Sensors and Actuators B 196 (2014) 440–449 Contents lists available at ScienceDirect Sensors and Actuators B: Chemical jo u r nal homep age: www.elsevier.com/locate/snb Off-on type fluorescent NBD-probe for selective sensing of cysteine and homocysteine over glutathione Dnyaneshwar Kand, Tanmoy Saha, Pinaki Talukdar ∗ Department of Chemistry, Indian Institute of Science Education and Research Pune, 411008, India a r t i c l e i n f o Article history: Received 10 December 2013 Received in revised form 4 February 2014 Accepted 6 February 2014 Available online 15 February 2014 Keywords: Thiol Probe UV–vis Fluorescence Bioimaging a b s t r a c t NBD-chloride is widely used as an efficient probe for selective labelling of thiols in proteins due to for- mation sulfur-substituted NBD under physiological conditions. Selective conjugation involving thiolate group is favoured over amines of proteins because, amino-substituted NBD can be formed only under more basic and elevated temperature conditions. Sulfur-substituted NBDs generally display weak fluo- rescence properties compared to amino-substituted derivatives. However, a sulfur-substituted NBD can be converted to corresponding amino-substituted derivative via S-N Smiles rearrangement. Theoretical calculations predicted off-fluorescence state for either the probe or the sulfur-substituted NBD formed upon addition of cysteine. On-fluorescence state was predicted for corresponding amino-substituted NBD derivative. Based on UV–vis and fluorescence spectroscopic studies, most efficient rearrangement was observed for cysteine. The rearrangement was relatively slower for homocysteine and not feasible for glutathione. Detection of cysteine and homocysteine by the probe resulted in 1599- and 760-fold off-on fluorescence enhancements, respectively. Sensing of cysteine by the probe provided a detection limit of 2.0 × 10 -8 M. The sensing of intracellular cysteine by the probe was also demonstrated by live cell imaging. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Reactive oxygen species (ROS), when present in moderate lev- els are responsible for processes such as proliferation and survival. However, elevated levels of ROS cause differentiation and apopto- sis inducing cell death [1–5]. Perturbation of normal ROS level also contributes to variety of diseases for example, liver damage [6], Alzheimer’s disease, cardiovascular disease, etc. [7,8]. Low molec- ular weight thiols (LMWTs) such as cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) play crucial role in scavenging ROS via the nonenzymatic thiol-disulfide redox process [9,10]. A low Cys level reduces the ability of an individual to prevent free rad- ical damage and may result in impaired function of the immune system. On the other hand, high levels of intracellular Cys pro- mote oxidative DNA damage [11]. Determination of the altered levels of Cys has been the promising approach for early detec- tion of these diseases. However, interference from other sulfhydryls often limits the proper determination of a particular diseased state. Great efforts have been made for the selective and sensitive detec- tion Cys using fluorescent probes [12–33]. However, limited Cys ∗ Corresponding author. Tel.: +91 20 2590 8098; fax: +91 20 2589 9790. E-mail address: ptalukdar@iiserpune.ac.in (P. Talukdar). specific probes which are capable of discriminating other sulfhydryls (Hcy and GSH) are known [34–39]. Among diverse fluorescence-based approaches, thiol mediated Michael addition on maleimide [17,28,40] and aromatic nucle- ophilic substitution reactions (SNAr) on 2,4-dinitrophenylsulfonyl (DNs) [22,41] have been used for developing thiol probes. How- ever, the selectivity of these probes could not be predicted prior to experimental evaluation. More predictive approach for selective Cys sensing involves the formation of thiazolidine from aldehyde [42]. Involvement of–SH and–NH 2 groups contribute to better selectivity although, Hcy is reported to compete in the sensing process by forming thiazinane product. Recently, an alternate two- step strategy has been reported by Yang and co-workers [34,39] which involves the addition (rate = k 1 ) of thiol on the nonfluo- rescent species 1 leading to thiol-conjugate 1(S) as a kinetically controlled product (Fig. 1A). Subsequent conversion of 1(S) to ther- modynamically controlled amine-conjugate 1(N) is characterized as S-N Smiles rearrangement [43]. The rate k 2 of the rearrangement is determined by the covalent-length of the spacer between the sul- fur and the nitrogen atoms. The rearrangement is more feasible for Cys because, it involves cyclic five-membered transition state. For Hcy, the rearrangement would proceed at slower rate due to for- mation of a cyclic six-membered transition state. Corresponding rearrangement involving GSH is not feasible due to the formation of http://dx.doi.org/10.1016/j.snb.2014.02.023 0925-4005/© 2014 Elsevier B.V. All rights reserved.