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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 H6G6 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.