensing platform provides a robust, sensitive, and specific method for PNK activity detection.
- The platform demonstrates successful application in analyzing cell extracts and screening for PNK inhibitors.
- This nanotechnology-enhanced biosensor holds significant potential for biochemical analysis and drug discovery.
