Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Label free phosphate functionalized semiconducting polymer dots for detection of iron(III) and cytochrome c with application to apoptosis imaging Mojtaba Shamsipur a,* , Ammar Chabok b,1 , Fatemeh Molaabasi c,1 , Amir Seyfoori c , Behnam Hajipour-Verdom d , Behnaz Shojaedin-Givi e , Mosslim Sedghi d , Hosein Naderi-Manesh d , Ali Yeganeh-Faal f a Department of Chemistry, Razi University, Kermanshah, Iran b Department of Chemistry, Faculty of Basic Sciences, Tarbiat Modares University, Tehran, 14115-154, Iran c Department of Biomaterials and Tissue Engineering, Breast Cancer Research Center, Motamed Cancer Institute, ACECR, Tehran, Iran d Department of Biophysics, Faculty of Biological Sciences, Tarbiat Modarres University, Tehran, 14115-154, Iran e Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, 14115-154, Iran f Department of Chemistry, Faculty of Basic Sciences, Payame Noor University, Tehran, Iran ARTICLE INFO Keywords: Phosphate-functionalized polymer dots Fluorescent nanoprobe Apoptosis imaging Fe(III) Cytochrome c ABSTRACT We report on facile synthesis and characterization of phosphate-functionalized polymer dots (PDs) by doping tributyl phosphate (TBP) in a semiconducting polymer poly[9,9-dioctylfluorenyl-2,7-diyl)-co-1,4-benzo-{2,10- 3}-thiadiazole)] (PFBT). Then, the prepared TBP@PFBT PDs were used to develop a very high sensitive probe for detection Fe 3+ , Cu 2+ ions and Cytochrome c based on aggregation induced fluorescence off mechanism. The PDs exhibited a linear dynamic range for Fe 3+ from 0.1 to 2 nM with a detection limit of 30 pM and for Cu 2+ from 2.0 to 50.0 nM with a detection limit of 0.35 nM. Meanwhile, this probe showed a linear dynamic range for Cyt c from 175 to 1750 pM with a detection limit of 32.7 pM. The TBP@PFBT PDs is a simple, one-step, fast, non- invasive, label-free, and inexpensive probe that is capable of online apoptosis monitoring response to drugs with an ever-present opportunity to contribute in a variety of in-vitro and in-vivo biological applications. We also obtained sharp, specific 2D and 3D imaging results for early stage apoptosis in breast cancer cells. Moreover, this technique possesses the advantage of rapid determination of Fe 3+ ion in biological or environmental samples. Importantly, this label-free assay provides short determination time of only a few min, easy operation and very low LOD allowing 100–4000 times increased in sensitivity over previously reported probes, together with high selectivity without need to using biorecognition elements like enzymes, antibodys and/or aptamers. Such ex- cellent features make the TBP@PFBT PDs an excellent probe for successful apoptosis imaging in live cells. 1. Introduction Iron and copper are two essential metals in manycrucial biological functions of cells. Because these two metals are used extensively in agriculture and industry, a trace amount of their ions may exist in natural waters. The free Fe 3+ and Cu 2+ ions are known to induce cy- totoxicity that causing serious problems such as liver and kidney dis- ease and even death (Georgopoulos et al., 2001; Kozlowski et al., 2006; Ryan and Ray, 2004; Torrado et al., 1998). Also, it is great motivators to develop very high sensitive approaches to analysis metals in a range of ppb or lower in some industries as measuring trace contamination in semiconductor-grade silicon (Fabry et al., 1994). Analysis methods at this levelneed expensive instrumentation and time-consuming routs and as a result, there is a great effort to develop, simple and easy methods based on cheap instrumentation. Apoptosis is a crucial programmed cell death, which refers to pathways that regulated cellular homeostasis and finally lead the cells to differentiation or death(Sun et al., 2010). Apoptosiscan interestingly track various biological processes including pathogenesis of different malignancies and autoimmune diseases, anticancer drug screening, normal cell turnover and etc.(Ruan et al., 2012) Generally, apoptosis can be followed by a change in cell morphology, mitochondrial function https://doi.org/10.1016/j.bios.2019.111337 Received 14 January 2019; Received in revised form 6 May 2019; Accepted 17 May 2019 * Corresponding author. E-mail address: mshamsipur@yahoo.com (M. Shamsipur). 1 These authors have the same contributions in this work. Biosensors and Bioelectronics 141 (2019) 111337 Available online 20 May 2019 0956-5663/ © 2019 Elsevier B.V. All rights reserved. T