Hybridizing Carbon Nitride Colloids with a Shell of Water-Soluble

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Hybridizing Carbon Nitride Colloids with a Shell of Water-Soluble Conjugated Polymers for Tunable Full-Color Emission and Synergistic Cell Imaging Qianling Cui, Jingsan Xu, Guizhi Shen, Chao Zhang, Lidong Li, and Markus Antonietti ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13212 • Publication Date (Web): 27 Nov 2017 Downloaded from http://pubs.acs.org on November 29, 2017

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ACS Applied Materials & Interfaces

Hybridizing Carbon Nitride Colloids with a Shell of Water-Soluble Conjugated Polymers for Tunable Full-Color Emission and Synergistic Cell Imaging Qianling Cui,† Jingsan Xu,* ‡ Guizhi Shen,# Chao Zhang,ǁ Lidong Li,* † and Markus Antonietti§ †

State Key Laboratory for Advanced Metals and Materials, School of Materials Science and

Engineering, University of Science and Technology Beijing, Beijing 100083, China ‡

School of Chemistry, Physics and Mechanical Engineering, Queensland University of

Technology, Brisbane, QLD 4001, Australia #

Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, China

ǁ

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of

Materials Science and Engineering, Donghua University, Shanghai 201620, China §

Department of Colloid Chemistry, Max Planck Institute of Colloids and Interfaces, Potsdam

14424, Germany

KEYWORDS: carbon nitrides, water-soluble conjugated polymers, colloidal hybrids, full-color fluorescence, cell imaging

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ABSTRACT: We present the preparation of a new multi-color emission system constructed from two complementary conjugated materials that are highly photoluminescent, i.e. phenylmodified carbon nitride (PhCN) colloids as the core and water-soluble conjugated polymers (WSCPs) adsorbed as the shell. The fluorescence bands of the PhCN and WSCPs effectively complement each other and the overall emission can be simply adjusted to fully cover the visible light spectrum, with white light emission also accessible. Photophysical insights imply that the interactions between PhCN and WSCPs preserve the binary system from emission distortion and degradation, which is essential to delicately tune the overall fluorescence bands. Notably, the continuously tunable emission color is achieved under single-wavelength excitation (365 nm). This hybrid shows a synergistic permeation performance in cell imaging, i.e. PhCN nanoparticles help the WSCP to enter the cells and therefore multicolor cellular imaging achieved.

1. INTRODUCTION Conjugated polymers are macromolecules with π-conjugated structures in which the electrons are delocalized across the whole molecule and can be optically excited.1-3 Water-soluble conjugated polymers (WSCPs) generally consist of two parts: (1) the π-conjugated backbones that show light-absorption and emission, and (2) the hydrophilic side chains that enable the polymers to dissolve in aqueous solution.4 Up to now, WSCPs have been reported having intriguing functions and promising applications in many fields. For example, taking advantage of their excellent photoelectric properties and water processability, WSCPs have been used to construct diverse optoelectronic devices, such as organic light-emitting diodes and solar cells.5-8 Due to the unique molecular-wire effect which can magnify the fluorescence signal significantly, WSCPs have been proved ideal chemical sensors for detection of various molecules or ions,


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which largely lower the detection limit and improve the sensitivity.9-11 Furthermore, by virtue of their excellent biocompatibility and ability to combine with different biomolecules, WSCPs have shown incomparable performance on biological sensing of nucleic acids, proteins, and other biomolecules, even on monitoring some complicated process like protein capsid assembly.12-17 In addition, their applications have also been explored in biological and medical field, including cellular imaging,18 diagnosis,19 drug delivery,20 photodynamic therapy,21 and photodynamic inactivation of bacteria.22 However, they often suffer from undesired variation of the optical behavior (spectral position shift, fluorescence quenching, etc) as they are strongly interacting with other substances in aqueous solution, especially species with partial hydrophobicity and/or opposite charges.21 This usually requires extensive manipulation of positioning and careful fluorescence selection when WSCPs are used for building hybrid luminescent materials. Polymeric carbon nitride (noted as CN), which is built up by stacked, planar tri-s-triazine units, can be regarded as a rather unique conjugated covalent solid.23,24 In the past few years, CN has been extensively studied as a metal-free material for optical energy conversions, such as catalyzing hydrogen/oxygen evolution25,26 and CO2 reduction27,28 under illumination. Recently, researchers start to pay attention to CN optimized for photoluminescence that shows high potentials in optoelectronics,29-32 optical sensing33 and biological imaging,34,35 etc. Strategies involving chemical etching and cutting are employed to manufacture CN nanoparticles with high fluorescence intensity and outstanding optical properties. For instance, Xie and co-workers carried out acid etching (H2SO4/HNO3 mixture) and subsequent hydrothermal treatment to prepare single-layered CN dots that exhibited enhanced one-photon blue emission under UV light as well as two-photon fluorescence under NIR excitation.36 Alternatively, our group developed a supramolecular-preorganization approach to incorporate phenyl moieties into the


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conjugated structure of CN, acquiring CN colloids (noted as PhCN) with enhanced π-electron delocalization across the framework.37 This method resulted in significantly improved fluorescence yields and avoided the etching and hydrothermal processes. However, to date, the emissive colors of CN are mostly limited to blue and green corresponding to the CN band-toband emission, which restricts many of its further applications. Therefore, approaches to endow the CN fluorophores with broader emission coverage, ideally, across the entire visible-light spectrum, are highly welcomed. Up to now, several approaches have been reported to develop fluorescent materials with multicolor emission, which are promisingly useful in display devices and biological applications. For instance, conjugated polymers with elaborately designed backbone structures can be synthesized and adjusted for variable band gaps and thereby multi emission wavelengths.38 For semiconductor quantum dots, whose photoluminescence properties derive from size-related quantum confinement effect, full-color emission can be obtained by adjusting their sizes.39 Recently, multi-color emissive carbon dots were also obtained by tuning surface states and heteroatom contents.40,41 However, to prepare multi-color or even full-color emissive materials by a simple and convenient approach is still highly welcomed and challenging. In this work, we combine the PhCN colloids with newly-designed WSCPs to construct luminescent systems that emit bright light ranging from red to purple upon photo-excitation (Figure 1). The fluorescence of the PhCN/WSCP hybrids can be simply adjusted by varying the ratio of the two components to cover the full visible-light spectrum, taking advantage of their complementary emission spectra. Notably, white light emission is also accessible for this system. It is worth mentioning that the continuous spectral tenability is achieved under single-wavelength (365 nm) excitation. The interactions between PhCN and WSCPs result in strong hybrid entities


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which in consequence are less sensitive to other optical interferences arising from binding and aggregation, while interestingly the influence of mixing on the emission behavior remains rather weak, which overall allows a priori-adjustment of the fluorescence of the hybrids avoiding spectral distortion. This result is essential to tune the final fluorescence bands with high precision and reliability. The stability of the composed nanoparticulate fluorophores enables multicolor cell imaging with different fluorescence channels.

Figure 1. (a) Idealized structure and fluorescence picture of PhCN aqueous dispersion. (b) Chemical structures and fluorescence pictures of PFDBT-BIMEG and PFDBT-N water solutions. (c) Full-color emission photographs of PhCN colloids (0.1 mg/mL) combined with PFDBT-N and/or PFDBT-BIMEG (0, 5, 10, 17.5, 25, 50, 100 µM). All the fluorescence is excited with a 365 nm UV lamp. 2. RESULTS AND DISCUSSOIN 2.1 Microstructure of PhCN Nanoparticles and Its Impact on the Fluorescence


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The structural characterizations (X-ray diffraction, FTIR and solid state NMR) of the PhCN are shown in Figure S1. The microtexture of the PhCN nanoparticles is characterized with transmission electron microscopy (TEM). The particles have sizes in the range of 50 ~100 nm and show irregular shapes (Figure 2a). Very interestingly, TEM images further illustrate that the nanoparticles present a unique lamellar superstructure (Figure 2a,b). The distance between two lamellar layers is determined to be ~3.5 nm. This superstructure can be further confirmed by the small-angle XRD pattern, which shows a peak at 2.35º (2θ) corresponding to an average packing distance of 3.7 nm (Figure 2c). We propose that this phenomenon arises from a liquid-mediated growth and demixing process as discussed before.31 When heated, the precursor will turn to homogeneous liquid which acts as the intermediate phase before transforming to a solid product. We suppose that the liquid phase can allow the molecules to move freely and arrange themselves into the favorable orientation. This self-assembly process is most likely directed by the phenyl groups in the precursor molecules. The phenyl groups interact with the other conjugated CN planes via π-π stacking, which brings together the neighboring CN layers and results in the organized, lamellar structure in the final product, without the assistance of any external templates. The presence of phenyls was verified by solid state NMR measurement (Figure S1b). For comparison, we also studied a similarly treated carbon nitride powders made from the cyanuric acid-melamine (CM) complex. CM has a very similar molecular constitution as CMp, but without phenyl substituents.42 Here, the TEM picture and small-angle XRD (Figure S2) show that the as-obtained material possesses only the typical CN sheet-like morphology, while the nano- and microstructure of the PhCN nanoparticles is completely different and domainorganized. Other researchers have adapted a range of precursors including cyanimide,43


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melamine44 and urea45,46 to manufacture textured CN nanomaterials, but the present lamellar microphase structure in the ca. 3 nm range is specific as such.

Figure 2. (a) TEM images of PhCN nanoparticles, (b) TEM image of an individual PhCN nanoparticle. (c) Small-angle XRD pattern of PhCN. (d) The normalized fluorescence spectrum of individual PhCN colloids, PFDBT-N and PFDBT-BIMEG aqueous solutions. The excitation wavelength is 365 nm. It is fair to mention that in our previous report the observation on the microstructure of the PhCN dots was restricted by the resolution of the TEM we were able to use (see Supporting Information of the reference).37 In the previous paper, it was postulated that the structural cooperation of the phenyl groups improved the delocalization of the π-electrons in the conjugated carbon nitride framework, which is the reason for a very high, environment independent fluorescence yield (48%). On the basis of the current finding, we believe that apart


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from the electronic effect due to phenyl substitution, the high organization of the lamellar structure also contributes to the improvement of the fluorescence intensity and insensitivity of the PhCN. This microstructure might even reduce the fluorescence loss that is caused by excitonphonon interaction, one major path of non-radiative recombination of the photo-induced charge carriers. 2.2 Fluorescence of WSCPs The two synthesized WSCPs have the same conjugated backbone (PFDBT, Figure 1b) and thus exhibit the same emission bands centered at 420 and 655 nm (Figure 2d), corresponding to the PF part and DBT part, respectively. Intramolecular and/or intermolecular fluorescence resonance energy transfer (FRET) is accessible between the two luminescence centers, i.e. PF as the donor and DBT as the acceptor upon photoexcitation. The highly hydrophilic BIMEG side chains allow the molecules to dissolve easily in water revealed by DLS results (Figure S3a), and correspondingly intermolecular FRET is small for PFDBT-BIMEG. In contrast, for PFDBT-N the intermolecular FRET is significant, pointing to the molecules’ tendency to form micelles in water due to the lower steric hindrance and hydrophilicity of its side chains (a quaternary ammonium salt) (Figure S3b). Here, intramolecular FRET is rather weak, owing to the low relative content (5 mol%) of DBT in the backbone.47 As a result, for PFDBT-BIMEG the 420 nm emission band is more dominant whereas for PFDBT-N the main emission peak is the 655 nm one (Figure 2d). The distinct photophysical behavior of the current WSCPs enables them to serve as candidates to be integrated into the present multi-color emissive platform, here based upon PhCN. 2.3 PhCN/WSCPs Hybrids for Full-Color Fluorescence and the Photophysical Study


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In this regard, titration of WSCP solutions (fixed-volume with concentration from 5 to 100 µM) to the PhCN colloid (~ 0.1 mg/ml) was carried out for achieving full-color emission. As known, the PhCN nanoparticles are highly negatively charged,37 while the WSCPs are positively charged due to the sidechain groups.47 Zeta potential measurement shows that the surface charge of the colloids changed dramatically from -33 mV to +32 mV with the input of WSCPs (Figure S4), indicating the electrostatic complexation between the PhCN dots and the polymer molecules. All the fluorescence spectra of the PhCN/WCSP systems were measured and recorded under 365 nm light excitation, consistent with the irradiation wavelength of the laboratory-used UV lamp (UV-vis absorption shown in Figure S5). It can be seen that the fluorescence spectra are –unexpectedly but rather nicely- the combinations of the emission bands of PhCN and WCSPs (Figure 3a), up to showing something close to an isobestic point. The emission spectra of the WSCPs remained in their behavior largely undisturbed, i.e. the peak and shape of the curves did not show noticeable shifts due to the PhCN addition, except the following: In the adsorption shell, the PFDBT-N’s peak intensity at 655 nm (I655) increased significantly with concentration accompanied by the 420 nm peak intensity (I420) decreasing, due to the strong intermolecular FRET mentioned above. For PFDBT-BIMEG, in contrary, I420 rise with increased concentration and meanwhile I655 showed only a slight increase (Figure 3b). Accordingly, the emissive color of the PhCN-WSCPs system can be well tuned by adjusting the concentration of WSCP, therefore covering the full visible light spectrum as displayed in Figure 1c (CIE coordinates shown in Figure S5b). Moreover, by titrating both PFDBT-BIMEG and PFDBT-N to the PhCN suspension in an appropriate ratio, a broad and strong emission spectrum covering the spectrum from 400 to 800 nm can be obtained (Figure 3c) which hence results in bright white light emission (Figure 1c). These fluorescent hybrids demonstrate reasonably high


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quantum yields (QYs), with a typical QY of 26% for the white emission system. The QY of individual component, PhCN, PFDBT-BIMEG and PFDBT-N, was previously reported as 48%, 24% and 22%, respecitively.37,47 It is noteworthy that the full-color fluorescence was achieved under single excitation wavelength (365 nm), whereas in some other fluorescent systems, e.g. carbon dots, usually varied excitation wavelengths are required for this purpose.48 Moreover, these hybrids remain well dispersed for at least one week in aqueous solution, without noticeable fluorescence degradation.

Figure 3. The fluorescence spectra of PhCN colloids (0.1 mg/mL) combined with different amount of (a) PFDBT-N or (b) PFDBT-BIMEG solutions (0, 5, 10, 17.5, 25, 50, 100 µM). (c) The fluorescence spectra of the PhCN (0.1 mg/mL)/PFDBT-N (10 µM)/PFDBT-BIMEG (10 µM) ternary system with white emission. The excitation wavelength is 365 nm. The corresponding fluorescent photographs are shown in Figure 1c. It is well established that FRET can act as a spatially local measurement tool and reveal molecular structure and interactions in chemical systems.49 To seek more understandings on the PhCN/WSCP system, the extent of FRET, typically for PFDBT-BIMEG, was quantified by comparing the relative peak intensity of I655/I420. For sole PFDBT-BIMEG at low solution concentrations (5 to 25 µM), wherein the molecules could freely move, the I655/I420 remained


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constant at around 0.047 (Figure 4a), meaning only very weak intramolecular FRET occurred at this stage. After the concentrations reached 25 µM and higher, the value of I655/I420 increased linearly to 0.085, indicating the gradual onset of intermolecular FRET owing to aggregation. On the other hand, the I655/I420 evolved in a completely different manner in the presence of PhCN dots. A comparably high value of 0.15 was already achieved when the PFDBT-BIMEG concentration was set to 5 µM. This fact clearly can be attributed to the rather tight and dense adsorption of the polymer molecules on the PhCN nanoparticle surface, which indirectly simplified the processes of interpolymer photon/energy transfer to the DBT acceptor. Interestingly, the subsequent concentration increase (until 25 µM) led to a backdrop of the I655/I420 to 0.075, which we attribute to the saturation of the adsorption and then the occurring coexistence of free polymer fluorophores. Above 25 µM, intermolecular FRET was also triggered in solution without PhCN, i.e. the polymers in general were close to each other. Hence the I655/I420 value started to increase again.


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Figure 4. (a) Variation in the fluorescence intensity ratio (I655/I420) as a function of PFDBTBIMEG concentrations (5-100 µM) with/without PhCN (0.1 mg/mL). (b) Decay curves of excited PhCN (0.1 mg/mL) in the absence/presence of PFDBT-BIMEG (25 µM), monitored at 500 nm emission wavelength. Decay curves of excited PFDBT-BIMEG (25 µM) in the absence/presence of PhCN (0.1 mg/mL) monitored at (c) 420 nm and (d) 655 nm. The excitation wavelength is 365 nm. Fluorescence lifetime is monitored to further study the fluorescence behavior of the hybrid system. The decay curves monitored at 500 nm demonstrate that the fluorescence of PhCN relaxes much faster after combining with 25 µM of PFDBT-BIMEG (Figure 4b), while the fluorescence intensity showed only a minor decline (Figure 3b, 25 µM). We assume this phenomenon is associated with the trap states within the bandgap of PhCN. The presence of trap states could endow the PhCN nanoparticles relatively long fluorescence lifetime and meanwhile high quantum yield. After the adsorption of the polymer molecules on the PhCN surface due to electrostatic forces, the radiative electron-hole recombination in the trap states is accelerated, resulting in shorted lifetime. Meanwhile, the fluorescence intensity can largely remain as nonradiative recombination is not introduced in this case. On the other hand, the decay rates of PFDBT-BIMEG remained almost unaffected by the presence of PhCN monitored at 420 and 655 nm (slower for 655 nm, if any change) (Figure 4c,d). 2.4 Comparison with Other Fluorescence Materials To illustrate the very special character of the present system, we also examined whether mixtures of other fluorescent materials, typically organic dye and inorganic semiconductor quantum dot (QD), can be employed to construct multi-color emission system with the WSCPs.


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Fluorescein sodium salt (FSS) which is commonly used as a fluorescent tracer for many applications was chosen as an anionic dye. The QD we used was a commercial water-dispersible, core-shell CdTe@CdS@ZnS with surface decorated by carboxyl groups. Both the dye and the QD are highly negatively charged and have the emission peak centered around 520 nm (Figure S6a), in order to resemble the characters of PhCN colloids. As a quick test, the emission intensity of PhCN, fluorescein and QD was fixed at a similar level and then each species was separately added to the PFDBT-BIMEG solution, respectively. The resulted fluorescence spectra (Figure S6b) show that the main emission (420 nm) of PFDBT-BIMEG was com pletely quenched after mixing with fluorescein and QD. Concurrently, the fluorescence of fluorescein and QD significantly reduced or even fully degraded. This is typical for strongly interacting optical systems in close proximity. Only for the PhCN/PFDBT-BIMEG hybrid, very high levels of fluorescence were maintained for both agents, suggesting that electronic interaction only occurs to an at least rather minor level. More detailed studies by varying the amount of PFDBT-BIMEG revealed that the gradual quenching of fluorescein was owing to the FRET from the fluorescein molecules to the DBT acceptor, resulting from physical bounding along with the overlap between the emission spectrum of fluorescein and the absorption spectrum of PFDBT (Figure S7, S8). For QDs, the complete quenching after input of PFDBT-BIMEG should be attributed to the fact that photoluminescence of QDs is usually very vulnerable to the surface chemistry.50 PFDBT could easily bind on the QDs surface such that intermolecular FRET can take place which led to a red emission even at very low polymer concentration (Figure S9). Consequently, a full color set of otherwise very similar fluorescent particles cannot be generated on the base of as-selected the dye/WSCPs or QDs/WSCPs systems. 2.5 Synergetic Behavior of the Hybrid in Cell Imaging


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As one of major applications of emissive materials, fluorescence based bioimaging has attracted particular interest because of its high sensitivity, fast response and technical simplicity.51-53 Cell imaging based on fluroescence provides us much information on cellular structures, status and even variation, which is helpful to understand various physiological process and valuable on diagnostic and therapy.54-56 Compared to widely-used molecular probes, fluorescent nanoparticles feature some superior advantages for bioimaging, such as improved brightness, inertness to surrounding environment, and easy internalization by cells.57 Taking advantage of their tunable emission and high brightness, the PhCN/PFDBT-BIMEG hybrid nanostructures could be a good candidate for multicolor fluorescence imaging, which shows superior advantages to the single-color techniques, such as minor interference from autofluorescence of the organism due to repeatedly multi-channel corroboration.38,58 As a proof of concept, cell imaging applications of PhCN, PFDBT-BIMEG and their hybrid PhCN/PFDBTBIMEG were demonstrated by incubation with HeLa cells for 10 h, respectively. The treated cells were observed under a confocal laser scanning microscope (CLSM) under a laser excitation at 405 nm, and three channels (blue, green, red) were used for collecting the fluorescence signals. The fluorescence image of HeLa cells treated with only PhCN colloids (Figure 5a, green channel) showed obvious intracellular staining, indicating the good internalization by cells even though the colloids are negatively charged. In contrary, fluorescent images acquired from blue and red channels (Figure 5b) create the clear outlines of the living cells, resulting from the accumulation of PFDBT-BIMEG on the cellular surface (PFDBT-BIMEG alone was not able to enter the cells even up to 24h as shown in Figure 4 in our previous paper).47 The preferential location of the polymer at the plasma membrane can be attributed to its intrinsic structure: rigid and hydrophobic backbone carrying with flexible and highly positively-charged side chains,


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which have a strong interaction with the lipid bilayer of the plasma membrane.59-61 Additionally, the polymer dispersed in water as extended unimolecular chains due to its good water solubility, further strengthening the contact with the lipids and hence avoiding cellular uptake.47 Interestingly, when PhCN/PFDBT-BIMEG hybrid was used for imaging, strong fluorescent signals were acquired inside the cytoplasm from all three channels (Figure 5c), revealing that the macromolecules were internalized together with the nanoparticles, instead of bounding to the cellular surface. Although small amount of polymer (~8%) may remain unbound to the nanoparticles in the hybrid solution (Figure S10), high quality cell imaging was still achieved. Figure 5c showed that no accumulation occurred on the cellular surface, suggesting that the minor unbound polymers had negligible effect on the final cell imaging. Compared to the case of single polymer, the PhCN/WSCP nanostructure tends to cause the cell membrane to wrap around the hybrids (due to the presence of the PhCN nanoparticles) and takes them into the cell.62-64 Moreover, the higher rigidity of the PhCN core was supposed to significantly increase the cellular uptake rate. As reported by Jiang et al, more rigid nanoparticles can move across the cell membranes more smoothly.65 Therefore, the cellular uptake of the conjugated polymers through an endocytosis process alongside the PhCN nanoparticles can be significantly increased by combination with PhCN colloids,65 and meanwhile multi-color cell imaging was achieved owing to the green fluorescence of PhCN. This synergistic behavior of the PhCN/PFDBT-BIMEG hybrid was demonstrated to be applied for multi-color cell imaging. Considering these fluorophores’ relatively low cytotoxicity that has been demonstrated by a standard MTT (Figure S11-S13), we expect the as-made hybrid to have much wider applications in the bioimaging field.


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Figure 5. Confocal laser scanning microscopy (CLSM) images of HeLa cells stained with PhCN (0.07 mg/mL) (a), PFDBT-BIMEG (30 µM) (b) and PhCN/PFDBT-BIMEG (0.07 mg/mL/30 µM) (c) for 10 h. The excitation laser wavelength is 405 nm. The scale bars represent 20 µm. 3. CONCLUSION In summary, we facilely realize multi-color photoluminescence of nanoparticles by integration of two types of conjugated materials, namely lamellar-nanostructured PhCN nanoparticles as a core and WSCP molecules adsorbed as a shell, whose emission bands can complement each other. The overall emission can be facilely tuned to fully cover the visible-light spectrum under singlewavelength excitation. The photophysics interpreted from FRET and fluorescence decay indicate the absence of charge transfer from PhCN to WSCP upon irradiation. The interactions between PhCN and WSCP, including electrostatic attraction, only short the lifetime of the excited stet within the PhCN core, while the adsorbed polymer adopt the behavior of a high concentration species, however again without electronic interactions with the core. This rather unique behavior allows the hybrid materials to luminesce with high brightness and well-maintained band shapes


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and positions, also the base of the rational colors tuning. The control experiments of replacing PhCN with inorganic QDs or organic dye lead to uncontrollable fluorescence including severe quenching and spectral distortion. These results underline of the distinct characters of the PhCN/WSCP combination. Importantly, the PhCN/WSCP system show, due to the colloidal interactions, a synergistic permeation performance in cell imaging, i.e. PhCN nanoparticles help the WSCP to enter the cells in the adsorbed state through an endocytosis process and enable the multi-color cytoplasm imaging, while WSCPs alone only bind to the plasma membrane and stay externalized. We believe this work provides a new simple pathway to design and construct multicolor emissive platforms for a range of applications such as optical sensing and bioimaging. METHODS Materials Water-dispersible CdTe/CdS/ZnS QD decorated by 2-mercaptopropanic acid with emission at 520±5 nm was bought from China Beijing Beida Jubang Science & Technology CO., Ltd. Other chemical reagents were all purchased from Sigma–Aldrich. Ultrapure Millipore water (18.6 MΩ cm) was used throughout the experiments. The synthesis of the PhCN colloids were synthesized according to our previous report by a sonication-assisted carbon nitride exfoliation process.[8] The details are also given in the supporting information. Poly[(9,9-bis(6′-((N,N,N-trimethylammonium)hexyl)-2,7-fluorene)-co-4,7-di-2-thienyl-2,1,3benzothiadiazole] dibromide (PFDBT-N) and poly[(9,9-bis(6′-((N-(triethylene glycol methyl ether)-di-(1H-imidazolium)methane)hexyl)-2,7-fluorene)-co-4,7-di-2-thienyl-2,1,3-


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benzothiadiazole] tetrabromide (PFDBT-BIMEG) were synthesized based our previous work.[17] The details are also given in the supporting information. Preparation of the Hybrid Fluorescent Materials The stock solution of PFDBT-BIMEG was dissolved in water directly with a concentration of 1 mM (molar ratio of repeating units). The stock solution of PFDBT-N was dissolved in DMSO (50 mM) and diluted with water to a concentration at 1 mM (molar ratio of repeating units). All the stock samples were kept at 4 °C in dark. 1 mL of the PhCN colloidal suspensions (0.2 mg/mL) was added to 1 cm × 1 cm cuvette, and liquots of polymer stock solution (1 mM) were then added to reach the final target concentration, while maintaining the total volume of the sample at 2 mL by adding water. After mixing, the fluorescence spectra of the aqueous solutions were measured. The excitation wavelength was set at 365 nm, and the emission spectra were monitored from 375 to 800 nm. For the case of FSS, a stock aqueous solution was prepared with a concentration of 5 × 10-4 M, and 1 × 10-5 M was used as the final concentration for complexation with the polymer. In the case of CdTe/CdS/ZnS QD, 50 µL of QD dispersion was used for fluorescence measurement to keep similar fluorescence intensity as that of FSS and PhCN. Cellular Imaging Experiments HeLa cells were seeded in 35 × 35 mm culture plates and maintained for 24 h in DMEM containing 10% (v/v) fetal bovine serum (FBS) in a humidified incubator with 5% CO2 atmosphere at 37 °C. These cells were respectively incubated with the samples for 10 h, including PhCN colloidal dispersion (0.07 mg/mL), or PFDBT-BIMEG (30 µM), or PFDBT-N


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(30 µM), or PhCN (0.07 mg/mL) + PFDBT-BIMEG (30 µM), or PhCN (0.07 mg/mL) + PFDBTN (30 µM). Then, the medium was removed and the cells were washed with PBS, and the fluorescence images and phase contrast bright-field images were recorded on a confocal fluorescence microscope (Olympus FV1000-IX81). For observing the fluorescent signals, the excitation wavelength was set at 405 nm and was focused through a 60 × oil immersion microscope objective (Nikon, NA=1.0). For multi-color imaging, the fluorescence signals were collected from 425 to 475 nm (blue channel), 490 to 540 nm (green channel), and 575–625 nm or 625-675 nm (red channel). Cytotoxicity Assay by MTT Method The cytotoxicity of the samples was studied using a 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) cell-viability assay. HeLa cells, or MCF-7 cells, or A549 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS). Then, the cells were seeded into 96-well plates at a density of 1 × 104 cells in each well. After 24 h of incubation at 37 °C in a 5% CO2 humidified atmosphere, the cells were treated with different amounts of PhCN colloidal dispersions (1 mg/mL, 10 µL, 20 µL, 40 µL, 60 µL, 80 µL, 100 µL), and cultured for another 24 h. The final concentration of PhCN was 0.01, 0.02, 0.04, 0.06, 0.08, and 0.10 mg/mL respectively. For the test of PFDBT-N or PFDBTBIMEG, the polymer concentration used was 10 µM, 20 µM, 40 µM, 60 µM, 80 µM, 100 µM. For the test of PhCN/PFDBT-BIMEG, the sample was mixed with PhCN (2 mg/mL) and PFDBT-BIMEG (2 mM) in the volume of 7 µL+3 µL, 14 µL+6 µL, 28 µL+12 µL, 42 µL+18 µL, 56 µL+24 µL, 70 µL+30 µL, respectively. After pouring out the medium, 100 µL of freshly prepared MTT (1 mg/mL in PBS) was added to each well and incubated for 4 hours. After removing the MTT medium solution, the cells were lysed by adding 100 µL of DMSO. The plate


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was gently shaken for 5 minutes, and then the absorbance of purple formazan at 570 nm was monitored using a Spectra MAX 340PC plate reader. Characterization The hydrodynamic diameter through dynamic lights scattering (DLS) method, and zeta potentials of the particles were determined using a Nano ZS90 Zetasizer (Malvern Instruments Ltd., UK). The UV-vis absorption spectra were measured on a Hitachi U3900 spectrophotometer. Fluorescence spectra of the samples were recorded by a Hitachi F-7000 spectrometer. For the fluorescence measurements, excitation wavelength was set at 365 nm. Time-domain lifetime measurements were performed using an Edinburgh Instruments F900 spectrometer with an excitation wavelength of 365 nm. Transmission electron microscopy (TEM) images were captured on a transmission electron microscope (JEM 2010). The fluorescent photos were captured by a Nikon D-7000 camera under a hand-held UV lamp illumination (365 nm). The absolute quantum yields of the hybrid dispersions were determined using a spectrofluorometer (FLSP920, Edinburgh Instruments LTD) equipped with an integrating sphere. The excitation wavelength was set at 365 nm. The scattering spectral range of blank and sample was set from 360 nm to 370 nm, and the emission spectral range was from 400 to 850 nm. Small angle XRD was performed on Xeuss SAXS/WAXS system (Xenocs, France). ASSOCIATED CONTENT Supporting Information. Detailed experiments, XRD pattern, NMR spectrum, absorption and FL spectra, optical pictures, cell imaging and cytotoxicity assay. These materials are available free of charge on the ACS Publications website.


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AUTHOR INFORMATION Corresponding Author * [email protected]; [email protected] Author Contributions Q.C. and J.X. designed the project. Q.C. and C.Z. conducted the materials synthesis, characterization and measurement. G.S. carried out cell imaging and cytotoxicity test. J.X. and L.L. supervised the project and M.A. gave useful suggestions. Q.C., J.X., L.L. and M.A. wrote the manuscript. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT Q.C. and L.L. are grateful to the National Natural Science Foundation of China (51503015, 51373022). J.X. is grateful to Discovery Early Career Researcher Award (DECRA) by Australian Research Council for financial support (DE160101488). REFERENCES

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