FINDER converts zero-background kinetic fingerprinting into area-scalable attomolar biomarker detection

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• Analytical sensitivity is limited by background noise, which increases with sensor area.
• False positives accumulate with larger sensor areas, hindering detection performance.
• Existing methods struggle with sensitive, rapid molecular classification under low-background conditions.

Purpose of the Study:

• Introduce FINDER (Fluorogenic INstantaneous Digital Enumeration and Recognition), a novel single-molecule platform.
• Develop a method for rapid molecular classification and amplification-free quantification.
• Overcome limitations of background noise for enhanced analytical sensitivity.

Main Methods:

• Utilize kinetic fingerprinting and fluorogenic transient probes for molecular classification.
• Implement near-zero-background conditions by suppressing solution and surface-associated noise.
• Employ multidimensional kinetic filtering for discrimination of single-nucleotide variants.

Main Results:

• Achieved attomolar detection limits for miRNA cancer biomarker hsa-miR-16.
• Demonstrated amplification-free quantification with sensitivity scaling with sensor area.
• Successfully classified individual molecules within seconds, enabling rapid per-field analysis.
• Generalized the platform for HPV16 DNA detection, RNA/DNA co-profiling, and EGFR variant discrimination.

Conclusions:

• FINDER enables sensitive, amplification-free biomarker quantification by converting kinetic specificity into area-scalable sensitivity.
• The platform facilitates semi-automated attomolar biomarker counting in practical workflows.
• FINDER offers a rapid and versatile approach for molecular diagnostics and analysis.