A reaction-based near-infrared fluorescent sensor for Cu²⁺ detection in aqueous buffer and its application in living cells and tissues imaging

Highlights

• A reaction-based sensor for Cu²⁺ detection with NIR excitation and emission.

• The detection limit for Cu²⁺ is as low as 29 nM in aqueous buffer.

• The probe shows special selectivity for Cu²⁺ over other metal ions.

Abstract

Copper (II) is one of the most of important cofactors for numerous enzymes and has captured broad attention due to its role as a neurotransmitters for physiological and pathological functions. In this article, we present a reaction-based fluorescent sensor for Cu²⁺ detection (NIR-Cu) with near-infrared excitation and emission, including probe design, structure characterization, optical property test and biological imaging application. NIR-Cu is equipped with a functional group, 2-picolinic ester, which hydrolyzes in the presence of Cu²⁺ with high selectivity over completed cations. With the experimental conditions optimized, NIR-Cu (5 μM) exhibits linear response for Cu²⁺ range from 0.1 to 5 μM, with a detection limit of 29 nM. NIR-Cu also shows excellent water solubility and are highly responsive, both desirable properties for Cu²⁺ detection in water samples. In addition, due to its near-infrared excitation and emission properties, NIR-Cu demonstrates outstanding fluorescent imaging in living cells and tissues.

Introduction

Copper is a redox-active metal and an essential trace element for life (Boal and Rosenzweig, 2009, Ridge et al., 2008). It helps to generate red blood cells and maintain the health of immune system, blood vessels, bones and nerves (Davis and O'Halloran, 2008, Dethloff and Bailey, 1998, Giavaresi et al., 2005, Prohaska and Gybina, 2004, Ross et al., 2001, Thiele and Gitlin, 2008). In particular, the potent redox capacity of copper, mainly in Cu²⁺ and Cu⁺ forms, is utilized for a wide array of neuronal activities, such as neurotransmission, synaptogenesis, neurogenesis, neurite outgrowth, neurotransmitter biosynthesis, oxidative phosphorylation and oxygen transport (Birkaya and Aletta, 2005, D'Ambrosi and Rossi, 2015, Ishida et al., 2013, Opazo et al., 2014, Peters et al., 2011). In particular, recent studies have shown that abnormal concentration of copper can damage the neuronal system, resulting in neurodegenerative diseases such as Alzheimer's disease (Eskici and Axelsen, 2012, Marx, 2003), Parkinson's disease (Davies et al., 2016, Lovati et al., 2009) and Amyotrophic lateral sclerosis (Gaggelli et al., 2006, Trumbull and Beckman, 2009). Therefore, chemical tools and approaches that can detect copper activities in living specimens are highly important.

Among various approaches, fluorescence-based methods have shown to be cost-effective, sensitive, and non-invasive to the samples. However, copper responsive fluorescent probes for biological models remains underdeveloped due to significant challenges in overcoming issues of metal special selectivity and specificity for different valences of copper. For example, fluorescent probes for Cu²⁺ detection has recently been reported with high sensitivity and selectivity (Aron et al., 2015, Carter et al., 2014, Cotruvo et al., 2015, Que et al., 2008). However, the vast majority of reported fluorescent probes for Cu²⁺ detection display turn-off response (Huang et al., 2009, Jung et al., 2013, Sarkar et al., 2015, Xu et al., 2014), especially for nano-sized fluorescent probes (Chen et al., 2009, Wang et al., 2014, Zong et al., 2011). Despite the fact that several probes have been reported with fluorescence enhancement properties and Cu²⁺ specificity, they still have major drawbacks: (1) high susceptibility to interfering ions such as Hg²⁺, Zn²⁺ and Fe³⁺ (Ding et al., 2013, Liu et al., 2013, Liu et al., 2012, Liu et al., 2015, Yu et al., 2008); (2) lack of good water solubility, and require organic solvents as test condition (Kowser et al., 2016, Tang and Cai, 2012, Wu et al., 2013); (3) excitation wavelength at ultraviolet light, which at high intensities, may damage biological samples (Cao et al., 2015, Hu et al., 2016, Xu et al., 2010, Yuan et al., 2015). Therefore, developing probes with good water solubility and NIR excitation and emission for Cu²⁺ detection remains a significant challenge.

To the best of the author's knowledge, only one probe has been reported for Cu²⁺ detection with near-infrared (NIR) emission (Li et al., 2011). This probe, however, suffers from low sensitivity, showing only a 10-fold fluorescent enhancement. In addition, the working mechanism of the probe is based on chelate coordination between Cu²⁺, nitrogen and oxygen atoms. One key disadvantage of this design is that Cu²⁺ is ‘consumed’ by the probe and that, when the concentration of Cu²⁺ is low, even if the sample is put under a long incubation time, the sensitivity is low.

In this paper, we report a reaction-based NIR excitation and emission probe, NIR-Cu, for Cu²⁺ detection in aqueous buffers, living cells and tissues. The paper describes the probe design, synthesis, characterization and spectral properties. This reaction-based fluorescent probe displays fluorescence enhancement by reacting with Cu²⁺ to yield fluorescent products with little affinity to Cu²⁺, which can avoid the shortcoming of probes with binding and cooperating affection. NIR-Cu has an ester bond between the NIR dye and 2-picolinic acid, and it hydrolyzes in the presence of Cu²⁺ with high selectivity in the presence of other metal ions. Moreover, detection limit for Cu²⁺ is as low as 29 nM, which translates to high sensitivity. In addition, excellent water solubility and fast response widen its utilities in biological samples. As a proof of concept, we have develop the ability of NIR-Cu to monitor Cu²⁺ with confocal fluorescence imaging in tissues as deep as 200 µm, which demonstrates the suitability of the probe in practical applications involving biological specimens.