Scalable synthesis of fluorescent organic nanodots by block copolymer templating

Abstract

Water-soluble fluorescent organic nanodots with narrowly distributed size are synthesized by using self-assembled block copolymers of poly(ethylene oxide)-b-poly(styrene-co-acrylonitrile) as templates and precursors in a scalable method. Advancements in fluorescence imaging techniques and the development of fluorescent probes have enabled scientists to gain detailed information about cellular processes and has aided in disease diagnosis.1-3 Many fluorescent probes have been developed, such as fluorescent dyes and semiconductor quantum dots, but there is still room for improvement; the low absorptivity and poor photostability of fluorescent dyes are problematic,4, 5 while semiconductor quantum dots are potentially cytotoxic due to the presence of heavy metals.6 Thus, there is an ongoing demand to explore metal-free, photostable fluorescent probes. Conjugated polymer dots (Pdots) and carbon dots represent two promising candidates.7-9 Advantages of Pdots include their brightness, photostability, and nontoxicity.2, 10-13 However, synthesis of the fluorescent conjugated polymers is challenging and requires specialized monomers, and due to the hydrophobic nature of the conjugated polymers, the preparation of Pdots requires surface modification or incorporation of hydrophilic functionalities to allow for dispersibility in water for bioimaging applications.14-18 Furthermore, the size of the Pdots, which is critical for their targeting ability and transport through biological systems,19-21 is difficult to finely control. Carbon dots also display bright fluorescence, are generally nontoxic, and have good photostability.22-25 Their bright emission relies on surface passivation, typically achieved through surface modification with poly(ethylene oxide) (PEO).26, 27 A drawback of carbon dots is that their synthesis can be energy-intensive, as common methods include pyrolysis and hydrothermal carbonization, both requiring long reaction times at elevated temperatures.28-30 This work aims to address some of the limitations in the synthesis of Pdots and carbon dots by introducing a method for synthesizing fluorescent organic nanodots (FONs) from a block copolymer (BCP) template. FONs represent a middle ground between Pdots and carbon dots, as their structure consists of a conjugated carbon framework derived from a polymer template. Their synthesis is scalable and involves milder thermal treatment conditions than those used to obtain carbon dots. The templating method presented here also ensures that the FONs are well dispersed in water, without the need for additional surface modification procedures. Importantly, the polymer synthesis is accomplished without the use of specialized monomers for conjugated polymers, and the size and size distribution of the FONs can be controlled readily due to the thermodynamically predominant BCP self-assembly. Polyacrylonitrile (PAN) is well known as precursor to fabricate carbon materials. For example, Tang and coworkers obtained carbon nanoparticles by using the BCP of poly(tert-butyl acrylate)-b-PAN.31 However, silicon wafers were used as substrates to preserve the morphology of discrete nanoparticles during pyrolysis, which was not economic for scalable preparation. Furthermore, PAN and its BCPs can only be soluble in a narrow range of solvents with high boiling point, which limits their application. Herein, the synthetic route is based on a poly(styrene-co-acrylonitrile) (PSAN)-containing BCP, which can be soluble in many solvents with low boiling point. Previous work has shown that under thermal treatment at 280 °C in air, the acrylonitrile (AN) units of PSAN undergo cyclization and crosslinking.32-34 Nitrogen originating from AN is simultaneously incorporated into the obtained polymer network. This nitrogen enrichment has been exploited to modify the chemical and electronic properties of these resulting partially conjugated materials, such as surface reactivity and accessibility and electronic band structures, important for electrochemical applications.35-37 A finding that is more relevant to the use of a PSAN precursor for FONs is that the emission wavelength of the FONs is tunable by changing their nitrogen content, that is, by changing the AN content of the BCP.38, 39 Additionally, compared with other methods of incorporating nitrogen species into carbon materials, such as chemical-vapor deposition40 or chemical treatment with nitrogen-containing chemicals,41 using a nitrogen-containing polymer as a carbon precursor results in a more uniform nitrogen distribution and avoids an additional postcarbonization treatment step.33, 42 In this paper, we report a facile method to synthesize water-soluble N-doped FONs from self-assembled PEO-b-PSAN in the presence of tetraethoxysilane (TEOS). The overall synthetic procedure is described in Scheme 1. Diblock copolymer PEO-b-PSAN was synthesized via atom-transfer radical copolymerization of styrene and AN using a PEO-containing macroinitiator (see Supporting Information Fig. S1 for synthetic details). The chemical composition was quantified by 1H-NMR spectroscopy (Supporting Information Fig. S2) to be PEO90-b-P(St35-co-AN23), where the subscripts stand for the degrees of polymerization of corresponding monomers. A narrow molecular weight distribution with a dispersity (Đ) of 1.18 was observed in gel permeation chromatography (Supporting Information Fig. S3). The obtained PEO-b-PSAN was then dissolved in THF, followed by sequential addition of HCl solution and TEOS. Assisted by the interaction between PEO and TEOS, slow evaporation of the solvents promoted phase segregation of PEO-b-PSAN to form spherical micelles with PSAN core and PEO shell enriched with organosilica species.43 With further evaporation of the solvent, these micelles can self-assemble into ordered structures. Subsequent pyrolysis at 280 °C under air and etching of the resulting silica matrix provided discrete FONs [Fig. 1(c)]. The ordered phase separation was confirmed by the sharp scattering peaks in the SAXS pattern and corroborated by TEM [Fig. 1(a,b) and Supporting Information Fig. S4]. It should be noted that the neat PEO-b-PSAN yielded a poorly ordered morphology, suggesting the critical role of TEOS in the co-assembly process. A SAXS pattern with indices of 1: √2: √3: √4: √5 indicated that a body-centered cubic (BCC) nanostructure formed upon calcination. Compared with the sample before heat treatment, the primary scattering peak became sharper and shifted to a higher q value, that is, a smaller d-spacing, owing to shrinkage in the framework during the heat treatment process. FONs with an average size of 12 nm [TEM and DLS Fig. 1(c,d)] were released from the composites by etching the silica matrix in a NaOH solution. A product yield of 75.5% was obtained, based on TGA (see detailed calculation in Supporting Information and Supporting Information Fig. S5). In contrast with a conventional one-step strategy that removes the sacrificial matrix in situ during the pyrolysis, the two-step method reported here, that is, calcination followed by post-etching, eliminates interparticle fusion and nanostructural collapse, as the silica matrix separates the FONs during the heat treatment. FTIR spectroscopy and X-ray photoelectron spectroscopy (XPS) were employed to study the functionalities present in the FONs (Supporting Information Figs. S6 and S7). The abundance of O─H and N─H bonds was confirmed by the strong and broad stretching vibrations at 3680–3030 cm−1 in the FTIR spectrum (Supporting Information Fig. S6)44 The stretching at 2934 and 2853 cm−1 corresponded to C─H. Other functionalities, such as CO, C─N, and C─O, were observed at 1653, 1348, and 1050 cm−1, respectively.44-46 The atomic ratios of N/C and O/C were calculated to be 1.9% and 39.3%, respectively, according to the deconvoluted high-resolution XPS.21, 47, 48 The nitrogen functionalities were primarily contributed by the pyridinic (399.0 eV) and pyrrolic (400.0 eV) groups (Supporting Information Fig. S7b).1 An aqueous solution of the synthesized FONs emitted a bright blue light under 365-nm UV irradiation [Fig. 2(a)]. Figure 2(b) shows the PL behaviors at different excitation wavelengths. Using quinine sulfate as a reference, the PL quantum yield was calculated to be 2.2%.8, 40 The FON solution with a concentration of 0.2 mg/mL showed the highest PL intensity (Supporting Information Fig. S9). With further increase in the FON concentration, up to 1 mg/mL, the intensity decreased, possibly due to aggregation-induced fluorescence quenching. FON aqueous solutions (up to a concentration of 0.2 mg/mL) were found to exhibit long-term stability without any noticeable precipitation at room temperature (Supporting Information Fig. S10), and the isolated FONs could be repeatedly dispersed in water. PEO-b-PSAN with different AN/St ratios were synthesized for studies of the effect of nitrogen content on the PL properties. Nitrogen-free FONs derived from PEO90-b-PS53/TEOS composites provided maximum emission under UV irradiation at the wavelength of 320 nm (Supporting Information Fig. S15a), while this wavelength increased to 360 nm for FONs derived from PEO90-b-P(St35-co-AN23). The observation that introduction of N-dopant narrowed the bandgap was also reported previously in other systems.49, 50 FONs with higher nitrogen content (N/C ratio = 5.26%, Table S1) were synthesized from ordered PEO90-b-P(St20-co-AN23)/TEOS composites (Supporting Information Figs. S11 and S12b). While the maximum emission was also realized with 360 nm UV absorption, the PL peak shifted from 452 nm to 418 nm (Supporting Information Fig. S15b). The blueshift could be ascribed to the increased electron affinity in the system with a larger number of N atoms.35 The pH-dependency and ion sensitivity of PL, which are critical to the application of FONs in biosensing, were investigated using FONs derived from PEO90-b-P(St35-co-AN23)/TEOS composites (Supporting Information Fig. S16a). The maximum PL intensity was observed in the neutral solution, and the intensity decreased by 63.3% from pH = 7 to pH = 0 and by 42.2% from pH = 7 to pH = 14. Additionally, a redshift of PL was observed when pH was increased from neutral to basic conditions, while the fluorescence emission peaks remained almost unchanged in acidic solutions. Furthermore, the fluorescence of the FONs could be quenched by Fe3+ ions (Supporting Information Fig. S17a). The Fe3+-quenched fluorescence can be recovered by adding a strong chelator such as ethylenediaminetetraacetic acid disodium salt (Supporting Information Figs. S18 and S19). Therefore, the obtained FONs can be classified as a reversible chemosensor. The nonspecific interactions between carboxylic groups of FONs and other metal ions such as Zn2+, Cu2+, and Fe2+ led to less-efficient quenching compared with the Fe3+-based system.51 Low cytotoxicity and good biocompatibility of FONs were demonstrated by the colony-forming unit enumeration assay study. Figure 3 shows the confocal microscopy images of E. coli cells labeled with the obtained FONs. The fluorescence in E. coli cells retained a high intensity, and the excitation-dependent PL property caused multicolor imaging under a wide range of excitation wavelengths (458, 488, and 514 nm, Fig. 3). In summary, a facile and scalable approach to well-defined N-doped FONs was developed. The key to the successful synthesis relies on the solution coassembly of PEO-b-PSAN with TEOS and the preservation of FONs by an in situ formed silica matrix. The prepared FONs exhibited blue fluorescence in aqueous solution, and the PL properties could be tuned by varying the nitrogen content and pH value. Potential applications of FONs in bioimaging and as a chemosensor probe capable of detecting Fe3+ ions were demonstrated. This work was primarily supported by Yale University Setup Funds. S.C. is thankful for the financial support from China Scholarship Council (CSC No. 201706020014) for his visit at Yale University. A.C. acknowledges financial support from the National Natural Science Foundation of China (51472018, 51272010), Beijing Nova Program (XX2013009), the Research Fund for the Doctoral Program of Higher Education (20121102120001), and the Fundamental Research Funds for the Central Universities (YWF-16-BJ-Y-75). A.N.L. thanks the National Science Foundation Graduate Research Fellowships. We thank Prof. Jaehong Kim and Prof. Menachem Elimelech for help with the photoluminescence characterization and cell culture experiments, respectively. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.