Evaluating the dual target binding capabilities of immobilized aptamers using flow cytometry

Area of Science:

• Biochemistry
• Molecular Biology
• Analytical Chemistry

Background:

• DNA aptamers are increasingly used as recognition elements in biosensors.
• Understanding how immobilization and processing affect aptamer binding is crucial for assay development.
• Vascular Endothelial Growth Factor (VEGF) is a key biomarker in various diseases.

Purpose of the Study:

• To quantify the binding activity of particle-immobilized DNA aptamers to both nucleotide (DNA) and non-nucleotide (VEGF protein) targets.
• To investigate the impact of processing steps, such as annealing and binding history, on aptamer binding capabilities.
• To assess the recovery of aptamer binding activity after target displacement.

Main Methods:

• Utilized flow cytometry to quantify binding events of DNA and VEGF targets to functionalized particles.
• Compared binding affinities of VEGF-binding aptamers against an ampicillin-binding aptamer and a non-aptamer DNA probe.
• Investigated binding under various conditions: single target incubation, co-incubation, serial incubations, and post-displacement.

Main Results:

• Demonstrated that processing steps significantly influence the binding activity of immobilized DNA aptamers.
• Showed that DNA target binding activity can be fully recovered after displacement from aptamer:DNA duplexes.
• Observed partial recovery of protein target (VEGF) binding activity following displacement.

Conclusions:

• Immobilization and processing protocols critically affect aptamer performance in binding assays.
• Full recovery of DNA target binding and partial recovery of protein target binding are achievable.
• These findings provide insights for optimizing aptamer-based detection strategies for both nucleic acid and protein targets.