One pot hydrothermal synthesis of fluorescent NP-carbon dots derived from Dunaliella salina biomass and its application in on-off sensing of Hg (II), Cr (VI) and live cell imaging

Highlights

• This is first report of synthesis of fluorescent carbon dots from Dunaliella salina.

• A-NPCDs were used as fluorescent probe for selective detection of Hg (II), Cr (VI).

• A-NPCDs offer excellent multi-color cell imaging.

• Limit of detection for Cr (VI) and Hg (II) were 0.018 μM.

• MTT assay showed that carbon dots were biocompatible and slightly cytotoxic.

Abstract

The impact of hazardous chemicals and toxic metal ions in the environment played havoc to the ecosystem. In the present work green chemistry approach was applied for one-step hydrothermal synthesis of nitrogen, phosphorus dual doped carbon dots, utilizing green precursor i.e, biomass of halophilic microalgae Dunaliella salina (algal derived nitrogen phosphorous carbon dots i.e A-NPCDs). Synthesized A-NPCDs were characterized through TEM, FT-IR, P-XRD, DLS and XPS. It showed appreciable optical properties with significant fluorescence quantum yield (8%)and exhibited blue color under UV – light (λ_ex = 365 nm). A-NPCDs acted as fluorescent turn off sensor for toxic metal ion such as Hg (II) and Cr (VI) with good selectivity and sensitivity. Interestingly, ANPCDs detected Cr (VI) up to 0.018μM which was below the permissible level of Cr (VI) in drinking water. Such sensing resulted because of combination of inner filter effect and dynamic quenching mechanism. Moreover, it also showed good selectivity (0.018μM) for Hg (II) via dynamic quenching mechanism. MTT assay of A-NPCDs on HEK-293 cell line showed biocompatibility with negligible cytotoxicity. Therefore, these were successfully employed for live cell imaging and intracellular detection of Hg (II) and Cr (VI) in HEK-293 cell line. Thus, green synthesized A-NPCDs may be a good alternative for chemically derived CDs in intracellular detection of Hg (II) and Cr (VI) of a complex biological environment.

Introduction

Carbon dots (CDs) are the new class of zero dimension discrete quasi spherical nanomaterials with size less than 10 nm. CDs have tremendously attracted scientific community due to its outstanding physicochemical properties such as, tunable excitation emission properties, broad excitation spectra, low cost synthesis, less toxicity, good membrane permeability, biocompatibility, ecofriendly, excellent water solubility and easy surface functionalization ability [1], and broad application in various field including cell imaging [2,3], sensing of heavy toxic metal ions and biomolecules [4,5], drug delivery [6], solar cell and photo catalysis [7]. During last five years many of the research groups have synthesized carbon dots using varieties of chemical precursors with different methodology such as chemical oxidation [8], arc discharge [9], laser ablation [10], hydrothermal and microwave irradiation [11]. These methods were cost intensive and required toxic chemicals along with multi step synthesis making it more tedious. Therefore, the hydrothermal method using cheap and ecofriendly biomass derived precursor have gained more attention because of their green chemistry nature. The approach was cost effective because of low energy consumption, easy control of reaction, no use of toxic chemicals and self-passivation of CDs without any post treatment. At present several research groups have utilized a green precursor such as bamboos leaves [12], Sargassum fluitans [13], green tea [14], Prunus avium [15], Phyllanthus acidus [16], Magnolia liliflora [17] Durian shell waste [18], Osmium sanctum [19], Prunus persica [20] Hylocereus undatus [21], Chionanthus retusus [22] Garlic [23], and eutrophic algal bloom [24], for the synthesis of CDs, which showed various practical applications such as detection of toxic metal ions, bio imaging and drug delivery. Many of the research groups have been reported the detection of heavy toxic metal ion by carbon quantum dots such as Rao et al. [25] reported the detection of Hg (II) from chemically derived N-CDs, Libo et al. [26] synthesized N-doped carbon quantum dot-graphene oxide hybrid for the selective sensing of Cd (II) and Pb (II), Edison et al. [15] reported the detection of Fe (III) in aqueous medium form green synthesized N-CDs, Liu et al. [27] synthesized highly fluorescent nitrogen-doped carbon dots for detection of Cr (VI). Moreover, detection of heavy toxic metal ion through carbon dots is good for human welfare as well as environment because most of the conventional techniques required sophisticated instruments and trained operator which are expensive and time consuming process.

Among heavy metals Hg (II) is non-biodegradable highly toxic to human beings and converted to methyl mercury (highly toxic) by microorganisms in aquatic systems. Its bioacculation in food chain ultimately affects the human body even at very low concentration [[28], [29], [30]]. The mercury ion poisoning caused adverse effect on liver, kidney, cardiovascular and central nervous system of the human body [31]. On other hand, chromium is constantly released in water bodies from the leather industries and anthropogenic processes. Chromium showed variable oxidation states i.e. Cr (III) and Cr (VI) in aqueous system. Among these Cr (VI) is highly toxic to human health because of its great mobility and carcinogenic properties [[32], [33], [34], [35]]. Therefore; it is urgent need to develop an efficient, cost effective and ecofriendly technique to detect these toxic metal ions at very low concentration in drinking water as well as in living cells.

In the present work it was demonstrated that a green precursor (algal biomass) and ecofriendly route (hydrothermal) synthesized nitrogen, phosphorous dual doped carbon dots (A-NPCDs) without using any chemical and passivating agent in one step method (Scheme 1). Dunaliella salina a unicellular wall-less alga isolated from Sambhar lake Rajasthan, India was cultivated in lab. This strain is halophilic in nature and has unique property to tolerate adverse environmental conditions such as high salinity, low temperature, high irradiance and nutrient limitations (micro and macronutrients). D. salina cells can accumulate large amount of ß-carotene [36] and glycerol [37] in specific conditions. In hypersaline conditions photosynthetic activity of D. sailna enhanced while in case of photosynthetic plants and microbes the photosynthetic efficiency decreased at similar salinity levels. Hasegawa et al. [38], reported that the growth of majority of plants inhibited at 0.2 M NaCl. Microalgal cultivation utilizes natural light, nonarable land, brackish or saline waters and minimal nutrients for growth and does not compete with agricultural farming [39]. Synthesized A-NPCDs were well dispersed in aqueous medium and showed excellent photo physical properties that led their practical applications in sensing of heavy toxic metal ions (Hg (II) and Cr (VI)) and cell imaging. Using various physical techniques it was documented that green synthesized carbon dots (A-NPCDs) showed multivariate application such as cell imaging and sensing of toxic metal ions Hg (II) and Cr (VI) in aqueous solution at physiological pH 6.8.