Enhancing Target Detection: A Fluorescence-Based Streptavidin-Bead Displacement Assay.

Area of Science:

• Biotechnology
• Analytical Chemistry
• Molecular Diagnostics

Background:

• Fluorescence-based aptasensors are innovative analytical tools for detecting analytes in medicine and therapeutics.
• Conventional aptasensor designs rely on target-induced DNA aptamer structural changes for fluorescence signals, but not all aptamers exhibit sufficient conformational changes.
• This limitation hinders the development of sensitive and reliable aptasensor assays.

Purpose of the Study:

• To develop a novel fluorescence displacement assay using complementary antisense strands to enable fluorescence measurements.
• To overcome the limitations of conventional aptasensor designs where target-induced structural changes are insufficient.
• To create a versatile model assay for detecting and quantifying various targets of interest.

Main Methods:

• A streptavidin-aptamer bead complex was designed utilizing a DNA aptamer specific to the SARS-CoV-2 receptor binding domain.
• A fluorescence displacement assay was employed, where a complementary antisense strand facilitates fluorescence measurement upon target binding.
• The assay's performance was evaluated for target detection and quantification.

Main Results:

• The developed assay demonstrated a linear detection range from 50 to 800 nanomolar (nM).
• The limit of detection (LOD) was calculated at 67.5 nM, and the limit of quantification (LOQ) was determined to be 204.5 nM.
• The assay proved effective for detecting and quantifying the target analyte.

Conclusions:

• The proposed fluorescence displacement assay provides a robust method for analyte detection, overcoming limitations of traditional aptasensors.
• This 'fit-for-purpose' model assay can be adapted for detecting and quantifying diverse targets by utilizing specific DNA aptamers and their complementary antisense strands.
• The study highlights the potential of antisense strand-mediated strand displacement in advancing aptasensor technology for various applications.