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.

Scheme 1. Synthesis of fluorescent probe CTAP-3 (±)-2.

Fig. 1. (A) Titration of CTAP-3 (4 μM) in pH 6 buffer (10 mM MES, 100 nM MCL-1) with 0.4 μM aliquots of Cu(i) generated in situ by reduction of Cu(ii)SO₄ with 100 μM sodium ascorbate (λ_ex = 365 nm). (B) Fluorescence response of CTAP-3 (4 μM) in pH 7 buffer (10 mM PIPES, 100 nM MCL-1) to divalent cations (20 μM transition metals, 10 mM Ca(ii) and Mg(ii)) and to Cu(i) generated by in situ reduction of 5 μM Cu(ii) with 100 μM sodium ascorbate. The probe responded normally to Cu(i) in the presence of all competing ions (not shown). Excitation at 365 nm. (C) Titration of CTAP-3 (2 nM) in ascorbate buffer (200 μM ascorbic acid, 170 μM KOH, 100 pM MCL-3) with 15 pM (0.95 part per trillion) aliquots of Cu(ii)SO₄ reduced in situ to Cu(i). Excitation at 355 nm (10 nm monochromator bandpass) with 370/36 nm sharp cutoff bandpass filter. Emission collected at 455 nm (10 nm bandpass). Error bars represent the standard deviation over 6 successive 5 second measurements for each titration point.

Fig. 2. (A) Fluorescence response of CTAP-2 (2 μM, λ_ex 380 nm) upon saturation with Cu(i) in the presence of DMPC/DMPG liposomes (4 : 1 mixture, 100 μM) in pH 7.0 buffer at 25 °C (10 mM PIPES, 0.1 M KClO₄, 100 nM MCL-1). (B) Fluorescence response of coppersensor-3 (CS3, 2 μM) under the same set of conditions (λ_ex 530 nm). Inset: time-dependent evolution of the fluorescence intensity at 550 nm prior to addition of Cu(i). (C) Fluorescence response of CTAP-3 (2 μM) under the same set of condition used in (A) for CTAP-2 (λ_ex 365 nm).