Azide–Alkyne Click Conjugation on Quantum Dots by Selective Copper Coordination

Abstract

Functionalization of nanocrystals is essential for their practical application, but synthesis on nanocrystal surfaces is limited by incompatibilities with certain key reagents. The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is among the most useful methods for ligating molecules to surfaces, but has been largely useless for semiconductor quantum dots (QDs) because Cu+ ions quickly and irreversibly quench QD fluorescence. To discover non-quenching synthetic conditions for Cu-catalyzed click reactions on QD surfaces, we developed a combinatorial fluorescence assay to screen >2000 reaction conditions to maximize cycloaddition efficiency while minimizing QD quenching. We identify conditions for complete coupling without significant quenching, which are compatible with common QD polymer surfaces and various azide/alkyne pairs. Based on insight from the combinatorial screen and mechanistic studies of Cu coordination and quenching, we find that superstoichiometric concentrations of Cu can promote full coupling if accompanied by ligands that selectively compete the Cu from the QD surface but allow it to remain catalytically active. Applied to the conjugation of a K+ channel-specific peptidyl toxin to CdSe/ZnS QDs, we synthesize unquenched QD conjugates and image their specific and voltage-dependent affinity for K+ channels in live cells.

Optical measurement of voltage sensing by endogenous ion channels

“…GxTX (Mann et al 2018). These probes are expected to respond to Kv2 channel voltage sensor 21 activation with similarly interpretable fluorescence changes, yet offer orders of magnitude 22 improvements in signal to noise ratio.…”

Electrostatic Repulsion Controls Efficiency of Cu‐Free Click‐Reaction with Azide‐Modified Semiconductor Quantum Dots

“…Fast hydrolysis of carbodiimide in water limits the time of reaction requiring a large excess of the reagent for satisfactory reaction yield. CuAAC reaction is not easily applicable to conjugation of semiconductor QDs like CdSe because of quenching of QDs PL by Cu ions . Although several papers demonstrate utilization of CuAAC for QDs conjugation, this method requires fine tuning of Cu catalyst concentration and other reaction parameters for different QDs samples.…”

“…CuAAC reaction is not easily applicable to conjugation of semiconductor QDs like CdSe because of quenching of QDs PL by Cu ions . Although several papers demonstrate utilization of CuAAC for QDs conjugation, this method requires fine tuning of Cu catalyst concentration and other reaction parameters for different QDs samples. Nowadays, SPAAC reaction seems to be the most perspective method for QDs conjugation because of its relative simplicity and the absence of Cu ions …”

“…Conjugation efficiency can be improved also by increasing the number of the functional groups on the QD surface. However, indirect quantitative analysis of the functional groups load on QDs via two‐step derivatization cannot provide high accuracy . SPAAC reaction allows achieving close to 100% yield at long reaction time without reagents degradation thus opening the way for better quantification of the QDs functional groups.…”

“…However, indirect quantitative analysis of the functional groups load on QDs via two-step derivatization cannot provide high accuracy. [9,16] SPAAC reaction allows achieving close to 100% yield at long reaction time without reagents degradation thus opening the way for better quantification of the QDs functional groups. Although, SPAAC-assisted conjugation of water-soluble QDs has been demonstrated earlier, [29] QDs encapsulated with polystyrene-co-maleic anhydride were modified via cleavage of anhydride groups of polymer by amino groups of amino-PEGs which did not allow precise control for the number of azide groups on the surface of QDs.Ref.…”