Picogram quantitation of total DNA using DNA-binding proteins in a silicon sensor-based system

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Biology

Background:

• Accurate quantification of DNA is crucial for various biological and medical applications.
• Existing methods for DNA quantification may lack sensitivity or reproducibility at very low concentrations.

Purpose of the Study:

• To develop a rapid, reproducible, and highly sensitive method for quantifying total DNA at picogram levels.
• To utilize high-affinity DNA-binding proteins and a semiconductor sensor for sensitive DNA detection.

Main Methods:

• A sandwich assay was constructed using single-stranded DNA-binding protein (SSB) conjugated with biotin and a monoclonal anti-DNA antibody conjugated with urease.
• The assay involves denaturing DNA, incubating it with a reagent containing SSB-biotin and anti-DNA-urease conjugates, and forming a complex with streptavidin.
• The DNA complex is captured on a biotin-coated membrane, and signal generation is achieved by measuring pH change catalyzed by urease using a light-addressable potentiometric sensor.

Main Results:

• The developed assay can detect as little as 2 picograms (pg) of DNA.
• The method demonstrates a quantitation coefficient of variation of less than 10% within the 10 to 200 pg range.
• The assay is both rapid and reproducible, suitable for high-throughput applications.

Conclusions:

• This novel method provides a sensitive and reliable approach for DNA quantification at the picogram level.
• The combination of high-affinity DNA-binding proteins, a sandwich assay, and semiconductor sensing offers significant advantages for molecular diagnostics.
• The assay's speed, reproducibility, and sensitivity make it a valuable tool for various research and clinical settings.