In-capillary probing QDs and HAT tag self-assembly and displacement using Förster resonance energy transfer

Area of Science:

• Bioconjugation chemistry
• Nanomaterials science
• Analytical chemistry

Background:

• His affinity tag (HAT) is a popular fusion tag but its positive charges hinder self-assembly with quantum dots (QDs).
• Developing methods to analyze interactions between charged ligands and QDs is crucial for nanomaterial applications.

Purpose of the Study:

• To synthesize ATTO 590-labeled HAT peptide for self-assembly with QDs.
• To develop an online capillary electrophoresis with fluorescence detection (CE-FL) method for analyzing QD-peptide interactions.
• To investigate the binding and displacement interactions between QDs and charged ligands.

Main Methods:

• Synthesis of ATTO 590-labeled HAT peptide.
• Sequential injection of QDs and labeled peptide into a capillary.
• Online self-assembly, separation, and detection using CE-FL.
• Analysis of Förster resonance energy transfer (FRET) signals.
• Investigation of displacement interactions using imidazole and H6G6 peptide.

Main Results:

• Successful online self-assembly of QDs and ATTO 590-labeled HAT peptide within 10 minutes.
• Obvious FRET signal detected, confirming energy transfer.
• Demonstration of imidazole and H6G6 peptide competing with HAT tag for QD surface binding.
• Clear separation of displacement intermediates using CE-FL.

Conclusions:

• Online CE-FL is a powerful tool for analyzing binding interactions between charged ligands and QDs.
• The developed method enables rapid, online self-assembly, separation, and detection.
• Understanding these interactions is key for advancing QD-based technologies.