Hydrothermal synthesis of gelatin quantum dots for high-performance biological imaging applications

Highlights

• Non-toxic gelatin quantum dots (GeQDs) have been synthesized via hydrothermal method.

• The GeQDs were used for the cell imaging of bacterial cells including E. coli and S. aureus.

• The GeQDs were used for the cell imaging of yeast cells including C. albicans, C. krusei, C. parapsilosis, and C. tropicalis

• The GeQDs were used for the cell imaging of mycelial fungi including A. flavus and A. fumigatus cells.

• The GeQDs were used for the cell imaging of A549, HEK293, L929 cell lines.

Abstract

In the present study, we are reporting a one-pot synthesis of gelatin quantum dots (GeQDs) by the hydrothermal process. The synthesized GeQDs were characterized by fourier transform infrared spectroscopy, nuclear magnetic resonance, ultraviolet-visible and photoluminescence spectroscopic techniques, and also by using high-resolution transmission electron microscopy. The GeQDs showed a high level of photoluminescence quantum yield (PLQY) with significantly higher stability for up to 6 months and presented similar fluorescent intensity as the initial PLQY without any precipitation and aggregation at ambient condition. The cell imaging ability of synthesized GeQDs was examined using cells belonging to diverse clinical backgrounds like bacterial cells including Escherichia coli and Staphylococcus aureus, yeast cells including Candida albicans, C. krusei, C. parapsilosis, and C. tropicalis, mycelial fungi including Aspergillus flavus and A. fumigatus cells, cancer cell lines A549, HEK293 and L929. The results demonstrated that the GeQDs illuminates the cells and can be utilized as potential cell labeling non-toxic biomarkers. In conclusion, it can be said that the gelatin stabilized QDs are a promising candidate for stable and long-term fluorescent imaging of different types of cells.

Introduction

In recent years, researchers have shown a keen interest in the fabrication of nanoparticles such as nanowires [1], nanodiamonds [2], luminescent graphene [3,4], quantum dots [5,6], nanorods [7], nanotubes [8], nanofilms [9,10], etc. The excellent features of carbon-based nanomaterials [[11], [12], [13]] make them promising candidates for diverse and unique applications in several important fields, such as catalysis [14,15], capacitors [16], bio-imaging [17,18], medical diagnosis [19], and photovoltaic devices [20]. Additionally, upconversion nanoparticles can be utilized for biosensing and bio-imaging applications [21,22]. Nanometer-sized crystals are an advanced class of tiny oxygenous nanoparticles having <10 nm size, denoted as quantum dots (QDs) and also display outstanding properties like wide absorption spectra, size-dependent optical properties, narrow photoluminescence spectra with good chemical and photostability [[23], [24], [25]]. The luminescence behaviour of QDs depends upon particle size of the QDs, which can be explained by the relation between the particle size and band gap. It can be explained by density-functional theory (DFT) [26,27]. It is suggested that the higher the particle size smaller will be the band gap (HOMO-LUMO), indicating the higher emission wavelength. Hence, QDs has the capability to emit light ranging from blue-green (2.9 eV) to orange-red (2.05 eV) depending upon particle size. In 1998, Alivisatos et al. and Nie et al. described the use of QDs as novel fluorescent probes for biological detection and imaging [28,29]. In recent years, the excellent electronic and optical properties, quantum edge effects and confinement, has revived interest in luminescent QDs as promising candidates to substitute traditional phosphor materials [14,15,[30], [31], [32]].

However, the conventional semiconductor QDs synthesized by using metallic substances, like CdS, PbSe, CdSe, and Ag₂S more or less exhibit hydrophobicity, toxicity, and high cost, restricting their practical application [23]. Therefore, the use of carbon quantum dots (CQDs), a novel category of the nanocarbon family, has gained momentum with utility for diverse applications due to their significantly higher photoluminescent properties, low toxicity, high chemical stability, biocompatibility, and easy functionalization [33,34]. Various studies have demonstrated different paths for the synthesis of CQDs, like plasma treatment [35], arc-discharge [36], laser ablation [37], microwave heating [38], combustion [39], electrochemical oxidation [40], and the hydrothermal method [41,42]. Apart from these impressive advancements, there is still an urgent need for the rapid and reliable synthesis of high-quality CQDs by an eco-friendly and easy approach with cheaper raw materials. Nowadays, as a conventional soft chemical synthesis pathway, the water-based hydrothermal synthesis of CQDs is accepted as perhaps the most cost-effective and simple approaches due to the inexpensive apparatus, low energy consumption, simple manipulation, good selectivity and product synthesis in a one-step process without any complicated controls [23,43]. Several studies have adopted this method for the green synthesis of CQDs. For example, Lu et al. demonstrated an economical, simple, and green approach based on the hydrothermal strategy for the production of water-soluble and fluorescent CQDs with a lower level of quantum yield (~6.9%) by utilizing pomelo peels as a raw carbon source and applied as the fluorescent probes for Hg²⁺ detection [44]. Another study documented the synthesis of photoluminescence CQDs from grass by using the hydrothermal process with only 6.2% of quantum yield [45]. However, the photoluminescence quantum yield (PLQY) depends on the size of the quantum dots, the technique of synthesis process (top-down, bottom-up), method of preparation (electrochemical, exfoliation), using of solvents (water, DMF, DMSO, THF etc.) and time. Mastronardi et al. first reported the effect of the quantum dots size on the photoluminescence quantum yield [46]. It has been suggested that the quantum yield gradually decreases with the decrease of the particle size of the QDs. The most likely pathways are (i) surface defects and traps, and (ii) vibration relaxation. Sahu et al. prepared photo luminescent CQDs form orange juice with a quantum yield of 26% and applied for cell imaging [47]. Apart from the methods of CQDs synthesis, the selection of an appropriate precursor as a carbon source is an additional necessary factor that should be under consideration. Recently, the study of Hsu and Chang noticed that materials containing both carboxyl and amino groups are advantageous for the synthesis of CQDs with a significantly higher photoluminescence quantum yield [47]. With this background, we postulated that gelatin could be a promising candidate for the synthesis of CQDs as it contains abundant carboxyl and amino groups. Gelatin contains many prolines, 4-hydroxyproline residues, and glycine residues. The basic building block of gelatin is -Ala-Gly-Pro-Arg-Gly-Glu-4Hyp-Gly-Pro-. Although there are available reports regarding the synthesis of gelatin-based QDs, their utilization as a versatile cell biomarker in different clinically available cell lines is not reported yet. Stable photoluminescent property and rapid detection of the biological samples are the foremost requirements for clinical diagnostics and biological assays. Additionally, the assessment of intracellular and intercellular structures by using light microscopy is essential in diverse disciplines, like pathology diagnostics and studies on cell biology, etc.

Herein, we are reporting a simple and cost-effective hydrothermal synthesis of fluorescent CQDs by utilizing gelatin as raw material (GeQDs). The synthesized photo luminescent GeQDs are nano-sized (4.8 ± 0.27 nm), highly water-soluble and emitted bright blue fluorescence. The surface chemistry, size, and morphology of GeQDs were characterized fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (¹H NMR), ultraviolet-visible (UV–vis), photoluminescence spectroscopic techniques (PL), and also by using high-resolution transmission electron microscopy (HR-TEM). The multicolor fluorescent GeQDs presented significantly higher stability in contrast with organic dyes and utilized as a promising fluorescent probe for biological imaging of different bacteria (Escherichia coli and Staphylococcus aureus), yeasts (Candida albicans, C. krusei, C. parapsilosis, and C. tropicalis), molds (Aspergillus flavus and A. fumigatus), and cell lines including A549 (human lung epithelial cell line), HEK293 (human embryonic human kidney cell line), L929 (murine endothelial fibroblast cell line) cell lines.