T4 DNA Polymerase-Proofread DNA Binding Identifier for Sensitive Homogeneous Immunoassays

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Aptamer-based homogeneous immunoassays offer broad applicability in bioanalysis and biodiagnosis.
• Nonspecific aptamer allostery causes signal leakage, limiting assay sensitivity.
• Existing methods struggle to overcome signal interference for enhanced detection.

Purpose of the Study:

• To develop a novel strategy to overcome signal leakage in aptamer-based immunoassays.
• To enhance the sensitivity and universality of homogeneous immunoassays.
• To investigate the role of T4 DNA polymerase in signal amplification and background reduction.

Main Methods:

• Development of a T4 DNA polymerase-proofread DNA binding identifier (ReID).
• Utilizing the dual-enzymatic activity of T4 DNA polymerase to eliminate leaked signals.
• Integrating target-induced aptamer allostery with polymerase chain reaction (PCR) signal amplification.
• Exploring the regulatory mechanism of dNTPs concentration on T4 DNA polymerase activity.

Main Results:

• Achieved an ultrasensitive protein detection limit of 8 fg/mL.
• Demonstrated highly sensitive detection of small molecules, confirming universality.
• Validated the effectiveness of the ReID proofreading approach in reducing background noise.

Conclusions:

• The ReID strategy significantly advances sensitivity and universality in homogeneous immunoassays.
• This approach offers a novel perspective for developing high-performance diagnostic tools.
• Potential applications include multiple proteomic assays and improved bioanalytical methods.