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

HAT tag, a natural His affinity tag, is one of the most popular fusion tags. However, HAT tag containing three positive charges limited its self-assembly with quantum dots (QDs). Herein, ATTO 590-labeled HAT peptide was synthesized to self-assemble with QDs inside the capillary. QDs and ATTO 590-labeled HAT peptide were sequentially injected into the capillary and probed by fluorescence-coupled CE (CE-FL), showing an obvious Förster resonance energy transfer signal. Online self-assembly, separation, and detection were achieved within 10 min. CE-FL further revealed that imidazole and H₆ G₆ peptide could partially outcompete with HAT tag on the QDs surface inside the capillary. The displacement intermediates were separated clearly using CE-FL. Our study demonstrates the power of online CE-FL in analyzing the binding interaction between ligands containing positive charges and QDs.