Real-Time Visualization of Perylene Nanoclusters in Water and Their

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Real Time Visualization of Perylene Nano-clusters (PNCs) in Water and Their Partitioning to Graphene Surface and Macrophage Cells Xuejun Guo, Xin Jin, Xiaofang Lv, Yingying Pu, and Fan Bai Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b01880 • Publication Date (Web): 09 Jun 2015 Downloaded from http://pubs.acs.org on June 17, 2015

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Environmental Science & Technology

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Real Time Visualization of Perylene Nano-clusters (PNCs) in Water and Their Partitioning to Graphene Surface and Macrophage Cells

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Xuejun Guo†*, Xin Jin†, Xiaofang Lv†, Yingying Pu‡, Fan Bai‡*

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†

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Normal University, No. 19 Xinjiekouwai Street, Beijing 100875, China

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‡

State Key Laboratory of Environment Simulation, School of Environment, Beijing

Biodynamic Optical Imaging Center, School of Life Science, Peking University, No. 5

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Yiheyuan Road, 100871, China

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*Corresponding author

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Guo, X. Email: [email protected]; Phone: 86-10-5880-7808 Fax: 86-10-5880-7808;

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Bai, F. Email: [email protected]; Phone: 86-10-6275-6164 Fax: 86-10-6275-6164.

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Guo X, Jin X, and Bai F contributed equally to this work.

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ABSTRACT Hydrophobic organic chemicals (HOCs) are of special ecotoxicological

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concern because they can be directly incorporated and bio-concentrated in living

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organisms. However, the effects of self-clustering of HOCs on their environmental

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behavior and toxicology have not yet received enough attention. With the use of a

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recently developed technique, single-molecule fluorescence microscopy, the motion and

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distribution of perylene nano-clusters (PNCs) formed in water at very low concentration

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(1 µM) were visualized with high temporal and spatial resolution. The liquid-solid

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interface process of PNCs adsorbing onto graphene was also recorded. Instead of the

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traditional view of HOCs adsorption as a single molecule, our study revealed the

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characteristic of irreversible adsorption of perylene onto the carbonaceous surface in the

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form of nano-clusters, exhibiting random sequential ‘car-parking’ events. More

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interestingly, the transport of PNCs across the cell membrane was also captured in

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real-time, demonstrating that they entered macrophage cells by endocytosis.

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Supplementing the well-recognized routine of passive diffusion through a membrane

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lipid bilayer, the uptake of HOCs in the form of nano-clusters by endocytosis is proposed

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to be an additional but important mechanism for their uptake into living cells. HOCs

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distributing in the environmental systems in the form of nano-clusters, exemplified by

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PNCs in this study, may have significant implications for understanding their

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environmental fate and potential toxicological effects.

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TOC abstract


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INTRODUCTION

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Hydrophobic organic chemicals (HOCs) are of special ecotoxicological concern

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among numerous anthropogenic chemicals due to their capacity to incorporate directly

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into the tissues of living organisms1-2. Resistant to biodegradation, many HOCs may be

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bio-concentrated as a result of continuous exposure to a contaminated environment.3

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Furthermore, HOCs’ accumulation may occur in predators and humans by

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biomagnification along food chains due to the ingestion of contaminated foodstuffs.4-5

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The species of a given HOC is extremely important because it determines behaviors

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such as persistence, transport, transformation and toxicology in the environment.6-7 When

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addressing the species of HOCs in environment, classic books and previous studies

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usually place emphasis on the chemical structure of these molecules and their


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consequential environmental behaviors.8 Scientists generally consider HOCs are

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uniformly distributed within a solid phase in single molecular form, for instance, a plant

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cuticle or mammalian adipose tissue.9-11 Consequently, all environmental studies of

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partitioning, toxicity and modeling are based on the assumption that the partitioning

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processes of environmental contaminants are uniform behaviors of single molecules,8,12

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although with no direct evidence supporting such hypothetical prerequisite. The effects of

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an important physical state, namely the self-clustering (also as self-aggregation or

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self-coiling) of HOCs, on their environmental behavior and ecotoxicology have not yet

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received attention, and relatively few papers paying attention to this subject have been

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published.9,11

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However, in vivo visualization of the HOCs taken up into living vegetation and other

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matrixes showed molecular clustering within specific plant cellular structures.10-11 Further

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studies revealed that HOCs can form nano- and micro-clusters over time in compatible

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lipid media such as eicosane, dotricontane and even polymeric lipids in leaf cuticle.9,11

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Actually, aggregation of HOCs to molecular clusters and nano-aggregates is an important

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phenomenon in many regions, such as in the ambient troposphere, natural waters, and

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even in the interstellar medium.13-17 Hydrophobic-lipophilic interaction (HLI), is one of

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the most important and essential driving forces for the formation of these micro- or

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nano-sized molecular clusters, aggregates, micelles, and vesicles, which play important

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roles in affecting the chemical reactivity and bioprocesses of organic substrates.18-19

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Many HOCs (such as polycyclic aromatic hydrocarbons (PAH), heterocyclic compounds,


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and organometallic compounds) are strongly hydrophobic and will form nano-clusters in

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water even at very low concentration, given that the partition coefficients of HOCs

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between themselves and water can reach the order of 109.9, 19-21

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The exciting development of single-molecule fluorescence microscopy (SMFM)

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makes in situ visualization of fluorescence chemicals possible. In contrast with ensemble

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methods, SMFM enables the scientist to examine individual members of a heterogeneous

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population and discover the information on distributions and time trajectories of

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observables that would otherwise be hidden. 22-23 By performing SMFM, many biological

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process, such as the infection of individual viruses, endocytosis of nano-particle, RNA

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catalysis and folding, and transcription factor dynamics can be detected, sorted, and

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quantitatively compared among their subpopulations.24-28

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Employing SMFM, this study used perylene as a chemical probe to investigate HOC

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self-clustering and partitioning in real time between water, graphene and macrophage

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cells. The high sensitivity of SMFM made it possible to detect single or

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low-copy-number of perylene naon-clusters (PNCs), with a time resolution on

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millisecond scale.29 SMFM can also be used to real-time visualize the partitioning

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process of PNCs between different phases. Perylene, a five ring PAH, was selected as the

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probe reagent. Perylene primarily derives from organic matter diagenesis and

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anthropogenic processes (e.g. incomplete combustion due to cigarette smoke and engine

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exhaust).30-32 It is considered as an important PAH in estuarine sediments.33 It has been

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widely distributed in many natural waters (0-520 ng/l), wastewaters (0.03-3.0 µg/l) and


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sewage sludges (140-6400 µg/kg).34 Moreover, its high quantum yield (0.94) and

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excitation spectra at visible light region make perylene an ideal probe for fluorescence

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microscope. Carbonaceous media is considered as the major sink for HOCs in

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environment.8 Graphene was used as a simplest surrogate of carbonaceous media in

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interfacial behavior of PNCs between water and condensed carbonaceous phase.

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Graphene is a single atom thick sheet of carbon, which allows the light to freely pass

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through.35 In addition, graphene shows excellent performance and potential future in

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adsorptive removal of many HOCs and their derivatives.36-37 In cell enrichment process,

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murine macrophage cell line was chosen since macrophage is a “first responder” to all

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foreign materials and is widely used for studying various aspects of foreign materials

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uptake and potentially related toxicity.38-40 In summary, the formation of PNCs in water,

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its characteristic adsorption onto graphene, and the new pathway of uptake by cell, which

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were to be revealed in this study, may have significant implications for understanding

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HOCs environmental fate and potential toxicological effects.

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MATERIALS AND METHODS

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Chemicals and Reagents. Perylene (Sw =1.59 nM; logKOW=5.82) and the dye DiO were

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purchased from Sigma. Dulbecco’s modified eagle medium (DMEM), fetal bovine serum

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(FBS), phosphate buffer solution (PBS), 5% trypsin, and phenol red free DMEM were

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purchased from Gibco. Graphene was obtained from Jichang Nano Company (China).

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Highly oriented pyrolytic graphite (HOPG) was purchased from NT-MDT Company

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(Russia). Ultrapure water was used throughout this work. The stock solution of 0.1 mM


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perylene was prepared using ethanol and stored at 4 °C. Before use, the perylene solution

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was prepared by diluting the stock solution 100 times using ultrapure water.

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Emission spectra of perylene in water solution

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The fluorescence spectra of perylene solutions (0.1 nM, 1 nM, 0.01µM, 0.1 µM, 1 µM, 5

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µM) were recorded using a fluorescence spectrophotometer (Hitachi, F-4600). The

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solutions (1.0 mL) in a 4 mL tube were excited at 405 nm, and the emission spectra at

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420-700 nm were recorded.

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Visualization of PNCs in water

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Fluorescence microscopy. The real-time Brownian movement of perylene clusters in

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1µM of perylene solution (1% ethanol) was observed using a fluorescence microscope

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(Zeiss, Axio Observer). Perylene clusters were excited by a 405 nm laser (Coherent OBIS

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405), and monitored with the filter set (dichroic mirror 405 nm, emission 570-640 nm).

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The fluorescent emission was collected by an 100× oil-immersion objective with

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numerical aperture of 1.46 and imaged onto an electron multiplying charge coupled

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device (EMCCD) camera (Photometrics, Evolve 512). Image series were recorded at 20

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frames per second using Epi-illumination.

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To determine the distribution of the molecule number of individual PNCs, we first

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calculated the fluorescence contribution of one perylene molecule, I0. One microliter of

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perylene solution was evenly coated onto a 5mm×5mm area of the coverslip. The

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coverslip was air dried avoid of light, and was observed using the Zeiss microscope.

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The calculation of the molecular number of an individual PNC was based on the


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assumption that the fluorescent contribution of each individual perylene molecule in

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different sized PNCs is identical. The fluorescent contribution of free perylene molecules

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was neglected here, since free-state molecules only accounted for 0.1 percent of the total

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perylene molecules. The fluorescence intensity of one perylene molecule in the cluster, I0

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was calculated from the following equations of (1) and (2):

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I0 = (

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N = N ACV

(1)

A × I)÷ N A'

(2)

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Where N, NA, c, and V present total molecular number of perylene on the coverslip,

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Avogadro's constant (6.02×1023), the concentration of perylene solution (1µM) and the

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volume of perylene solution smeared on the coverslip (1 µL); A, A’, and I refer to the area

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of the smeared place on coverslip (25 mm2), the area of the captured images by

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microscope (0.64 mm2), and the averaged fluorescence intensity of the captured images,

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respectively. The average fluorescence intensity of the captured image (80µm×80µm)

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was obtained by randomly recording 100 different images in the coated area, and then the

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average value was taken.

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Then the images of PNCs in 1 µM of perylene solution were captured using the

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same fluorescence microscope. The fluorescence intensity of each PNC was extracted,

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and presented as Ii. A control experiment showed that immersing in water or drying in air

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has not effect on the fluorescence intensity of a PNC. Thereby, the number of molecules

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(Ni) for one individual PNC (Ii) in water can be calculated using the equation (3).


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Ni =

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Ii I

(3)

0

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Atomic force microscopy. Atomic force microscopy was used to further characterize

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the shape and size of PNCs. Perylene solution (1 µM) was added dropwise onto highly

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oriented pyrolytic graphite (HOPG) substrate. The HOPG surface was loaded with

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isolated PNCs via incubating for 1.0 h. Bruker MultiMode 8.0 was used to record the

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images in tapping mode using a J-scanner and Veeco NP cantilevers with a spring

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constant of 0.06 N·m-1. Images were recorded under aqueous conditions with a silicone

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rubber O-ring.

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Visualization of PNCs adsorption onto graphene. Graphene (1 cm×1 cm) was coated

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onto a glass coverslip using the method from Suk et al.,41 the Raman spectra of which is

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shown in Fig. S4. The coverslip with graphene was put on the stage of fluorescence

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microscope (Zeiss, Axio Observer). Subsequently, a drop of 1 µM perylene solution was

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added onto the top of graphene. Then the adsorption process was recorded by the

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fluorescence microscope. Different from visualization of PNCs in water, the illumination

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was switched to total internal reflection (TIRF) by using Laser TIRF 3 of Zeiss, to avoid

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the interference of floating PNCs in solution. Image series were recorded at 20 frames per

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second for analyzing individual PNC adsorption behavior.

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Visualization of PNCs uptake by macrophage cells

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Cell cultures. The murine macrophage J774A.1 cell line was purchased from Cell

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Resource Center, Chinese Academy of Medical Sciences & Peking Union Medical


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College. The cells were cultured in DMEM supplemented with 10% FBS in an incubator

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at 37 ºC with 5% CO2.

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Real-time tracking the endocytosis of PNCs by the macrophages. Before the

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fluorescence imaging, cells grown in the Petri dish with glass coverslip on the bottom

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were washed with PBS. One milliliter of phenol red free DMEM was added to the dish.

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The process of endocytosis of PNCs was recorded by the fluorescence microscope (Zeiss,

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Axio Observer) after 1 mL of phenol red free DMEM containing 1µM perylene was

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added. Illumination was switched to TIRF using Laser TIRF 3 of Zeiss. Image series

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were recorded at 1 frame per 30 seconds for 15 minutes using time lapse imaging.

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Colocalization. Cells grown on the Petri dish with glass coverslip on the bottom

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were washed with phosphate buffer solution and incubated with 16 µM DiO for 15 min at

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37°C to label the cell membrane. After that, cells were washed with PBS and incubated in

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phenol red free DMEM containing 1 µM perylene for 15 min. Then the cells were

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washed again with PBS. The DiO labeled cells were imaged by laser confocal

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microscope (Zeiss, LSM 710 NLO & DuoScan System). The PNCs were excited with a

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405 nm laser, and monitored with the filter set (dichroic mirror 405 nm, em 570-640 nm).

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DiO was excited with a 488 nm laser, and monitored using another filter set (dichroic

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mirror 488 nm, em 500-520 nm). The fluorescent emission was collected by an 40×

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objective.

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PFA fixed cells. To inhibit metabolism, cells were incubated with 4% PFA for 30 min

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at 37°C. After that, the treatment was withdrawn and cells were washed using PBS. Cells


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were then incubated in phenol red free DMEM containing 1 µM perylene for 15 min.

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Before fluorescence imaging, the cells were washed with PBS. Images were recorded

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using Axio Observer Zeiss microscope with Epi-illumination (excitation 405 nm;

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emission 570-640 nm for PNCs, 435-485 nm for single molecular perylene).

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RESULTS AND DISCUSSION

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Characterization of PNCs in water. In our study, SMFM was used first to visualize the

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PNCs formed in water by precipitation. When the perylene concentration in water is well

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above 1.59 nM of its water solubility at 25 ºC, the molecular forces exhibited between

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conjugate aromatic molecules, such as π-π interaction and hydrophobic-lipophilic

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interactions, will lead to self-clustering of these molecules and formation of PNCs (Fig.

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1A). As indicated in Fig. 1B, the PNCs exhibited unique optical properties differing from

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single-molecular perylene by a new emission peak at 550~650 nm, while the emission

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peak of single molecular perylene is around 435~470 nm. This new emission peak

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(550~650 nm) was identified as a characteristic peak for real-time monitoring PNCs in

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our study. The red-shift of emission maxima of PNCs compared with individual perylene

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molecule was also observed in previous studies.21, 42


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Figure 1. Characterization of PNCs. (A) Ball-stick model of perylene and the simulated

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nano-cluster. (B) Fluorescence spectra of perylene solution at different concentrations

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(0.1 nM, 1 nM，0.01µM, 0.1 µM, 1 µM, 5 µM) excited at 405 nm. The emission peak

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ranging from 550 nm to 650 nm is the characteristic peak of PNCs. (C) Atomic force

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microscopy image of PNCs (1 µM) loaded on HOPG surface. Image was recorded under

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aqueous conditions. The inset is the section of one typical PNC (with white arrow) on

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HOPG. The scale bar is 1 µm. (D) Distribution histogram of the height and radius of


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PNCs deposited onto HOPG. (E) A representative Brownian motion trail of PNC (2.0 s)

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extracted from continuous imaging of PNCs in water (20 frames per second). (F)

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Distribution histogram of the number of molecules in PNCs (1 µM). The results are

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calculated from the analysis of single PNC’s fluorescence intensity using equations (1-3).

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The Brownian motion of PNCs in water was real-time recorded by SMFM (Movie 1).

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The fluorescent intensity for an individual PNC was presented as maxima as it moved

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within the focusing plane of microscope. When it moved vertically out of the focal plane,

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the PNC particle in image became fuzzy with decreasing fluorescent intensity, and finally

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disappeared. The Brownian motion trail of one typical PNC in water was captured, and

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shown in Fig. 1E. From the Brownian motion, we calculated the mean hydrodynamic

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radius (RH) of PNCs in 1 µM perlylene solution based on the Stoke-Einstein relation:24, 43

RH =

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K BT 6πηD

(4)

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Where KB = Boltzmann constant (1.38×10-23 J/K); T = Temperature (298K), η =

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viscosity of the solvent (0.8937×10-3 Pa·s). D is a factor of proportionality with direct

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relationship to the solubility and molecular weight of the molecule. The slope of the

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following equation gives the value of D:

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4 D(ti − t 0 ) = [(R(ti ) − R(t 0 )]2

(5)

Where t0 = initial time, and ti = time after t. R refers to a two-dimensional vector indicating the spatial location of the center of mass of the cluster.


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The mean hydrodynamic radius (RH) of PNCs in 1 µM perlylene solution was

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calculated as 42.3 nm from capturing 30 individual clusters. Atomic force microscopy

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(AFM) images of PNCs at the same perylene concentration deposited onto HOPG (Fig.

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1C) showed the mean radius of 49 nm, which is in agreement with the average value of

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RH obtained from calculation. The distribution histogram of the height and radius of

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PNCs deposited onto HOPG is shown in Fig. 1D. AFM provided further evidence of the

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disk-like shape of PNCs deposited on the HOPG, with average thickness of 4.03 nm.

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The distribution of molecular number of PNCs can be calculated from equation

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(1)-(3) built above in the experimental section. Obtained from analysis of at least 100

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individual PNCs, the distribution histogram of the molecular number of PNCs in 1.0 µM

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perylene solution is shown in Fig. 1F. The histogram was well fitted with a Gaussian

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distribution and the mean molecular number per PNC was 1.03×105. After the

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verification of cluster form of perylene in water, the settleability of PNCs was determined

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by centrifugation at different rotating speed. Even with 5000 rpm of rotating rate, the

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fluorescence intensity of the supernatant derived from PNCs only decreased 12%,

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demonstrating a high stability of PNCs formed in water (Fig. S1).

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The formation and aggregation state of HOCs in the aquatic environment should be

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dependent on a multiple of influencing processes and factors, including the equilibrium

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between clustering and dis-clustering, the chemical structure of different HOCs, the

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different HOC sources, the broad range of HOC concentrations and solubility, complex

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water chemistry, water mixing and so on.44


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Using SMFM, we visualized in real-time and characterized the PNCs formed in

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1 µM perylene solution, the content of which is well above its water solubility. PNCs in

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0.1 µM and 0.01 µM (only 6.3 times of its solubility) perylene solution were recorded too,

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although with significant decrease in counting number of PNCs in SMFM images. This

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observation is reasonable and predictable, given the water solubility of perylene is at

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nanomole level (Sw =1.59 nM). The total concentration of perylene can be up to

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0-2.06 nM in virgin natural water body, 11.9 nM for wastewaters and 25.4 nM from

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sewage sludges.34 It was reported that the total PAHs concentration in many rivers

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reached a few or tens of thousands of nanograms per liter, and generally PAHs occurrence

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was even more prevalent in sediment of these rivers.45-48 Considering the partition

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coefficients of many HOCs between themselves and water can reach the order of 109, we

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propose here that most of these HOCs are essentially distributed as homogeneous or

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heterogeneous forms of micro-aggregates and nano-clusters in these aqueous systems due

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to hydrophobic-lipophilic and π-π interactions between themselves and each other.9, 19

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These HOCs clusters in the aqueous environment and their associations with some other

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carbonaceous and inorganic solid phases are likely to account for a significant fraction of

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HOC-suspended solids (HOC-SS), which are generally considered as the major species of

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most HOCs distributed in natural water columns.

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The occurrence and distribution of these HOCs micro- and nano-clusters in the

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aqueous systems should have important implications for understanding their

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environmental fate and their potential toxicological effects. Here, the PNCs formed in


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water by a simple method of precipitation were visualized and characterized using

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SMFM, which thereby stimulated us to address the important implications of molecular

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clustering phenomenon of HOCs in fields of environmental partitioning and related

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ecotoxicology. The adsorption process of PNCs from water onto graphene, and a specific

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cell enrichment process, endocytosis uptake of PNCs by macrophage, were presented in

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the following sections.

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Adsorption process of PNCs from water onto graphene. After the characterization of

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molecular clusters was identified, the adsorption behavior of perylene onto the graphene,

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as presented in Movie 2, was exhibited as the irreversible adsorption (or deposition) of

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colloids or particles onto a carbonaceous surface instead of the traditional

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single-molecular adsorption model. Fig. 2 shows the representative images of PNCs

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sequestered by graphene at different time, which clearly revealed a random sequential

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adsorption of PNCs onto the substrate. The time course of the PNCs adsorbing onto

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graphene was like a ‘car-parking’ event, neither with further PNCs migration on the

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surface nor immediate desorption, indicating a strong sticking-force exhibited between

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PNCs and graphene substrate. The increase in the number of adsorbed PNCs on graphene

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and the change of total fluorescence intensity was recorded over a focal area of 80 µm ×

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80 µm with time-lapse, the typical results of which are shown in Fig. 3A. The adsorption

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of PNCs was rapid at the first 10 seconds, and then slowed down considerably. The total

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fluorescence intensity was positively correlated with the number of adsorbed PNCs,

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exhibiting an obvious stepwise growth curve as captured by SMFM with high temporal


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resolution. Interestingly, the increase in PNCs counts with time was not a monotonically

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increasing curve, probably due to a phenomenon of overshooting generally observed in

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colloid and polymer adsorption.49-50

304 305

Figure 2. Representative fluorescence images of PNCs (1 µM) adsorbed onto graphene

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surface in solution for 0 s, 5 s, 10 s, 15 s, 20 s, and 25 s. All Scale bars are 10 µm.

307 308

Owing to the high sensitivity of time resolution offered by SMFM, the individual

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adsorption behavior of PNC can be visualized. Fig. 3B recorded the irreversible and

310

independent adsorption of an individual PNC onto the surface of graphene. We captured

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2-4 images during the processes of a PNC moving close to the graphene surface,

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modifying its position during the process until final adsorption. This adsorption process


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of PNCs on graphene typically took 100-150 ms. Although most of PNCs were adsorbed

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stably (Fig. 3B), desorption process of a small fraction of PNCs was also captured.

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Fig 3C recorded one PNC shot the graphene substrate and then escaped immediately. In

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Fig. 3D, the PNC was recorded respectively with 2 times of steep rise in fluorescence

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intensity, spaced by gradual or sharp decline of fluorescence intensity at intervals. It is

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speculated that the steep rise in fluorescence intensity was caused by the overlapped PNC

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adsorption. The sharp decline of fluorescence intensity for an adsorbed PNC was due to

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the detachment of smaller PNCs away from the original one. The gradual decline of the

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original was probably as a result of gradual dissolution or dis-aggregation of PNC from

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the substrate.

4

80 Count

(B)

100

60

40

40 Count Intensity

20 5

10 Time (s)

15

Intensity(X10 )

60

6

(A)

20 0 20

4

2

0

0

3.0

323

2.4

4

Intensity(X10 )

4

Intensity(X10 )

0.8 0.4 0.0

1000 1500 2000 2500 Time (ms)

(D)

(C) 1.2

500

0

500

1000 1500 2000 2500

1.8 1.2 0.6 0.0

0

Time (ms)


2000

4000

6000

Time (ms)

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Figure 3. (A) Adsorption kinetics of perylene adsorbed on graphene. The green squares

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show the number of adsorbed PNCs on graphene surface. The pink circles show the total

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fluorescence intensity of adsorbed PNCs on graphene surface. Original fluorescence

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image series were recorded at 20 frames per second. (B-D) Adsorption process of

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individual PNCs on graphene extracted from fluorescence images. The figures describe

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the dynamic fluorescence intensity of three individual PNCs deposited on graphene

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during the imaging process.

331 332

Adsorption to the condensed phase is affects the transport, degradation,

333

bioavailability, fate and ecological risk presented by HOCs in the environment. Most, if

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not all the previous investigations of HOCs adsorption onto the solid phases were

335

performed in an almost fixed condition: the concentration range of HOCs in the aqueous

336

phase was below the solubility of that compound in water.51 Under this restricted

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paradigm, the adsorption process of HOCs is artificially simplified to a uniform behavior

338

of dissolved single molecule partitioning between the aqueous and the solid phase,

339

without considering the effect of self-clustering of HOCs. Obviously, this simplified

340

experimental frame does not reflect the truth because the discharged level of HOCs into

341

the accepting water body is very often beyond this dissolubility limit.44 As verified in our

342

study, nano-cluster forms of perylene could exist in aqueous solution even at 0.01 µM.

343

With high spatial and temporal resolution, SMFM recorded here the liquid-solid interface

344

process of PNCs with the characteristics of colloids adsorption (deposition) and


19


Page 20 of 33

345

desorption (detachment), but not the uniform behavior of single molecules as generally

346

assumed.8, 12 As a consequence, taking into account HOC clustering in water and its effect

347

on adsorption will provide a more precise framework for building models that quantify

348

the dynamic behavior of organic chemicals in environmental systems.

349

Observation of PNCs formed in water and their random and irreversible deposition

350

onto the carbonaceous surface has important implications in predicting HOC behaviors in

351

the aqueous environment. Clustering of HOCs in aquatic environment would lead to the

352

increase in the discreteness and heterogeneity of HOCs distributed in the liquid-solid

353

interface. Secondly, traditional view of single molecular HOC adsorption/desorption

354

(A/D) onto the sediments cannot sufficiently explain the impact of hydrodynamic force

355

and perturbation on HOCs release from the sediments into the water column.52-54 The

356

colloidal property of HOCs clusters depositing onto the solid phase might partly explain

357

the significant release of HOCs from sediments by the external forces, such as waves,

358

tides, currents, dredging, trawling and ship traffic. Furthermore, A/D hysteresis is

359

considered as a general phenomenon for HOCs adsorbed onto the solid phases.55-56

360

Besides the previously well-proposed mechanisms,57-59 perylene self-clustering and the

361

resulting irreversibility of PNCs adsorbed onto graphene shown here might present a new

362

but important interpretation to A/D hysteresis.

363 364

PNCs transport through cell membrane. It is generally considered that HOCs pass

365

through the lipid bilayer by passive diffusion.51 However, the formation and stability of


20

Page 21 of 33


366

PNCs in water even at very low concentration intrigued us to investigate if perylene as a

367

typical HOC can be transported into the cell by endocytosis. Laser scanning confocal

368

microscopy was used to image whether the PNCs were incorporated into the macrophage

369

(Fig. 4A). The cells were first labeled with DiO, a fluorescent lipophilic dye that is

370

commonly used to image cell membranes. The image confirmed that PNCs were located

371

in the intracellular space.

372 373

Figure 4. (A) Merged image of PNCs (red) and DiO (green). Images on right and bottom

374

represent the intrinsic 3D sectioning of the image. The scale bar is 10 µm. (B) Merged


21


Page 22 of 33

375

images of the uptake and transport of PNCs (red) in J774A.1 cell. The trails of PNCs are

376

marked by blue lines. (C-D) Merged image of PNCs (red) and single molecular perylene

377

(blue) in living cells (C) and 4% PFA fixed cells (D). (C) and (D) are taken under the

378

same imaging conditions. Scales bars are 10 µm. Obvious signals of PNCs could be seen

379

in the living cells (C) but not in the fixed cells (D).

380 381

Using SMFM, we also investigated the transport of PNCs in cells by tracking single

382

PNCs in living cells. Total internal reflection (TIRF) illumination was used here to avoid

383

the interference from PNCs floating in solution or adsorbed onto the cell membrane.

384

Movie 3 shows the processes of PNCs entering and transferring in the macrophage during

385

15 min of real time visualization. Fig. 4 (B) traced the trajectory of an individual PNC

386

moving from the apical domain to the midst of cell.

387

If the incorporation of perylene into the macrophage was primarily due to

388

endocytosis, it should be significantly disrupted by metabolic inhibitor paraformaldehyde

389

(PFA), since endocytosis uptake of macromolecules or particles is an energy dependent

390

process. To confirm this assumption, the uptake of PNCs by the macrophage was studied

391

with the pretreatment of 4% PFA or not. Since PNCs is characterized by the new red shift

392

emission peak, PNCs and single molecular perylene can be captured separately. Fig.

393

4(C–D) shows the fluorescence image of PNCs (em 570-640 nm) and single molecular

394

perylene (em 435-485 nm) in the living (Fig. 4C) and fixed (Fig. 4D) cells incubated with

395

1 µM of perylene for 15 min. As expected, intracellular accumulation of PNCs in 4% PFA


22

Page 23 of 33


396

pretreated cells was completely blocked compared with the living cell. The accumulation

397

of single-molecular perylene was also dramatically reduced for the PFA-pretreated cells

398

(Fig. S2-3). Note here that the significant reduction in uptake of single-molecular

399

perylene by the PFA-pretreated macrophage should not be explained by the blocking of

400

spontaneous diffusion of small molecule through lipid bilayer because PFA does not

401

affect on this. This was perhaps due to the obstruction of endocytosis uptake of PNCs.

402

These were to be dis-aggregated to single-molecular perylene with the involvement of

403

various intracellular components (i.e., lipoproteins and nonpolar protein domains), and

404

subsequently partitioned into different subcellular compartments.

405

Passive diffusion through the lipid bilayer is usually accepted as the primary route

406

for HOCs incorporation into organisms.60-61 This is certainly a reasonable assumption

407

when the concentration of a specific HOC is less than its solubility in that liquid matrix,

408

because HOC in single molecular form is compatible with the lipid bilayer and can

409

diffuse freely through the cell membrane. However, in the blood plasma, most HOCs (i.e.,

410

Benzo(a)pyrene

411

lipoprotein-associated physiological form, the cellular uptake of BaP and other HOCs

412

was demonstrated to be a simple and spontaneous diffusion through the cell

413

membrane..53,A wide variety of organism cells, such as epithelial cells of the skin,

414

digestive tract and lungs, are exposed to the aquatic environment in the absence of

415

lipoproteins and some such dynamic carriers.60 These cells act as basic physical barriers,

416

through which only various contaminants can enter into the body from an outside aquatic

(BaP))

are

noncovalently

associated


with

lipoproteins.

In

23


Page 24 of 33

417

environment. Additionally, many single-celled eukaryotic species, such as algae and

418

protozoa, live in water environment.62 As shown in this study, PNCs were stably formed

419

in water and accessible to cell via endocytosis uptake even at very low incubated doses.

420

Therefore, the endocytosis uptake of cluster forms is proposed here to be an additional

421

but important mechanism for HOCs bio-concentration into living cells. Our speculation

422

on endocytosis uptake of HOCs was supported by recent research from Fayeulle et al.

423

showing F. solani used an ‘endocytosis-like’ process for BaP uptake dependent on

424

energy.63 This is contrary to the general consensus of BaP uptake by diffusion.

425

The endocytosis uptake of HOCs in cluster form, as PNCs observed here, would

426

enhance the quantity of pollutant incorporated into cells even though these compounds

427

have very low water solubility in aquatic environments. This process may permit faster

428

incorporation of HOCs into cells and may further influence their intracellular localization

429

and toxicological properties. Further investigations are required to address the dynamics

430

and mechanisms of intracellular distribution, transport, dis-aggregation and partitioning

431

of these clusters between different cell compartments.

432

Supporting Information Available

433

Fluorescence intensity of perylene in supernatant after centrifugation with different

434

rotating speed, supplemented images of PNCs (red) and single molecular perylene (blue)

435

in living and fixed cells, comparison of the fluorescence intensity from single molecular

436

perylene in living and fixed cells, Raman spectra of graphene on coverslip, the movie of

437

Browain motion of PNCs, the movie of PNCs adsorbing onto graphene, the movie of


24

Page 25 of 33


438

uptake and transport of PNCs in J774A.1 cell. These materials are available free of

439

charge via the Internet at http://pubs.acs.org.

440

Acknowledgments

441

This work was supported by National Natural Science Foundation of China (NSFC)

442

(Grant Nos. 41371440) and the National Key Basic Research Program of China

443

(2013CB430406). FB also acknowledge the financial support from the Recruitment

444

Program of Global Youth Experts.

445 446

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33

Real-Time Visualization of Perylene Nanoclusters in Water and Their

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