A carbazole-based “turn-on” two-photon fluorescent probe for biological Cu²⁺ detection vis Cu²⁺-promoted hydrolysis

Highlights

• CuCM showed a 66-fold fluorescence enhancement response to Cu²⁺ in aqueous solution.

• The Cu²⁺-promoted C=N bond hydrolysis sensing mechanism was fully confirmed.

• CuCM can be used to detect Cu²⁺ in living cells under two-photon excitation.

Abstract

A carbazole-based “turn-on” two-photon fluorescent probe (CuCM) was successfully designed and characterized, which can be used for Cu²⁺ detection with high selectivity, low detection limit (32.8 nM), good water solubility, large Stokes shift (117 nm) and two-photon absorption cross sections (1328 GM at 860 nm). The fluorescence enhancement was attributed to the Cu²⁺-promoted C=N bond hydrolysis sensing mechanism, which is fully confirmed by the UV–vis absorption, fluorescence, ¹H NMR titration and MALDI-TOF mass analysis. Bio-imaging study revealed that the new probe CuCM could be used as an effective two-photon fluorescent probe for detecting Cu²⁺ in living cells.

Introduction

Cu²⁺ ions, as the third most abundant transition metals in the human body, play important roles for both environmental and biological systems [1], [2]. However, excessive Cu²⁺ ions will cause imbalance in cellular processes and lead to cell death and neurodegenerative diseases including Alzheimer's disease, Wilson's disease, Parkinson's disease, and prion disease [2], [3], [4], [5]. Therefore, it is highly desirable to develop efficient detection methods for Cu²⁺ ions in environmental and biological samples.

Fluorescent probes have been evaluated as powerful tools to detect Cu²⁺ ions due to their several outstanding advantages such as the high sensitivity and selectivity, real-time detection and easiness of manipulating [6], [7], [8], [9], [10], [11]. One general approach in the design of fluorescent probes for Cu²⁺ detection is to attach a recognition group to a fluorophore to form a Cu²⁺ complex. Chelating of Cu²⁺ with probe usually induces intrinsic fluorescence quenching due to the paramagnetic property of Cu²⁺, but fluorescence “turn-off” signals are usually less sensitive and offer limited spatial resolution [12], [13], [14]. Some ones have been developed to exhibit “turn-on” fluorescence enhancement signals upon binding with Cu²⁺ [15], [16]. Alternatively, reaction-based fluorescent probes have been achieved emission enhancements by reacting with Cu²⁺ to yield fluorescent products, which could avoid the challenge of paramagnetic characteristics of Cu²⁺, and satisfy the rational design of Cu²⁺ probes for practical applications. Up to now, a few efficient fluorescent probes based on the irreversible chemical reactions promoted by Cu²⁺ have been reported, such as the hydrolysis [17], [18], [19], [20], [21], [22], [23], [24], [25], [26] and oxidation reaction [27], [28], [29], [30], [31], and some of them can realize Cu²⁺ imaging in living cells. However, those probes still encounter some problems, such as poor water solubility [17], [28], small Stokes shift [25], and high temperature [20], and most of them are designed based on single-photon fluorescence technology. Two-photon microscopy (TPM), which employs two photons for the excitation in the near-infrared wavelength (ca. 700–1100 nm), provides an opportunity to overcome the problems originated from the single-photon fluorescence technology, such as excitation with short-wavelength light (ca. 350–550 nm), photo-bleaching, photo-damage, and cellular auto fluorescence [32], [33], [34], [35], [36], [37], [38]. Therefore, the development of two-photon fluorescent probes for Cu²⁺ would be of great value. So far, only a very limited amount of fluorescent probes have realized the two-photon imaging and tracking of Cu²⁺ through the use of TPM [39], [40], [41], [42]. Chao group developed a dinuclear Ru^II polypyridyl complex (RuH₂bpip) to act as a two-photon luminescent probe for biological Cu²⁺ detection, which shows a significant two-photon absorption cross sections (400 GM) but a “turn-off” two-photon emission signals [40]. Other two-photon probes used to detect Cu²⁺ are still limited to the relatively weak two-photon emission intensities and smaller values of two-photon absorption cross sections [41], [42]. Hence, it's of our great interest to design a reaction-based two-photon fluorescent (TPF) probe for Cu²⁺ detection, which emits strong “turn-on” TP-excited fluorescence (TPEF) and has large two-photon absorption cross sections.

Herein, we report a carbazole-based “turn-on” two-photon fluorescent probe (CuCM) for Cu²⁺ with good water solubility, high selectivity, low detection limit, large Stokes shift and two-photon absorption cross sections. A donor-π-acceptor (D–π–A) two-photon fluorophore based on electron-donating carbazole and electron-withdrawing cationic pyridinium moiety was utilized, and this excellent intermolecular charge transfer (ICT) system will guarantee the good two-photon activity of the probe [43], [44], [45], [46], [47], [48], [49], [50]. Cationic pyridinium moiety also enabled the probe with good water-solubility. 2-Picolinyl hydrazide was chosen as a chelating moiety of Cu²⁺, which were easily conjugated with the carbazole-based fluorophore by the formation of C=N bond. Interestingly, we found that the C=N bond of CuCM is selectively hydrolyzed by the reaction with Cu²⁺ under mild conditions to generate the corresponding aldehyde derivative (compound 4) (Scheme 1), which was fully proved by the UV–vis absorption, fluorescence, ¹H NMR titration and MALDI-TOF mass analysis. We further demonstrated that the membrane-permeable probe can react with intracellular Cu²⁺ and exhibit bright fluorescence in living cells under two-photon excitation.