Rational design of a water-soluble, lipid-compatible fluorescent probe for Cu(i) with sub-part-per-trillion sensitivity

Abstract

Fluorescence probes represent an attractive solution for the detection of the biologically important Cu(I) cation; however, achieving a bright, high-contrast response has been a challenging goal. Concluding from previous studies on pyrazoline-based fluorescent Cu(I) probes, the maximum attainable fluorescence contrast and quantum yield were limited due to several non-radiative deactivation mechanisms, including ternary complex formation, excited state protonation, and colloidal aggregation in aqueous solution. Through knowledge-driven optimization of the ligand and fluorophore architectures, we overcame these limitations in the design of CTAP-3, a Cu(I)-selective fluorescent probe offering a 180-fold fluorescence enhancement, 41% quantum yield, and a limit of detection in the sub-part-per-trillion concentration range. In contrast to lipophilic Cu(I)-probes, CTAP-3 does not aggregate and interacts only weakly with lipid bilayers, thus maintaining a high contrast ratio even in the presence of liposomes.

Glutathione limits aquacopper(I) to sub-femtomolar concentrations through cooperative assembly of a tetranuclear cluster

“…The three MCL-affinity standards form complexes with apparent dissociation constants ranging from 10 pM to 100 aM at pH 7 (17,18), thus covering the full range of Cu(I) affinities previously reported for GSH. Both the free ligands and their Cu(I) complexes are transparent across the visible and near-UV range, permitting spectroscopic observation of Cu(I) complexation equilibria in their presence.…”

Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells

“…These effects can produce large changes in fluorescence independently from the target analyte, making reliable interpretation of cellular staining patterns difficult if not impossible (20). Solubilized by two anionic sulfonate groups, CTAP-3 circumvents these effects to provide a high-contrast response to Cu(I) even in the presence of lipid bilayers (13).…”

“…With these results at hand, we further characterized the probe in aqueous buffer in the presence of model lipid bilayers to evaluate its suitability for cellular imaging (19). Since cellular lipid bilayers comprise a variable mixture of neutral and anionic head groups, we prepared liposomes using a 4:1 ratio of zwitterionic palmitoyl oleoyl phosphatidylcholine (POPC) to anionic palmitoyl oleoyl phosphatidylglycerol (POPG) in aqueous buffer (13). After equilibration with the liposome mixture, crisp-17 gave a 65-nm shift in emission maximum from 555 to 620 nm upon saturation with Cu(I) (Fig.…”

“…We recently devised a water-soluble Cu(I)-selective turn-on fluorescent probe, CTAP-3, which offers a detection limit in the subpart-per-trillion range and retains a high fluorescence contrast ratio even in the presence of lipid bilayers (13). Microinjection experiments of the brightly fluorescent probe-Cu(I) complex revealed an unexpected rapid dissipation of the fluorescence signal (see below), suggesting competitive chelation of Cu(I) by endogenous ligands.…”

Dual site‐controlled two‐photon fluorescent probe for the imaging of lysosomal pH in living cells

“…Fluorescence imaging is an important method for imaging cellular pH due to its marked advantages including in situ , real‐time and non‐invasive detection . To date, many one‐photon lysosome‐targeting fluorescent probes have been developed to detect pH changes .…”