Multicolor polymeric carbon dots: synthesis,separation and polyamide-supported molecular fluorescence

Multicolor carbon dots (CDs) have been developed recently and demonstrate great potential in bio-imaging, sensing, and LEDs. However, the fluorescence mechanism of their tunable colors is still under debate, and efficient separation methods are still challenging. Herein, we synthesized multicolor polymeric CDs through solvothermal treatment of citric acid and urea in formamide. Automated reversed-phase column separation was used to achieve fractions with distinct colors, including blue, cyan, green, yellow, orange and red. This work explores the physicochemical properties and fluorescence origins of the red, green, and blue fractions in depth with combined experimental and computational methods. Three dominant fluorescence mechanism hypotheses were evaluated by comparing time-dependent density functional theory and molecular dynamics calculation results to measured characteristics. We find that blue fluorescence likely comes from embedded small molecules trapped in carbonaceous cages, while pyrene analogs are the most likely origin for emission at other wavelengths, especially in the red. Also important, upon interaction with live cells, different CD color fractions are trafficked to different sub-cellular locations. Super-resolution imaging shows that the blue CDs were found in a variety of organelles, such as mitochondria and lysosomes, while the red CDs were primarily localized in lysosomes. These findings significantly advance our understanding of the photoluminescence mechanism of multicolor CDs and help to guide future design and applications of these promising nanomaterials.

Understanding the origin and sensitivity of carbon dot emission will improve their utility in various applications.

Since the accidental discovery of luminescent carbon fragments in 2004,¹ carbon dots (CDs) have attracted great research interest due to the diverse synthetic methods, tunable luminescence, and applicability in a broad range of fields, including bio-imaging,^2–4 sensing,^5,6 and light emitting diodes (LEDs).^7,8 Typically, CDs are fluorescent carbon nanostructures of sizes less than 10 nm, composed of carbon, oxygen, and nitrogen.^9–12 CDs can be produced through bottom-up methods, which involve small molecular precursors like citric acid, malic acid, urea, ethylenediamine, and so on.^13–15 In a high temperature reaction, polymerization and dehydration occur among various functional groups, and the resulting products are usually a mixture of small molecule residues, oligomers, and long chain polymers.¹⁶ The unclear fluorescence mechanisms and poorly understood internal structure of CDs limit the ability to understand, tune, and fully exploit their fluorescence properties.Fortunately, in recent years, breakthrough syntheses of multicolor CDs have been achieved.^17–19 Several different multicolor CDs have been synthesized with aromatic compounds such as phenylenediamine.^4,20–22 However, it should be noted that precursors such as aniline and phenol may have toxic effects on human health and the environment,^23,24 and thus should be avoided where possible. Syntheses of colorful CDs from non-aromatic compounds such as citric acid and urea often employ solvothermal methods. Utilizing different solvents such as formamide and dimethylformamide have been shown to play a significant role in tuning CD emission.^25,26 In addition, chromatographic post-treatment of as-made CDs plays a critical role in obtaining different colored fractions, using techniques such as anion-exchange column chromatography,²⁶ normal phase silica chromatography,²⁷ and reversed phase silica chromatography.¹⁵ Compared with high performance liquid chromatography (HPLC), the aforementioned column chromatography techniques help to separate CDs on a larger scale. These separations are based on charge²⁶ or polarity,²¹ and are efficient in isolating the desired fractions with distinct colors so that detailed structural characterization can be performed.To gain insight into the fluorescence mechanism of these multicolor CDs, researchers have considered three hypotheses: quantum size effects,²⁸ the inclusion of molecular fluorophores,²⁹ and surface state-induced emission.³⁰ For example, Rogach and coworkers developed solid-state CDs with tunable fluorescence via the seeded growth method. They attributed the tunable emission to the size of π-conjugated domains.³¹ Yang and coworkers synthesized CDs by hydrothermal treatment of citric acid and ethylenediamine. They identified a small molecule fluorophore, IPCA (1,2,3,5-tetrahydro-5-oxo-imidazo1,2-α]pyridiine-7-carboxylic acid) from CD column separation fractions, which contributed to the blue fluorescence.¹³ Xiong and coworkers synthesized CDs from urea and p-phenylenediamine that emitted a range of colors and separated them with silica column chromatography. They found the degree of carbon oxidation increased as the emission redshifted and thus, they endorsed the surface state hypothesis.²¹ In addition to the above mechanisms, computational methods such as density functional theory (DFT) have also been applied to analyze the fluorescence origins of CDs. The charge transfer between functional groups on the polymeric unit of CDs made from citric acid and ethylenediamine was found to facilitate blue emission.¹⁶The goal of present work is to understand the fluorescence origin of multicolor CDs. The model multicolor CDs were obtained by reacting citric acid and urea in formamide via a microwave-assisted hydrothermal treatment. An automated chromatographic apparatus was employed to separate as-made CD mixtures into distinct color fractions. The individual separation process took around 20 minutes, and the obtained CD fractions exhibit discrete illumination-induced emission throughout the visible region of the spectrum. Interestingly, the sizes of separated CD fractions are not statistically different from one another, suggesting that the quantum size effects are not the source of differential emission. Solvatochromism experiments showed that the blue and green fractions have similar fluorescence behavior as a function of solvent polarity, but the red fraction behaved differently. Using computational simulations, three models of the fluorescence origin were constructed and evaluated, showing that the formation of small blue fluorescent molecules is likely and pyrene analogs could be the origins for various emission colors. Moreover, two representative CD fractions, the blue- and red-emitting fractions, were chosen for subsequent cell imaging experiments. The localization pattern for the CD fractions differed: blue-emitting CDs were observed in a wide range of organelles, while red-emitting CDs were primarily enclosed in lysosomes. Understanding the origin and the sensitivity of CD emission will improve their utility in bioimaging applications.