L-Phenylalanine doped carbon quantum dots: Non-toxic, High Performance photoluminescent agent for in-vitro bioimaging applications

Abstract

As compared to fluorescent organic dyes, traditional fluorophores and conventional semiconductor quantum dots, Carbon dots are promising fluorescent nanomaterials which possess remarkable properties such as excellent biocompatibility, stability, low toxicity, high luminescence, large surface area to volume ratio, flexible surface for conjugations with various biomolecules, tuneable size and optical properties [1, 2]. These properties endow carbon dots with wide range of applications for diverse areas which include sensors, theranostic, diagnostics, photocatalysis and bioimaging [2]. These fluorescent biocompatible CDs are versatile materials which can be integrated with various functional groups for dual purpose of targeted drug delivery and simultaneous imaging [1]. They are capable of efficient cellular internalization and stimuli-responsive drug release [1]. Although, CDs show high photoluminescence (PL), often they exhibit relatively low quantum yield (QY), indicating weak emission fluorescence [1]. Since, low QY cannot be ignored, therefore, efficient strategies are required to improve the PL properties of CDs. With advances in nanotechnology research, nitrogen doping has emerged as one of the most effective strategies for the same [3]. The optoelectronic properties of carbon dots can be enhanced by nitrogen (N) doping [4]. N-doping introduces defects and subsequently increases the upward shift in the Fermi level with simultaneous increase in electrons in the conduction band. This introduces new energy states linked to N-functionalities and eventually enhances the PL emission. The hybridization between the dopant molecule and carbon backbone plays an important role in PL mechanism and also, the incorporation of amine group in molecules govern the molecular state of formation of CDs [4]. Amino acids being non-toxic and biocompatible, they have gained much attention in biomedicine and can serve as attractive doping agents. Among the amino acids, we selected phenylalanine for this study. Phenylalanine is nutritionally classified as an indispensable amino acid and can be converted to tyrosine by phenylalanine hydroxylation [5].

            In this study, L-Phenylalanine (P) amino acid was used as nitrogen dopant in CDs. One pot microwave assisted method was used to synthesize P-CDs from citric acid and phenylalanine. Microwave assisted method is rapid, provides uniform heating with energy saving and produces higher yield in shorter preparation time. Herein, citric acid and L-phenylalanine as precursor and nitrogen dopant, respectively were mixed and irradiated at 480 W for 15 min. The as-prepared CDs were physico-chemically characterized using UV-vis absorption spectrometer, X-ray diffractometer (XRD), Fourier Transform Infrared (FTIR) spectrometer, Energy Dispersive X-ray spectrometer (EDX), Photoluminescence spectrometer. The UV–vis spectrum of P-CDs showed two absorption peaks at 250 and 340 nm (Fig.1A). The strong absorption peak at 250 nm attributed to π-π* transition of C=C (benzene), while the characteristic small shoulder absorption peak at 340 nm revealed the n-π* transition due to p-π orbit between the N aromatic and conjugate structure [5]. PL-spectra showed strong emission maxima at 440 nm with excitation wavelength of 340 nm (Fig. 1B). The observed strong PL efficiency might be due to n-π* transition. Further, EDX spectrum revealed characteristic peaks for constituents of P-CDs (Fig. 1C) and the weight percentage of the constituents was carbon (40.38%), oxygen (48.37%) and nitrogen (11.25%). XRD profile of P-CDs showed the broad characteristic peak for amorphous carbon at 2?=22º (Fig. 1D). The P-CDs exhibited negligible toxicity against HeLa cell lines and microbial fungal pathogen (C. albicans), which demonstrated their suitability for in-vitro and in-vivo steady state and live imaging applications. P-CDs were readily internalized in the fungal cells, which was confirmed by the blue fluorescence visible inside the cells (Fig. 1E). It can be concluded from this study that P-CDs are promising, biocompatible, highly fluorescent nano-bioimaging agents, with high PL and tuneable optical properties, which can be used efficiently for in-vitro and in-vivo imaging in biotechnology and nanomedicine.