Health and Ecosystem Risks of Graphene - Chemical Reviews (ACS

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Health and Ecosystem Risks of Graphene Xiangang Hu and Qixing Zhou* Key Laboratory of Pollution Processes and Environmental Criteria (Ministry of Education), College of Environmental Science and Engineering, Nankai University, Tianjin 300071, China 3.5.4. Cytotoxicity Mechanisms 3.5.5. Challenges in Cytotoxicity 3.6. Pharmacokinetics and Nanotoxicity of Graphene in Organs 3.6.1. Translocation 3.6.2. Metabolisms and Bioresponses 3.6.3. Obscure Fate 3.7. Perspectives in Toxicity 3.7.1. Nanotoxicology in Vitro 3.7.2. Nanotoxicology in Vivo 3.7.3. Non-Negligible Knowledge in Toxicology 3.7.4. Challenges of Graphene Toxicology 4. Health Risks 4.1. Penetrating Biobarriers 4.1.1. Damage of Mucus 4.1.2. Cell Vision 4.1.3. Penetrating Phospholipid Bilayers 4.1.4. Skin Permeation 4.1.5. Across Blood−Brain Barriers 4.1.6. Across the Placenta 4.1.7. Glomerular Filtration 4.2. Special Attention to Health Risks 4.2.1. Human Cytotoxicity 4.2.2. Hematopathology 4.2.3. Pulmonary Responses 4.2.4. Pharmacokinetics 4.2.5. Diseases and Oxidative Stress 5. Ecosystem Risks 5.1. Problems for Environmental Purification 5.2. Subtle Ecological Fate 5.3. Unavailable Model Species 5.4. Bacterial Inactivation 5.5. Virus Photocatalysis 5.6. Ignored Phytotoxicity 6. Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments References

CONTENTS 1. Introduction 2. Properties of Graphene Determining Health and Ecosystem Risks 2.1. Size/Size Distribution of Graphene Determines Its Properties and Nanotoxicity 2.2. Bioresponses of Graphene Shapes 2.3. Edges and Nanoholes with Reactive Groups 2.4. Corrugation Influences Cell Adhesion 2.5. π Bonds Induce Nonspecific Binding 2.6. Conductivity Affects Cell Signal Transformation 2.7. Effects of Functionalization on Surface Charges and Hydrophobicity 2.8. Interrelationship between the Parameters 3. Interactions from Small Molecules to Organs 3.1. Adsorption of Metals and Small Organic Molecules 3.2. Hybrid and Cleavage of Nucleic Acids 3.2.1. Hybrid in Vitro versus in Vivo 3.2.2. Subtle Self-Assembly 3.2.3. Option between Protection and Cleavage 3.2.4. Effects of a Hybrid 3.2.5. Competitive Adsorption and Heterogeneous Electrochemistry 3.3. Affinity and Activity of Proteins 3.3.1. Interactional Forces 3.3.2. Affinity Locations 3.3.3. Health Risks of Graphene Nonspecific Affinity 3.3.4. Protein Corona 3.3.5. Conformation and Electronic Property 3.3.6. Challenges in Micro- and Macro-Bioresponses 3.4. Enzyme Activity 3.5. Cell Responses 3.5.1. Reliability of Assay Methods 3.5.2. Negative Cytotoxicity 3.5.3. Positive Cytotoxicity © XXXX American Chemical Society

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1. INTRODUCTION Graphene is defined as a two-dimensional crystal composed of monolayers of carbon atoms arranged in a honeycombed network with six-membered rings.1,2 Compared with zero-

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Received: February 6, 2012

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dx.doi.org/10.1021/cr300045n | Chem. Rev. XXXX, XXX, XXX−XXX

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surface coating. These parameters responsible for the toxicity of graphene have been summarized by Johnston and colleagues.16 Herein, this review focuses on the unique properties of graphene and the related challenges. The distinct properties of graphene are discussed, including size/shape, edge, nanohole, π−π stacking, surface charge, conductivity, hydrophilicity/ hydrophobicity, and functionalization, which significantly affect the environmental fate and bioresponses of graphene. Importantly, these physiochemical parameters are interdependent and are discussed at the end of this section.

dimensional fullerenes and one-dimensional nanotubes, twodimensional graphene exhibits exceptional properties, including doubled external surface area, easy modification, planar support for biomaterials, ballistic transporting capacity, chemical inertness, mechanical strength, high thermal conductivity, and optical transmittance.3,4 Intrinsic graphene is characterized as a zero-gap semiconductor with superhydrophobicity resulting in a high leakage current, instability, and inertia.2,5 Another interesting material with a range of applications is graphene oxide (GO), with oxygen-containing groups,6 a great transporting gap, and a well-polydispersed property at room temperature.7,8 The availability of several types of oxygencontaining functional groups on the basal plane and the sheet edge allows GO to interact with a wide range of organic and inorganic materials in noncovalent, covalent, and/or ionic manners, so that functional hybrids and composites with unusual properties can be readily synthesized.9,10 Throughout this review, graphene includes intrinsic graphene and graphene derivatives, such as GO. Recent advancements in graphene research have opened up a wide range of applications in electrochemical devices, energy storage, catalysis, cell imaging, photochemotherapy, drug delivery, biosensors, contamination purification, and extraction devices for chemical, biological, and environmental samples.10−12 Given the potential occupational and public exposure to graphene due to its versatile applications, scientists are directing more attention toward investigating the biosafety aspect of the nanomaterial.13−15 Research on the risks of graphene to health and the ecosystem is just beginning. The main agenda in the field of human health and ecology is to assess and understand the nature of and risks associated with exposure to graphene, including its chemical behaviors, environmental fate, transportation/translocation, persistence, transformation, biological responses (toxicity and natural defense), related diseases, and ecosystem effects. A fundamental understanding of biological interactions of graphene with metals, small modularities, cells, proteins, and organs is vital to the future design of safe graphene products. Generally speaking, the risks of ecological and human exposure to graphene are determined by its chemical behaviors, environmental fate, and ecological processes. Although the biocompatibility and toxicity of graphene are currently at the center of attention, its risks to health and the ecosystem remain largely unexplored. In particular, valid information related to health and ecosystem risks is overwhelmingly lacking. Although many reports are meaningful, some lack valid evaluation and actual confirmation. This baseless work increases the gap between laboratory tasks and actual facts about human bodies and various ecosystems. This review analyzes the recent work regarding the health and ecosystem risks of graphene, scrutinizes its potential risks, reduces the scientific “blind spots” and knowledge gaps, and attempts to identify future directions in which this field is likely to develop.

2.1. Size/Size Distribution of Graphene Determines Its Properties and Nanotoxicity

The size of graphene directly controls the physicochemical properties of graphene. A larger graphene size induces a smaller percolation threshold with changes of the thermal and mechanical properties of graphene.17 The thermal conductivity of graphene grows with increasing linear dimensions of graphene flakes.18 A strong size dependence of charge distributions was found in rectangular graphene sheets.19 The reduced size with increased edge-to-center ratio results in a higher charge density.20 Small GO flakes reveal superparamagnetic behaviors ascribed to the interaction between localized spins.21 As the size increases, the normalized energy density increases.22 As the size of graphene increases, the absorption in the visible light region intensifies and shoulders of the absorbance spectra in the UV region become prominent.23 When the size of a graphene photosensitizer decreases, the catalytic activity to pollutants can be remarkably enhanced owing to the quantum size effect and increased specific surface area.24 The physicochemical properties determined by the size of graphene are bound to induce the corresponding biological responses. The size of graphene is paramount to its performance in vivo.25 The nanomaterial size has a remarkable effect on the rate and route of translocation and clearance in vivo.26 The increase in size enhances the uptake of graphene in organs and reduces circulation in the blood.27 Clinical and experimental studies have revealed that the small size, and consequently large specific surface area, enhances the generation of reactive oxygen species (ROS).26 So far, the effect of size on the subcellular localization, tissue distribution, and clearance of graphene is obscure. The evidence to show size-dependent toxicity of graphene is limited. This lack of evidence is likely attributed to the difficulty in size determination as the particle aggregates and varies with the dispersion medium in vivo. As with other nanomaterials, directly synthesized graphene exhibits an innate size distribution. Although some researchers have provided the strategies to control the size of graphene,23,28 a relatively narrow size distribution still exists. Generally, graphene nanosheets show Gaussian size distributions.29,30 As previously discussed, the thermal conductivity, charge distribution, capacitance, energy gap, UV absorbance spectra, and photocatalysis of graphene are size-dependent.17−24 Correspondingly, the presence of a size distribution leads to inhomogeneous physicochemical properties. Fasolino and coauthors proposed that the size distribution influenced the fluctuations, Dirac spectrum, and electronic properties of graphene, and the thermal fluctuations exhibited a size distribution peaked around 80 Å.31 Given the effects on physicochemical properties, the size distribution is critical to the pharmacokinetics and toxicity of graphene. The presence of a size distribution probably induces inhomogeneous biological

2. PROPERTIES OF GRAPHENE DETERMINING HEALTH AND ECOSYSTEM RISKS Although the physical properties of the surfaces and edges of graphene have been extensively studied, there is very limited knowledge about the influence of characteristic parameters of graphene in posing health and ecosystem risks. The characteristic parameters include dissolution, chemical composition, size, shape, agglomeration state, crystal structure, specific surface area, surface energy, surface charge, surface morphology, and B


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2.4. Corrugation Influences Cell Adhesion

responses. The cytotoxicity, subcellular localization, blood circulation, organ uptake, and etiopathology of nanomaterials are affected by the size distribution.32−35 Importantly, the size distribution influences the nanotoxicity because the exposure dose of nanomaterials is related to mass and sizes in toxicology. The previous work has demonstrated that nanomaterials with a certain size distribution exhibited the greatest cytotoxicity and cellular uptake.36,37 This suggests that there is an optimum size range for uptake in terms of producing a biological response to nanomaterials as opposed to smallest to largest. Conclusively, it is not reasonable to study the toxicity and pharmacokinetics of graphene while ignoring the size distribution. Graphene synthesized within a very narrow size distribution with uniform properties is critical to the correct evaluation of the risks graphene poses to ecosystems and human health. To a large extent, the reasonable evaluation of ecosystem and health risks requires precise control of particle sizes and a very narrow size distribution.

Meyer et al.56 reported that graphene is not flat, but typically exhibits nanometer-scale corrugations. Graphene can envelop bacteria with noninvasive damage, thus implying that graphene is very flexible.57 Correspondingly, the adhesion energies are 0.45 J m−2 for monolayer graphene and 0.31 J m−2 for samples containing 2−5 graphene sheets, which are larger than those of typical micromechanical structures and comparable to solid− liquid adhesion energies.58 The results suggest that graphene is extremely flexible and likely liquid rather than solid. The liquidlike topography leads to intense adhesion on bacterial and cellular membranes and then induces the differentiation and growth of the cells. At this point direct data to support the hypothesis are emergent. 2.5. π Bonds Induce Nonspecific Binding

Graphene has hybridized sp2 bonding, which shows in-plane σ bonds/atoms and π orbits perpendicular to the plane. The strong σ bonds work as the rigid backbone of the hexagonal structure, while the out-of-plane π bonds control the interaction between different graphene layers, which pose the aggregation of graphene in the medium. Graphene is chemically inert (or stable) because its entire pz atomic orbit is strongly coupled and stabilized in a giant, delocalized, π-bonding system.50 On one hand, this π system precludes graphene from covalent addition with other compounds. On the other hand, as a π ligand, it renders versatile complexation reactions, for example, complexation with organic compounds and transition metals through π−π, H−π, and metal−π interactions. The metal−π interactions are widely used to interpret the interactions of graphene with metals.59−61 H−π bonds and π−π stacking are viewed as the main interactions of graphene with organic compounds, such as dyes, proteins, and DNA.62−64 Nonspecific binding is a random adsorption of biocomponents such as small molecules, proteins, bacteria, and viruses on noncomplementary materials. The π-stacking interactions induce the nonspecific binding of organic molecules with graphene. Most nucleic acids, proteins/enzymes, and other biomolecules have aromaticity. Therefore, the π-stacking interactions between biomolecules and graphene are nonignorable.65 For instance, there is considerable nonspecific adsorption of serum albumin proteins and lectins on a graphene layer.66−68 The other formation of nonspecific binding results from electrostatic interaction between the charged nature of graphene (and its derivates) and the charged biomolecules.69 For instance, Park et al.70 reported nonspecific binding from electrostatic interaction between bacteria and GO paper. As a result, the nonspecific binding triggers related bioresponses. However, the information about bioresponses from nonspecific binding is obscure. For instance, GO acts as a general enhancer of cellular growth by nonspecifically increasing cell attachments and proliferation.71 The nonspecific binding can produce completive adsorption, reduce the efficiency of applications, and trigger unexpected adverse effects, for example, blocking the ideal properties of graphene and competing with the targeted molecules. This is the reason that nanomaterials should pose less nonspecific binding and more specific affinity in therapy. To reduce nonspecific binding, the surface of graphene is modified with ligands. The functionalization of GO with polyoxyethylene sorbitan laurate reduced nonspecific binding of bacteria to GO surfaces.70 GO modified with an aptamer reduced nonspecific binding to small chemical molecules and biomacromolecules.72,73 More information on

2.2. Bioresponses of Graphene Shapes

Graphene has been synthesized in symmetric hexagon, asymmetric hexagon, rectangle, rhombohedron, regular triangle, ribbon, and Ω shapes.27,38,39 The tensile fracture stress tends to decrease as the length increases and the width remains constant.40 Different shapes with various electronic properties exhibit changes in hyperpolarizability or spinning multiplicity.41 The shape of nanomaterials affects the membrane-warping process during endocytosis or phagocytosis.42 The shapedependent toxicity of nanotubes and nickel, gold, and titanium (Ti) nanomaterials has been reported.43,44 Unfortunately, the influence of graphene shapes on human health and ecosystem risks is obscure, aside from the identification of the shapes. 2.3. Edges and Nanoholes with Reactive Groups

Defects such as structural imperfections and chemical impurities could unintentionally or unavoidably produce edges that disturb the reactive microenvironment and paths of bioresponses.45,46 Armchair and zigzag are viewed as two configurations of graphene edges, and the latter configuration is relatively stable.47 The zigzag and armchair configurations show different electrical properties and vibrational modes.47−49 The zigzag configuration is metallic, while the armchair configuration can be either metallic or a semiconductor.48 The armchair configuration has a higher Young’s modulus, tensile failure stress, and strain compared with the zigzag configuration at the same size.40 The effects of the two configurations on the environmental fate, toxicity, and bioresponses of graphene are still under debate. Obviously, it is difficult to characterize the dynamic structural and electronic properties and thus difficult to explain the related health or ecosystem responses. Generally, the edge of graphene is sharp and reactive with active groups.50 The dangling bonds at edge sites are highly reactive to guest atoms or molecules. As reported, the sharp edge can directly break the membranes of cells, bacteria, and viruses and pose physical damage to living organisms.51,52 Carbon is easily removed, and nanoholes form on the graphene plane when the initial hydroxyl and epoxy groups are close to each other.47,53 The reduction of GO promotes the formation of nanoholes by intense chemical methods and mild biological methods, even solar irradiation.54,55 The nanoholes with carbonyls are bound to affect water solubility, conductivity, rigidity, and affinity sites in environmental and biological media. More attention should be paid to this matter in the future. C


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Figure 1. Unique properities of graphene.

nonspecific binding is reviewed in section 3.3.3. Collectively, sufficient attention should be paid to the subtle bioresponses from nonspecific binding in the future.

Besides direct chemical groups, functionalization influences the surface charge and hydrophobicity of graphene by changing the degree of ionization. The effects of the surface charge of graphene on its toxicity and environmental behaviors are reflected in the adsorption of ions and biomolecules and its agglomerates by electrostatic interactions. In general, it is viewed that cationic surfaces are more toxic than anionic surfaces, and cationic surfaces are more likely to induce hemolysis and platelet aggregation, whereas neutral surfaces are the most biocompatible.81 The result may be ascribed to the affinity of cationic particles for the negative phospholipids or proteins. Arvizo et al.82 proposed that perturbation of the membrane potential was dependent on the surface charge of nanoparticles, as well as that the cellular membrane potential played a prominent role in intracellular uptake of nanomaterials. The membrane potential perturbations resulted in increased Ca2+ influxes, which in turn inhibited the proliferation of normal cells. The effect of graphene surface charges on the nanotoxicology is not well-known. Clearly, the surface charge of graphene has a strong impact on the disruption of red blood cell membranes, which is attributed to the strong electrostatic interactions between negatively charged oxygen groups on the GO surface and positively charged phosphatidylcholine lipids on the cell membranes.83 Compared with reduced GO (RGO) with limited oxygen groups, GO significantly induces platelet activation, release of intracellular free Ca2+, and activation of nonreceptor protein tyrosine kinases, suggesting that the prothrombotic character of GO is dependent on the surface charge distribution.84 Compared with GO, amine-modified graphene has absolutely no stimulatory effect on human platelets, nor does it induce pulmonary thromboembolism,85 confirming that the surface charge distribution is an important regulator of the physical interface between graphene and a biological system. Therefore, the functionalization of graphene modifies the surface charges and then affects the environmental

2.6. Conductivity Affects Cell Signal Transformation

Currents can travel on graphene for micrometers without scattering at room temperature. Graphene can sustain current densities 6 orders of magnitude higher compared to copper.2 Graphene stimulates the signal channels or related proteins and then affects the growth of cells via electron transformation.54,74 By supposing that graphene is similar to nanotubes as it can serve as an electronic bridge from cells to cells, graphene would have an influence on the signal transfer and ionic channel among cells and would disturb the function of the cells. The hypothesis needs to be verified in the future. Graphene as an electronic acceptor to eliminate electron pairs has exhibited synergy effects on the catalysis of analyzers in the environment.75,76 Thereby, the superlative conductivity leads to many uncommon biological responses and environmental behaviors. 2.7. Effects of Functionalization on Surface Charges and Hydrophobicity

Pristine graphene is relatively inert and exhibits a much lower chemical reactivity than fullerenes and nanotubes.77 The solubility of graphene is low in both aqueous and organic solutions, which inhibits its applications. Compared with pristine graphene, GO with various oxygen groups presents a relatively high solubility, is effective at functionalizing, and demands extensive attention. Block copolymer, polyethylene glycol (PEG), polyetherimide (PEI), or ssDNA binding to the oxygen groups of GO can enhance the biocompatibility.78 Functionalization with other amine compounds or proteins also reduces the nanotoxicity of graphene.79 Functionalization usually influences the retention time in bloods and the translocation in organs and then appeases or triages the toxicity in vivo.80 D


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graphene on natural metals and small organic molecules have not attracted enough attention in vivo. The interaction mechanisms of metals using graphene involve three aspects. First, graphene is modified, and then its water solubility increases the chance of contact with pollutants in solution.63 Next, the Lewis base from graphene and the Lewis acid from metals form electron donor/acceptor complexes. Finally, the metals enter into the interlayer space of graphene.91 π−π stacking and van der Waals forces are the main interactional mechanisms between graphene and organic pollutants.93 The adsorption kinetics show pseudo-secondorder kinetics and follow the Langmuir isotherm model.66 The thermodynamic parameters reveal that the adsorption process of graphene is a spontaneous and endothermic process.91,92

fate and nanotoxicity of graphene. Simultaneously, functionalization controls the hydrophobicity or hydrophilicity of graphene.86 Pristine graphene is hydrophobic. Our previous work demonstrated that a graphene-functionalized aptamer had enhanced hydrophilicity and presented an excellent capacity to adsorb toxins and viruses in aqueous solution.72,73 GO is relatively hydrophilic due to its oxygen groups. In fact, GO is an amphiphile with hydrophilic edges and a more hydrophobic basal plane. The amphiphile allows GO to easily enter the phospholipid bilayer.87 In summary, these parameters are related to each other, are interdependent, and should be considered together for the environmental fate and toxicity of graphene. 2.8. Interrelationship between the Parameters

3.2. Hybrid and Cleavage of Nucleic Acids

Various physicochemical properties of nanomaterials are interdependent. For example, the size of graphene can affect the density of active groups, conductivity, charge density, and solubility.47,76 Functionalization has an influence on the surface charge and hydrophobicity of graphene by changing the degree of ionization. The amphiphile of graphene is convertible with changes in pH and size.87 Because carbonyls likely exist along the edge,47 pH affects the degree of ionization at the edge. Therefore, the pH of the medium should be considered for its toxicity evaluation and environmental fate. In theory, smaller sizes with relatively more edges lead to a stronger hydrophilicity. It has been indicated that the conversions of hydrophobicity and hydrophilicity are possible as a result of pH and size variations affecting the bioresponses and environmental behaviors. It is better to consider the systematic properties and related microenvironment when discussing one specific property of graphene. In addition, these parameters are affected by the concentration of nanomaterials, reaction medium, and reaction time.88,89 The vital properties of graphene are summarized in Figure 1.

3.2.1. Hybrid in Vitro versus in Vivo. The order of the binding strength of bases with graphene is G > A ≈ T ≈ C, because G has stronger hydrophobic interactions and more hydrogen bonds.95−97 However, the A−T base pairs are more likely to interact with graphene than the G−C base pairs.98 DNA assembles on graphene into two distinct patterns, namely, small spherical particles and elongated networks, which are caused by a crossover in the competition between base−base π stacking and base−graphene interaction, rather than simple hydrophobic interactions.99 The existence of hydrogen binding, π−π stacking, and solvation is a fine balance in the thermodynamics of nucleobases with graphene.100 The shorter DNA is adsorbed more rapidly and binds more tightly to the surface of graphene, and the adsorption is favored by a lower pH and a higher ionic strength.63 Desorption of DNA from graphene obviously occurs when cDNA, surfactants, and complementary sequences are added; however, the temperature is not very sensible for desorption.63,101,102 Currently, most of the adsorption and desorption of DNA on graphene are conducted in vitro. The fate and behavior of graphene binding to DNA should be studied further in vivo. In addition, the effects of pH, ionic strength, and temperature on the interactions of graphene with nucleic acids should be considered. 3.2.2. Subtle Self-Assembly. The short DNA segments consisting of up to 12 base pairs can self-assemble on graphene and form a stable hybrid with forestlike and flat structures.98 Graphene is incapable of capturing dsDNA, but assembled graphene has the ability to bind dsDNA in electrolyte solution.103 Conversely, dsDNA can bind to GO spontaneously and form a complex in the presence of a certain ionic strength, while the complex is blocked by adding nonionic surfactants.102 Self-assembly of DNA on GO also transfers into a multifunctional hydrogel.104 DNA was assembled onto the graphene surface by physical adsorption and DNA released when it met the complementary sequences.101,105 Interestingly, cDNA promotes the aggregation of GO with DNA.106 Generally, the self-assembled experiments were carried out for one or two DNAs in a simple solution. The actual environmental conditions and biofluids are complex, where self-assembly of nucleic acids on graphene should be very sophisticated. 3.2.3. Option between Protection and Cleavage. Recently, it was discovered that the complex of DNA and GO protects DNA from enzymatic digestion.102 Therefore, graphene was used to prevent DNA from degradation during cellular delivery or other biotechnology.107,108 ssDNA adsorbed on graphene’s surface is effectively protected from DNase

3. INTERACTIONS FROM SMALL MOLECULES TO ORGANS The interactions of graphene with metals, small organic molecules, nucleic acids, proteins, enzymes, cells, or organs are critical to understanding the mechanisms of environmental and ecological responses and evaluating human health and ecosystem risks. 3.1. Adsorption of Metals and Small Organic Molecules

Graphene with a large surface area is a promising nanomaterial for the treatment of metals and organic pollutants in water. Water-dispersible magnetite/graphene was used to remove arsenate in drinking water or aqueous solutions with a wide pH range and high adsorption capacity.63,90 Hexafluorophosphatefunctionalized graphene can efficiently adsorb Pb(II) and Cd(II) from water with a high adsorption capacity.60,91 For trace organic pollutants, graphene also exhibits striking properties in the removal of aromatic compounds and dyes in solution, such as polybrominated diphenyl ethers (PBDEs).92,93 In addition, graphene as a novel coating for solid-phase microextraction has been applied as a means for extraction of organochlorine pesticides in aqueous samples with high recovery, a long life span, and a low cost.94 Interestingly, metals such as Ca or Mg are active sites of enzymes. Thus, the interactions of graphene with metals can affect the function of enzymes. Furthermore, small organic molecules can bind to graphene in vivo as occurs in artificial solutions. The effects of E


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strong electrostatic and π−π interactions between nucleic acids and graphene.108 However, the adsorbed DNA on graphene can be exchanged by the free DNA in solution.63 The questions are whether graphene binding to DNA is reversible or poses adverse effects and how much nonspecific binding occurs. By considering the nonspecific binding, the applications in biosensors, drug delivery, and imaging in vivo face health risks. An interesting application of graphene is its fast detection of the sequences of DNA. Because DNA can cross graphene nanopores/nanogaps and then produce a change of electrochemical signals,118,119 the sequences of DNA can be detected by graphene.120,121 Although the fast DNA sequencing with a graphene-based nanochannel device is encouraging,122 the potential challenges of this novel technique should be noted. Considering that the sequencing error is dependent on the nanogap width,120 a high spatial resolution and a complex algorithm are required.123 Correspondingly, DNA/RNA incorporation in nanopores/nanogaps could be an interesting environmental health topic. The incorporation of nucleic acids makes the electronic structure of graphene heterogeneous.124 The heterogeneous electrochemistry probably increases the uncertainty in its application and triggers health and ecosystem risks. This extrapolation should be considered in the future.

I.106,107 It is probable that there are two paths of graphene protecting DNA from nuclease. The steric hindrance could hinder the access of nuclease to nucleic acids due to the covalent immobilization and noncovalent π−π stacking between nucleic acids and graphene. Moreover, graphene may directly inactivate nuclease and then protect nucleic acids. Compared to the protective effects, the ending base pairs are broken apart when DNA assembles on graphene with flat structures.109 Furthermore, graphene can intercalate efficiently into DNA molecules at the major groove and initiate the scission of DNA with bivalent metal ions, leading to the breakage of DNA.110 Graphene serves as a shuttle for metal ions. Rather than radicals, the oxidative manner can be the mechanism of DNA cleavage.110 Given that the protective and unprotective effects of graphene on nucleic acids can simultaneously exist, future work should consider the balance between the two interactions. 3.2.4. Effects of a Hybrid. Graphene-assembled ssDNA by noncovalent π−π stacking under strong ultrasonication forms a water-dispersible graphene/DNA hybrid.111,112 Patil et al.113 proposed that self-assembly of graphene with DNA was a layered nanocomposite. As DNA is a natural composition of organisms and can block sharp edges and promote the solubility of graphene,112,113 the hybrid of DNA/graphene likely enhances the biocompatibility of graphene. Although patterning of DNA on graphene provides new avenues to applications, the current−voltage characterization of the hybrid DNA/graphene exhibits a shift of the Dirac point and “intrinsic” conductance.114 This suggests that the properties of graphene should be evaluated again after modification with DNA. In addition, the conformation, activity, and function of DNA also deserve sufficient investigation after DNA has immobilized on GO. The interactions and challenges of graphene with nucleic acids are presented in Figure 2.

3.3. Affinity and Activity of Proteins

Proteins consist of one or more polypeptides with globular or fibrous forms and facilitate biological functions. The interactions of graphene with proteins connect the biological applications (biosensors, biocompatibility, drug delivery, and therapy) with health risks (nanotoxicity).125−128 Herein, the related interactional forces, affinity location, nonspecific affinity, conformation, electronic property, and challenges are reviewed. 3.3.1. Interactional Forces. Graphene tends to aggregate due to π−π stacking interaction; thus, modifications with proteins become necessary to improve its stability and introduce unique functionalities. Qin et al.129 suggested that van der Waals forces rather than π−π stacking play a dominant role in the interaction of amino acids and graphene in aqueous solution. As for GO, besides the hydrophobicity and π−π stacking forces (on electron-conjugated subdomains), hydrogen bonding between the oxygen functional groups of GO and nitrogen/oxygen-containing groups of proteins might be counted.130 The peptide/graphene hybrid is influenced by pH and is preferred to acidic or neutral conditions.125 The constituents of the protein/graphene conjugation repel or attract each other, depending on the presence of charges.79 Molecular dynamics simulation approaches indicated that electrostatics and van der Waals forces play a dominant role in the interaction process.131 In addition, pristine graphene has a large water contact angle and could damage the hydrogen bonds of proteins by the dispersion and hydrophobic interaction. 3.3.2. Affinity Locations. Atomic force microscopy indicated that peptides preferentially localize and assemble close to the edges of graphene nanosheets.64 Peptides localize at the graphene edge with a weaker interaction energy (−109 kcal/mol), while peptides residing close to the planar center have a stronger interaction energy (−148 kcal/mol).64 The driving force for peptides binding to the edge or the planar surface could be attributed to electrostatic interaction or π−π stacking, respectively. The hydrogen-terminated graphene edges with positive charges can attract negative charges of amino acid residues. The aromatic amino acid residues can

Figure 2. Interactions and challenges of graphene with nucleic acids.

3.2.5. Competitive Adsorption and Heterogeneous Electrochemistry. An aptamer (ssDNA/RNA) is noncovalently immobilized on graphene to sensitively monitor DNA or proteins via fluorophore quenching, which has been applied in biosensors, drug delivery, and imaging in vivo.106,115 Aptamer/graphene nanocomposites are also directly applied in electrochemistry and electrocatalysis in the environment.72,116 Aptamer immobilized on graphene exhibits a low background signal and a selective adsorption to the ligands.117 The mechanisms of fluorophore quenching and adsorption are the F


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Figure 3. Opportunities and challenges of the biomolecule corona for graphene.

form π−π interactions with graphene surfaces. Moreover, the locations of peptides are pH-dependent. Peptides were found to be absent from the edges and instead to deposit on the planar surface of graphene under acidic conditions.64 This is useful information in explaining the behaviors of graphene in vivo or in the ecosystem. 3.3.3. Health Risks of Graphene Nonspecific Affinity. Recently, graphene and graphene-based materials have served as an ideal platform for biosensors and therapy.107,132,133 These applications are based on the specific binding of graphene to targeted proteins. There are various proteins in bloods and cell surfaces. Graphene nonspecifically binds to proteins in vivo by π-stacking interactions and electrostatic interactions.66,69 For instance, GO with reactive COOH and OH groups facilitates conjugation with various functional proteins, leading to irreversible denaturation (or activation) of proteins and potential long-term toxicity.134 In addition, nonspecific delivery (due to nonspecific binding) of chemotherapeutic agents by nanomaterials leads to undesired side effects on normal tissues and insufficient dosages to kill cancer cells.135 Therefore, a major challenge for cancer and other disease therapy using graphene is nonspecific affinity.136,137 The technical breakthrough is to enhance the specific affinity for target proteins. To reduce the health risk of graphene applications, functionalization with the ligands of specific proteins or blocking agents is used to avoid nonspecific affinity.136,137 However, graphene nonspecifically binds to various proteins, even when it is functionalized with a specific aptamer.138 The nonspecific affinity for proteins should be highly considered for nanotoxicity and nanotherapy. 3.3.4. Protein Corona. The protein corona is composed of an inner layer of specific proteins with a lifetime of several hours in slow exchange with the environment (hard corona) and an outer layer of weakly bound proteins, which are characterized by a faster exchange rate with the environment (the soft corona).139 Due to the long lifetime of the hard protein corona, it is now believed that the hard corona rather than the pristine nanomaterial surface interacts with cellular receptors and defines the fate of nanomaterials in an ecosystem or biological environment.140,141 Cells associate and take up only the proteins they need. Therefore, if a long-lived protein corona that presents the relevant receptor-binding sites is in contact with the cells long enough, it can activate the uptake machinery of the cells in a process known as endocytosis.142

Recently, Mahmoudi and coauthors comprehensively reviewed the protein corona, such as the formation, influencing factors, conformational changes, and analytical methods.143 A statistical analysis of the size of the nanoparticles results in a Gaussian distribution for protein-coated nanoparticles, with the average diameter being 5 nm larger for the coated nanoparticles than the bare ones.140 The modification of proteins enhanced the stability of nanoparticles in biological conditions, and then an electrolyte improved the dispersity of protein/nanoparticles.144 Monopoli et al.145 expounded the adsorption process (a competitive manner with the Langmuir adsorption isotherm) of nanoparticles with proteins and validated the feasibility of in vitro and in vivo extrapolations. The differential display of proteins bound to the surface of nanoparticles can influence the tissue distribution, cellular uptake, and biological effects of nanoparticles. It was observed that the uptake of FePt nanoparticles by HeLa cells was suppressed by the protein corona compared with the bare nanoparticles.146 Although deliberate attachment of protein ligands (for example, transferrin) can increase endocytic uptake, it is less clear how the spontaneous formation of a protein corona might contribute to tissue uptake in vivo. It is generally perceived that plasma protein binding is important in determining the organ distribution and clearance of carrier particles from the circulation.147 The primary work presented that the cytotoxicity of GO was greatly mitigated at 10% fetal bovine serum.126 Information on the protein corona with graphene is limited. To quantitate and understand the binding and exchange behavior of proteins on the surfaces of graphene are emergent. The primary work can focus on the effects of the physicochemical property on the protein corona, such as the size, shape, composition, charge, defects, and other surface properties of graphene. Subsequently, more attention should be paid to the conformational changes (reversible and irreversible) of proteins induced by graphene. Nanoparticles enhance the rate of protein fibrillation, which is related to protein misfolding diseases, such as Alzheimer’s disease and Parkinson’s disease.148 Therefore, the toxicity of the graphene/protein corona needs to be expounded. In addition, a nonprotein corona can form nanomaterials with ions and small biological molecules in a biological system. Xu and coauthors observed highly selective binding of the components of the cell culture medium (other than proteins) and phosphate-buffered saline to ZnO and CuO nanoparticles.149 Furthermore, nanoparticles form exceptionally G


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stable surface complexes with ligands incorporating a phosphate or phosphonate moiety.150 In summary, the biomolecule corona, especially for a protein corona, is critically important to the environmental fate and toxicity of graphene in vivo, and related research should be conducted urgently. The critical issues of the biomolecule corona are described in Figure 3. 3.3.5. Conformation and Electronic Property. Incubation with graphene can induce conformational changes of proteins. Generally, due to strong π−π stacking, it is surprisingly difficult for graphene to maintain the conformations of proteins and very easy for graphene to destroy the structure of proteins.79 The peptides are mostly dimeric and maintain an α-helical structure in solution, whereas they unfold and assemble into a morphous dimer at the graphene surface.151 The formation of an α-helical structure is driven by interpeptide hydrophobic interactions.151 The extent of helix unfolding depends on the interplay between peptide−graphene and peptide−peptide interactions. The extent of helix breakage induced by carbon nanomaterials is inversely proportional to their curvature, and the helix breaking tendency is remarkable for a planar graphene sheet compared with nanotubes.131 However, Zuo et al.152 proposed that proteins retain their structural intactness and biological activity on GO. This conclusion requires further proof. Compared with covalent binding, noncovalent modification of graphene has various advantages, including ease and reversibility of the decoration process with minimal risk of permanently altering its intrinsic structures and properties.86,113 Kodali et al.153 supposed that noncovalent modification of graphene did not perturb the electronic band structure of graphene, such as sp2 hybridization and π bands. However, edge-functionalized proteins of pristine graphene are expected to play an important role in the modulation of its electronic properties, because it can modulate the band gap properties of graphene by creating electron-scattering sites.64 Proteins binding to the edge affect the electronic structure of graphene more significantly than those binding to the planar surface of graphene.64 3.3.6. Challenges in Micro- and Macro-Bioresponses. Interactions with proteins have an influence on the biodistribution and toxicity of graphene;154 for example, polyL-lysine-functionalized graphene is relatively water-soluble and biocompatible.155 The interactions between graphene and proteins depend on the size, shape, and surface properties of the graphene and protein types. The π−π stacking interactions between graphene and aromatic residues (Trp, Phe, and Tyr) have not been sufficiently addressed. The superstructure of graphene-functionalized proteins should be explored by experimental and computational approaches. So far, related work is lacking. If graphene plugs into the hydrophobic core of proteins to form stable complexes, the plugging of graphene can disrupt and block the active sites of proteins, thus leading to the functional loss of proteins. Despite the limited knowledge of the interactional mechanisms and features, it is clear that proteins play an overriding role in human health and environmental risks of graphene. The research gaps between molecule-level responses and high-level bioresponses should be given attention in the future. For instance, the relationship between the response of proteins and the occurrence of diseases is not well-known for graphene. The interactions and challenges of graphene with proteins are summarized in Figure 4.

Figure 4. Interactions and challenges of graphene/enzymes with proteins.

3.4. Enzyme Activity

It is evident that GO possesses a number of reactive functional groups that can easily bind to the free amine terminals of the enzyme to result in a strong amine covalent linkage.156 The GO-immobilized enzymes have shown improved thermal stability and a wide active pH range, which is useful for practical applications in biosensors, medicine, and wastewater treatment.90 GO with dissolved dioxygen forms surface intermediates and then oxidizes glutathione (GSH) to dimer glutathione (GSSG) and minor oxidized species (GSxOy).157 Because the surface of GO is negatively charged under physiological conditions, enzymes bind to GO by their positively charged residues.133 GO acts as an artificial receptor and inhibits the activity of α-chymotrypsin, a serine protease, due to the coexistence of anionic, hydrophobic, and aromatic residues and a large surface area to mass ratio of GO.48 The enzyme−GO interactions are reversible without alteration of the native conformation of enzymes.32 For the application of enzymes on graphene, graphene should prevent the aggregation and denaturation of enzymes, but should not perturb the native conformation of enzymes in an undesired way. Moreover, the binding sites are not likely to be completely blocked by the tested enzymes. Therefore, random enzymes or proteins could interact with GO. Functionalized graphene can serve as an enzyme. Folic acidfunctionalized graphene possesses intrinsic peroxidase-like activity, and its catalysis is strongly dependent on th epH, temperature, and H2O2 concentration, similar to horseradish peroxidase.158,159 It has been reported that GO promotes enzymatic catalysis in organic solvents.160 Subsequently, enzymatic catalysis produces nanoholes in the planar graphene.38 Finally, the holes open up the band gap with a semiconducting property and produce additional oxygencontaining functional moieties. Generally, CO2 is the final product of complete enzymatic catalyzed oxidation. Given that the reactive sites of an enzyme are closer to GO than pristine graphene, the catalysis for GO is higher than that for pristine graphene.53 If graphene has a catalysis similar to that of enzymes, it can be used in contaminated water treatment and H


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to that found in vivo.174 Conjugation of quantum dots with graphene indicated a favorable biocompatibility to Hela cells by MTT assay.175 The cytotoxicity of graphene exhibits time/concentration/ solution dependency. The dynamic formation of the protein corona for nanomaterials is a key molecular event, depending on the incubation time.88 GO does not enter A549 cells and has no obvious cytotoxicity (morphology, viability, morality, and membrane integrity) depending on the concentration.89 Furthermore, the cell culture solution influences the dynamic formation of protein/graphene and the cellular response.88 It is significant that graphene enhancing the attachment and proliferation of mammalian cells is nonspecific,71 and the protein/GO complex can enter into living cells and form duplexes with cellular active matters, for example, ATP.176 The above two phenomena should be noted. 3.5.3. Positive Cytotoxicity. Compared with nanotubes, graphene has a low toxicity for MTT and LDH tests.169 Graphene is also more damaging than GO to mammalian fibroblasts.83 The growth rate of HepG2 cells (translation process and protein folding) might be disturbed after exposure to graphene by proteome analysis.166 GO exhibits obvious toxicity to normal human cells and tumor cells.177 Interestingly, the cytotoxicity is largely attenuated when GO is incubated with fetal bovine serum.127 TiO2/GO hybrids reduce the mitochondrial membrane potential, cell viability, and activity of superoxide dismutase (SOD), catalase (CA), peroxidase (POD), and glutathione-S-transferase (GST), as well as promote production of malondialdehyde (MDA).178 The sizes, chemical groups, and surface of graphene may have a strong impact on biological and toxicological responses. An increase of the nanotube dimension suppresses adhesion and enhances differentiation,179 but the effect of the size of graphene on the adhesion and growth of cells is not clarified. The smaller GO displays more efflux of hemoglobin from red blood cells and exhibits lower cytotoxicity.83 The cytotoxicity exhibits a graphene concentration and incubation time dependency. At low concentrations, graphene induces stronger metabolic cell activity, and this trend is reversed at high concentrations.180,181 Although the cytotoxicity increased with time, the cytotoxicity could also be reversed with time.182 For example, in cellular metabolism, the mild cytotoxicity of graphene was reversed after cells were incubated with a fresh medium.183 GO also produces cytotoxicity in concentrationand time-dependent manners and can enter into the cytoplasm (lysosome, mitochondrion, and endoplasm) and nucleus, which decreases cell adhesion and induces cell floating and apoptosis.177 3.5.4. Cytotoxicity Mechanisms. Currently, the uptake paths of graphene into cells are still obscure. Because graphene is taken up by cells with minimal temperature dependence, it is supposed that the cellular uptake of graphene possibly relies on direct penetration such as endocytosis, rather than energydependent pathways.183 In contrast, GO/gelatin nanosheets are taken up by MCF-7 cells through a nonspecific endocytosis mechanism.184 Li et al.74 described the signal transformation paths and related protein stimulation to enhance cell growth. Compared with strong π−π stacking on graphene, GO maintains the conformation of important proteins such as insulin and enhances stem cell differentiation.185 Graphene promotes neurite sprouting and outgrowth of hippocampal cells due to the stimulating expression of growth-associated protein43.186 An electrical signal stimulates growth of neuron cells and

also disturb the redox system in vivo when graphene enters into human and other biological bodies. Interestingly, GO facilitates the production of quantum dots and triggers the generation of reactive oxygen species,161 which are byproducts seriously harmful to human health and the ecosystem. The interactions of enzymes with graphene face challenges similar to those of the interactions of protein with graphene. 3.5. Cell Responses

Cell graphene-based tissue engineering devices, electrical sensors, and anticancer drugs have been proposed for in vitro or in vivo application. Graphene could influence cell morphology, alignment, adhesion, migration, proliferation, and cytoskeleton organization when it contacts cells.162−165 Herein, the assay methods, toxicological responses, and interactional mechanisms are reviewed. 3.5.1. Reliability of Assay Methods. Generally, lactate dehydrogenase (LDH) release is widely monitored to evaluate the cellular membrane damage induced by graphene and other nanomaterials. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay is conducted to quantify the activity of cellular mitochondria. However, the spontaneous reduction of MTT by graphene could result in a false-positive signal.83 Moreover, the common cytotoxicity assays on membrane damage, metabolic irregularities, or inflammatory responses, such as LDH or MTT, are not available for analysis of cell senescence, the cellular signal, or inherited alteration. Gene expression impacted by nanotopography is a somewhat intuitive representation of the effect on basic cell function.80 In addition, the proteomic array can compensate common cytotoxic tests and provide enormous information on cytotoxicity.166 To confirm the experimental results, genome or proteome analysis should reveal the metabolic pathway, redox regulation, cytoskeleton formation, and cell growth in future work. 3.5.2. Negative Cytotoxicity. Graphene has provided a good substrate for the adhesion and proliferation of the mouse fibroblast cell line (L-929).167 Similarly, L929 cells can adhere to and develop on graphene/chitosan composite films without cytotoxicity by MTT assay and obvious variance in morphology.168 A graphene hydrogel matrix can serve as a scaffold for proliferation of the MG63 cell line.162 Scanning electron microscopy revealed that osteoblast cells have a high ability to grow over graphene films without any observable variation in their morphology or size.169 Proteome analysis also indicated that graphene has a lower cytotoxicity than a singlewalled naotube.166 Likewise, RGO is more biocompatible with neuroendocrine PC12 cells, oligodendroglia cells, and osteoblasts than single-walled carbon nanotubes.170 Research on RGO indicated consistent cell growth and proliferation, with the cells retaining their native shape and without any sign of lysis.70 GO enables more favorable cell adherence and proliferation than pristine graphene due to the more abundant oxide groups.171 It is significant that graphene-functionalized organic or inorganic molecules have improved biocompatibility. Artificial peroxidase or extracellular matrix proteins enhance cell adhesion/growth on graphene.172 GO coated with chitosan nearly eliminates the hemolytic release from red blood cells.83 Fe3O4/GO composites possess a good physiological stability and low cytotoxicity to HeLa cells, depending on the concentration of the composites.173 Ca2CO3 immobilized on pristine graphene or GO induce the adhesion of osteoblast cells with high cell viability and provide a microenvironment similar I


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facilitates the propagation of cells.170 The genic responses for cell growth are related to the gene transfection efficiency of cells grown on graphene, which was improved up to 250% in comparison to that of cells grown on glass.187 Compared with the mechanisms of biocompatibility, the mechanisms of cytotoxicity have been sufficiently studied. Tremendous research has revealed that oxidative stress is a ubiquitous mechanism of cytotoxicity.74,188,189 Oxidative stress induced by nanomaterials has been systemically reviewed.189 Pristine graphene can deplete the mitochondrial membrane potential, increase the intracellular ROS, and trigger apoptosis by activation of the mitochondrial pathway (activation of signal proteins).74 Hu et al.127 suggested that the cytotoxicity of GO is induced by physical damage to the cell membrane from direct interactions between the cell membrane and GO nanosheets. The cell membrane damage is likely attributed to the strong electrostatic interactions between negatively charged (oxygen) groups of graphene and positively charged phosphatidycholine lipids of membranes.183 The possible mechanisms of graphene cytotoxicity are proposed as follows:177 graphene attaches to the surface of cells and provides a stimulus signal to the cells; the signal transduction inside the cells and nucleus leads to down-regulation of adhesion-associated genes and proteins, causing the cells to detach, float, and shrink; graphene enters into the cytoplasm and reaches the lysosome, mitochondrion, endoplasm, and nucleus, which disturbs the cell metabolism and gene transcription/translation and induces cell apoptosis or death. In addition, graphene has a valuable photothermal anticancer effect under near-infrared irradiation.165,178 The mechanisms of the graphene-mediated photothermal killing of cancer cells involve oxidative stress and mitochondrial membrane depolarization together with apoptotic and necrotic cell death.165 3.5.5. Challenges in Cytotoxicity. Little is known about the underlying mechanisms of graphene topographical cues on the cell function, in spite of the large body of work that has been done on cytotoxicity. Regarding the effects of graphene on immune and neural cells in vivo, the possible mechanisms are not clarified and need further research.177 In addition, cellular uptake and expulsion models and cell division redistribution remain unclear for graphene. Statistical analysis of nanoparticle delivery shows that the cells take up nanotubes and nanoparticles randomly and redistribute them to their daughter cells in a biased way.190,191 However, tracking two-dimensional graphene inside cells is a brand new approach. Recently, nanotubes have been revealed as conduits of cell-to-cell communication by an electrical couple influencing signal transformation.192,193 If planar graphene can act in similar roles, the effects of graphene on cell-to-cell communication have significant implications for human health and disease. The interactions and challenges of graphene with cells are described in Figure 5.

Figure 5. Interactions and challenges of graphene with cells.

translocation and biodegradation, which vary with time and chemical modification. Most GO was located (90 mg/kg, 24 h) in the liver, followed by the lung, spleen, kidney, stomach, and heart.194 In contrast, the maximum concentration of GO/ dextran accumulated in the spleen (20 mg/kg, 24 h), followed by the liver, lung, stomach, skin, kidney, heart, and bone.180 Zhang et al.182 suggested that GO was predominantly deposited in the lung, followed by the spleen, liver, stomach, kidney, heart, bone, and brain (1 mg/kg, 14 days). Wang et al.177 documented that GO entering into mice by vessel injection was primarily located in the lung (13.8 mg/kg, 30 days), liver, spleen, and kidney. Compared with the work of Wang et al.,195 most PEGylated graphene was located in the spleen (20 mg/kg, 30 days), followed by the liver, stomach, bond, thyroid, lung, kidney, heart, skin, muscle, and brain. The above reports imply that the exposure time and modification rather than dose of graphene significantly influence the biodistribution of graphene in vivo. 3.6.2. Metabolisms and Bioresponses. The clearance capacity of graphene by the kidney is limited.177 GO was introduced intravenously, entered into the circulatory system, stomach, and intestines, and was finally excreted through feces and urine.194 GO obviously cleared from the mouse body within a week, and no pathological changes were observed.181 Similar to GO, PEGylated GO gradually disappeared from 1 h to 60 days, and its metabolism was through both renal and fecal excretions.195 Yang et al.195 also proposed the degradation of GO by oxidative metabolic pathways in vivo. No obvious organ (function) damage was noticed except that the color of the liver and spleen turned brown due to the accumulation of graphene, but these organs changed back to normal color after 20 days.195 However, GO entering into lung tissues induces lung inflammation and granulomas, which are highly dependent on the injected dose.196 Duch et al.196 supported the fact that GO administered directly into the lungs of mice results in severe and persistent lung injury. 3.6.3. Obscure Fate. The biodegradation/clearance of graphene in organs (brain, lung, liver, kidney, etc.) is critical to understanding the fate of graphene in vivo. Related research and study of its mechanisms are just beginning. Furthermore, it is too early to draw a conclusion about the effects of the size and surface groups on the distribution of graphene in organs. Intravenous administration is widely used to study biodistribution in vivo, while the studies for oral administration, inhalation, and skin permeation are lacking. Notably, graphene uptake and toxicity in the brain are very important to human health, but it is still ambiguous whether the blood−brain barrier

3.6. Pharmacokinetics and Nanotoxicity of Graphene in Organs

3.6.1. Translocation. Compared with other carbon nanomaterials, GO exhibits a long blood circulation time (half-time 5.3 h) and low uptake in the reticuloendothelial system.181 In contrast, after intravenous injection, GO/dextran accumulated in the reticuloendothelial system, including the liver and spleen.180 Compared with the injected dose, the conduction time and chemical modification are two dominant factors for the distribution of graphene in organs. This is attributed to J


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obscures the association between nanomaterials and biological behaviors.199 In our opinion, a parameter representing the size and mass concentration could possibly be a dose metric for graphene. Compared with the studies in vivo, exploration of the mechanisms is easy for the tests in vitro due to the adjustable conditions and the limited influential factors. However, the toxicological mechanisms of graphene are still obscure, for example, signal transformation, protein stimulation, or genic expression. Moreover, it is not certain whether the uptake or toxicity of graphene is specific to target organs. The studies in vitro can potentially misidentify the systemic effect and target organs. 3.7.2. Nanotoxicology in Vivo. Given the complexity of the in vivo system, animal experiments are important for helping to avoid misleading results and conclusions in vitro.200 Limited work has been conducted in studying the primary biodistribution of graphene in mice/rats. Moreover, various administrated methods, especially for the time frame of the study, produce diverse results, as mentioned in section 3.6. Therefore, researchers should describe the detailed methods of animal experiments for the purpose of convenient comparison with other work. For future in vivo work, six issues should be considered. First, valid methods should be conducted for the translocation of graphene and its mechanisms. Second, the physicochemical properties of graphene should influence the biodistribution and toxicity in vivo. Third, animal models should be extended beyond rats and mice to evaluate health and environmental risks. Generally, the terms of acute and chronic toxicity of graphene for mice/rats are less than 90 days and from four months to a few years, respectively. Fourth, longterm exposure is essential for evaluating the translocation, metabolism, and toxicity of graphene, because the fate of graphene is intensively time-dependent in vivo. The retention time in biofluids and organs and translocation through barriers (biomembrane/pore, blood−brain barrier, placental barrier, etc.) are critical to human health. Fifth, pharmacokinetics should provide information regarding internal doses and secondary organs for the purpose of designing and interpreting in vitro and in vivo studies. Finally, although graphene is an attractive material for drug delivery, tissue engineering, and imaging in vivo, its effects on male/female reproduction and fertility are yet unknown. 3.7.3. Non-Negligible Knowledge in Toxicology. Four non-negligible issues in graphene toxicology were reviewed: valid dose, toxic acceptability, irreversible/nonadverse effect, and joint toxicity. (i) The concentrations of graphene involve exposure dose, adsorbed dose, and target dose. The target dose is a direct concentration related to bioresponses. However, a wealth of literature has used exposure dose to interpret dose− response relationship, which has led to a false spectrum of toxic effects. (ii) Acceptable toxicity does not mean that there is no toxicity. It is known that toxins are dependent on concentrations and time. For example, water can be a toxin with enough exposure and time. Moreover, humans can produce a natural defense to exogenous substances. As an example, although flour powder is commonly viewed as safe, flour powder can lead to pulmonary changes when it is taken in.201 To identify the toxicity, the acceptable daily intake/ maximum allowable concentration of graphene should be expounded as soon as possible. (iii) The reversible/irreversible and adverse/nonadverse effects of graphene are ambiguous. The immediate effects and delayed effects upon various target organs are unknown. An acute toxicity assay with one

inhibits graphene from entering into the brain. A diagram of graphene in vivo is described in Figure 6.

Figure 6. Critical fate of graphene in vivo.

3.7. Perspectives in Toxicity

3.7.1. Nanotoxicology in Vitro. Over the past few years, the majority of graphene toxicity research has focused on the interaction with nucleic acids, proteins/enzymes, or cells in vitro. Given the fact that one or a few biomarkers are insufficient to assess long-term toxicity, toxicoproteomic evaluation of nanomaterials is recommended in vitro.197 The primary purpose of the in vitro studies is to verify biocompatibility by one or two simple tests, which likely leads to false-negative or -positive results. Systematic toxicological data are necessary to support a solid result or conclusion and reduce the gaps between the studies in vivo and in vitro, as described in Figure 7. The recommended steps of

Figure 7. Challenges of graphene toxicity in vitro and in vivo.

toxic evaluation for nanomaterials in vitro were presented by Sharifi and colleagues.25 The particle size, size distribution, particle morphology, particle composition, surface area, and chemistry in the medium are not trivial elements and need to be defined for accurate assessment of nanoparticle toxicity.198 So far, there are no well-defined techniques for the characterization of graphene in biological samples. The medium of reaction could significantly influence the properties and bioresponses of graphene. The natural or close to natural medium is the best choice for assay. In addition, how to identify the dose metric of the nanomaterial is still an insurmountable question. The use of mass concentration alone as a dose metric K


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of graphene on the pore size, thickness, and functions of mucus barriers as a scavenger or lubricant. 4.1.2. Cell Vision. Cell vision defines the process by which a cell recognizes nanomaterials, which is a concept complementary to the protein corona.212 Cell vision refers to the first contact point (surface proteins and sugars and the phospholipid composition of cell membranes) of the nanomaterial surface with cells. Namely, the tolerance and means by which cells fight nanomaterials can reflect cell vision. For instance, various cell types show different ROS levels and genes associated with cell proliferative responses, depending on the pathways they use to defend themselves against foreign nanomaterials.213 Cell vision indicates that the uptake and toxicity of nanomaterials are dependent on the cell type.212,213 Therefore, cell vision influences the amount of uptake of foreign objects into cells as well as their fate in the intracellular environment. Compared to the physicochemical properties of nanomaterials, cell vision should be highly considered for nanotoxicity.212 Previous work showed the uptake of superparamagnetic iron oxide nanoparticles and their corresponding toxicity profile to be strongly dependent on the cell type.212,213 Other publications also confirmed the important role of cell vision in the uptake and toxicity of nanomaterials.25 The cell vision concept is useful to obtain reliable and reproducible nanotoxicology data and reduce the conflict of future toxicological reports. The emergent question is how the surface proteins, sugars, and phospholipid composition of cell membranes identify the nanomaterials or biomolecule corona. 4.1.3. Penetrating Phospholipid Bilayers. Phospholipid bilayers are flat sheets that form a continuous barrier around cells and keep ions, proteins, and other molecules located in the proper areas. Modified carbon nanotubes with alternation from relatively hydrophilic to hydrophobic can penetrate through phospholipid bilayers.214 Because GO is amphoteric,87 it is possible to cross through the bilayer of cell membranes. Titov et al.215 stated that graphene sheets can be hosted in the hydrophobic interior of amphiphilic phospholipid membranes. Given the sharp two-dimensional edge, excellent electrical conduction, and amphoteric property of graphene, the transportation of graphene through phospholipid bilayers and the related bioresponses should be observed. 4.1.4. Skin Permeation. The permeation of graphene from aqueous solution or air through human skin is of considerable environmental importance. However, studies of dermal deposition are very rare. So far, most dermal exposure research has implanted nanomaterials into subcutaneous tissues to detect the toxicity of cells or other bioresponses.216 Skin irritation tests have suggested that edema or erythema do not appear on skin that has been exposed to [email protected] Nanotubes have shown dermal penetration following deposition on exposed skin and then increased epidermal thickness and accumulation/activation of dermal fibroblasts.218 However, paths and mechanisms of the penetration were not discussed. The penetration of graphene through skin is not yet known. Herein, a possible route of graphene translocation in skin is supposed: graphene is absorbed through sweat pores, is transferred from the epidermis into the dermis and subcutaneous fatty tissues, and finally penetrates into the veins and arteries. This hypothesis needs verification. 4.1.5. Across Blood−Brain Barriers. The blood−brain barrier is a tightly protective reticulum surrounding the brain to restrict substances in the blood from entering the brain. The tight junctions of capillary endothelial cells with a semi-

administration could ignore the reversible or nonadverse effects. Unfortunately, a long-term test with multiple administrations is rare. (iv) Our previous work has indicated that there are significant differences between single toxicity and joint toxicity.202,203 Antagonist or synergistic effects of proteins, metal ions, or organic matters with graphene can occur. This issue of joint toxicity has not raised enough attention, which has likely led to overestimating or underestimating the risks of graphene. 3.7.4. Challenges of Graphene Toxicology. To compare the results from different researchers and laboratories, a standard assay, such as choice of model animals or set of exposure conditions, is necessary. To correctly evaluate nanotoxicity, one option is the development of a multilevel approach for evaluating the toxicity of nanomaterials.204 For instance, cytotoxicity tests involve viability assays, evaluation of apoptosis/necrosis, evaluation of mitochondrial toxicity, gene expression and morphology analysis, and assay of dynamic biological processes through nuclear magnetic resonance (NMR) based metabonomics.205,206 The recent modification and development of traditional methods also should be considered for the establishment of standard assays. Mahmoudi and coauthors proposed that the conventional in vitro examination method may contain large errors compared with the modified method and the modified method highlights the components, contact time, and refreshment of cell media in cytotoxicity.207,208 Krug and Wick summarized the unreliable methods and unrealistic test conditions, such as MMT assay lacking reliability and exaggerated particle doses with little relevance to human exposures.209 Subsequently, they recommended the points of eligible nanotoxicology tests, for example, sufficiently characterized nanomaterials and proper methodology. Given that there are no standard assay methods for graphene toxicity, a global control test should be addressed to avoid falsepositive or -negative results. For the cytotoxic evaluation of human health, human cells should be the priority. In addition, a long-term test evaluating graphene is lacking. The ecosystem and human bodies are exposed to nanomaterials at multiple rather than single doses.210 Therefore, multiple administrations are close to actual cases. Given the rapid appearance of modified graphene and the related new discoveries, the development of appropriate protocols to assess the potential toxicity of graphene exhibits a hysteresis.88 A quantitative structure−activity relationship (QSAR) is expected to be established to accurately and rapidly predict the toxicity of various types of graphene. The challenges of testing graphene toxicity are described in Figure 7.

4. HEALTH RISKS 4.1. Penetrating Biobarriers

There are four distinct routes by which nanomaterials can enter into the human body: inhalation, ingestion, dermal penetration, and injection or implantation for biomedical applications.15 Correspondingly, the biobarriers, including mucus, cell vision, phospholipid bilayer, skin, blood−brain, placenta, or glomerular filtration, are critical in appeasing the health risks of graphene. 4.1.1. Damage of Mucus. Mucus secretions typically protect the exposed surfaces of the eyes and the respiratory, gastrointestinal, and female reproductive tracts from foreign entities, including pathogens and environmental ultrafine particles.211 In the future, it will be worth studying the effects L


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graphene stimulates cell growth and differentiation.229 Other probable mechanisms involve an increase in gene expression and the delivery of electric currents to stimulate the Casignaling pathway or neurotransmitter-related proteins.226 So far, no significant discrepancies have been observed between human cells and animal cells for graphene inducing bioresponses. Long-term studies remain essential.230 4.2.2. Hematopathology. Hematology studies of graphene are almost unknown. There are four issues that should be addressed: circulation and long-term fate (clearance rate) of graphene in blood, interactions of graphene with the human blood protein/platelet/complement cascade, effects of graphene on the signals/ion channels in human red blood cells/ white blood cells/blood platelets, and evaluation of the immunotoxicity and related diseases. 4.2.3. Pulmonary Responses. Pulmonary responses to graphene are critical to human health due to possible inhalation, but the related studies are lacking. Four points should be considered in future work: interaction of graphene with pulmonary surfactant proteins, bioresponses of graphene by human bronchial epithelial cells, pulmonary tissue damage and functional loss, and downstream biochemical events. 4.2.4. Pharmacokinetics. The surfaces of nanomaterials are rapidly covered by selective sets of plasma proteins in blood, forming what is called the “protein corona”. Protein absorption extensively influences the fate of nanomaterials and their biodistribution in the body. On one hand, the binding of human serum albumin or apolipoprotein promotes a prolonged circulation time in blood.231 On the other hand, graphene affects the activity/confirmation/structure of proteins, which is reviewed in section 3.3 regarding the affinity and activity of proteins. Passivation, such as protein enwrapping and endocytosis, is also worth further investigation. The reported results of translocation of graphene in vivo are reviewed in section 3.6. The key to biodistribution is quantitatively mapping the location of graphene at different time points and at different doses as size, shape, aggregation state, and surface chemistry. Accurately quantifying graphene and quantifying its byproducts are two challenges to pharmacokinetics. Graphene modified with a fluorophore or radiation only represents the initial concentrations. The breakdown of graphene could produce new behaviors and elicit new bioresponses, and thus, a holistic understanding and cataloging of the quantity of graphene degradation as well as when is extremely important. Biodegradation is an overriding path to appease nanotoxicity,232 but the biodegradation of graphene is not clear in vivo. It has been shown that graphene can be biodegraded by bacteria and viruses to form RGO in vitro. 72,233,234 Furthermore, computational docking studies have revealed that horseradish POD catalyzes the oxidation of GO and results in the formation of holes on its basal plane.54 There are various enzymes in vivo, and their catalytic capacity could have a different story in terms of in vivo behaviors. Graphene could be metabolized in the liver through phase I and II metabolic pathways. Phase I reactions involve the formation of a new or altered functional group by oxidation, reduction, or hydrolysis reactions to increase reactivity or polarity. Phase II reactions involve conjugation of an endogenous compound, such as glucuronic acid or glycine, to ensure higher water solubility and lowered chemical reactivity. The elimination of graphene can occur via multiple routes, including perspiration, seminal fluids, mammary glands, saliva, and exhaled breath. It has been proven that urine and feces are

permeable property regulate the function of the blood−brain barrier where only small and lipid molecules can cross.219 The breakdown of the blood−brain barrier results in cell damage, neurodegeneration, and brain inflammation. Wang et al.177 concluded that graphene could not enter into the brain because of the blood−brain barrier, but more sufficient proof is needed. In fact, some literature has reported that graphene was detected in the brain by intravenous injection.181,195 There is almost no literature on the interactions of graphene and the blood−brain barrier. The current study involves the effects of the sizes and chemical groups of graphene on its translocation, potential transport mechanisms (diffusion or active transportation), and neurological disorders. In addition, blood−cerebrospinal fluid barriers should be investigated further. 4.1.6. Across the Placenta. The placenta system includes the maternal blood sinus, mononuclear trophoblast, syncytiotrophoblast, endothelium, and fetal blood capillary. The placenta connects the developing fetus and the uterine wall for nutrient uptake, waste elimination, and gas exchange via the mother’s blood supply. There has been a considerable focus on nanomaterials possibly crossing through the placenta into fetal bloods and thereby affecting fetal growth.220 The size of nanomaterials usually determines the translocation in organs. Less than 100 nm silica or titanium dioxide nanoparticles cause damage to the placenta and fetus.220 Generally, the size of graphene ranges from 20 to 500 nm, and GO and RGO tend to be a relatively small size. Therefore, graphene crossing through the placenta is definitely possible. Graphene could influence fetal health by direct translocation through the placenta or cause indirect disorder of placenta functions. The possibility, mechanism, and adverse effect of graphene across the placenta should be addressed in future work. 4.1.7. Glomerular Filtration. Although graphene can be excreted by urine,194 glomerular filtration and its mechanism are not yet understood. Glomerular filtration is composed of endothelial fenestrations (pore size 60 nm), glomerular basal membranes, podocyte slit membranes (pore size 5−20 nm), and podocyte slits (pore size 25−60 nm).221 The size of designed graphene usually ranges from 20 to 500 nm, which is not easy to filter directly. It is probable that the biodegradation of graphene in blood circulation leads to a significant amount of graphene crossing through the glomerular membrane. It is also likely that the pores of filtration expand when they contact graphene. Further investigation of glomerular filtration is necessary to understand the clearance of graphene in vivo. 4.2. Special Attention to Health Risks

In addition to the seven important biobarriers mentioned, the cellular, hemeal, and pulmonary responses, pharmacokinetics, diseases, and oxidative stress to nanomaterials are consistently relevant and connected to human health.222−224 4.2.1. Human Cytotoxicity. Graphene accelerates osteogenic differentiation of human mesenchymal stem cells.225 Laminin-coated graphene enhances differentiation of human neural stem cells into neurons.226 Similarly, GO exhibits good biocompatibility and has potential advantages with respect to human cell attachment and proliferation, especially for retinal pigment epithelium cells.227 In contrast, Vallabani et al.228 believed that GO induces cytotoxicity and apoptosis in human lung cells. The primary mechanisms of graphene enhancing human cell growth have been studied. Graphene substrates promote adherence of human osteoblasts and mesenchymal stromal cells because the excellent electrical property of M


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elimination routes of graphene.194 In summary, the pharmacokinetics of graphene in vivo is not clear. 4.2.5. Diseases and Oxidative Stress. The most serious health risks induced by nanomaterials are the related diseases. GO elicits aggregatory responses in platelets through the activation of Src kinases, and release of Ca from intracellular stores, as well as the extensive pulmonary thromboembolism, is induced in mice, which implies that thrombus can be formed by GO.84 Graphene is a potential material for drug delivery and imaging in vivo; however, the effects on male/female reproduction and fertility have not been examined.235 The effects of graphene on immune systems, reproductive systems, and neural systems have not yet been sufficiently investigated.230 Generally, oxidative stress with excess ROS is viewed as a dominant mechanism of pathological changes induced by graphene.15,236 Compared with fullerenes and nanotubes, graphene triggers only moderate oxidative stress. 89,166 Oxidative stress from graphene is responsible for mitochondrial damage and ensuing cell demise, apoptotic DNA fragmentation, and activation of caspases.165 Subsequently, the activation of MAPK, TGF-β, and mitochondrial apoptotic signal pathways regulate essential cellular events, including proliferation, differentiation, or apoptosis.25 Nanoparticle-induced oxidative stress affects cell signaling in three stages, as described by Rallo and colleagues.237 A low level of oxidative stress enhances the transcription of defense genes through transcription factor nrf2. A high level of oxidative stress activates inflammation signaling through NFkB, and a higher level is connected with the activation of apoptotic pathways and necrosis. The relationships and mechanisms between diseases and graphene need to be further explored. The health risks of graphene are presented in Figure 8.

Figure 9. Possible ecosystem risks of graphene.

Figure 10. Interactions and challenges of graphene with bacteria.

products or even in laboratories. The possible ecosystem risks of graphene are described in Figure 9. 5.1. Problems for Environmental Purification

Graphene sheets and their hybrids enhance the adsorption, degradation, and photocatalysis of metals, dyes, viruses, and volatile aromatic pollutants.72,90,243,244 There are four challenges for the related application in the ecosystem. First, various pollutants exist in the natural environment, such as persistent organic pollutants, heavy metals, toxins, and biowaste, whereas the current work only focuses on dyes and metals. Thereby, the target compounds need to be extended. Second, the reactive medium should be consistent with the natural medium, rather than a simple buffer. Third, the stability (dispersibility, integrity, and natural properties) of graphene in various media needs to be explored further. Finally, the reactive byproducts are unknown for graphene and could probably lead to serious adverse effects in the ecosystem.

Figure 8. Health risks of graphene.

5.2. Subtle Ecological Fate

Computational simulation suggested that horseradish POD is preferentially bound to the basal plane rather than to the edge; subsequently, the enzyme catalyzes the oxidation of GO and results in the formation of holes on its basal plane.54 There are various enzymes in the ecosystem where the behaviors of graphene are not clear. In addition, GO converts into RGO by bacteria under ambient conditions and at rapid time scales,112,113 but effects on the size and morphology of GO were not studied. Recently, our work has revealed that viruses could reduce GO into RGO under visible-light irradiation, thereby implying a degradation route of graphene in the environment.72

5. ECOSYSTEM RISKS The rapid rise of graphene is raising concerns about potential adverse effects on the ecosystem. Graphene has been proposed in photocatalysis and adsorption for the treatment of contaminated water.73,238,239 Wild organisms have been used to reveal the fate and behaviors of nanomaterials in the ecosystem.240,241 In addition, graphene with a large surface has been viewed as a promising sorbent for waste gas,242 which will lead to graphene exposure to air. The potential toxicity of graphene could be serious in aquatic and terrestrial environments when graphene is applied in industrial and consumer N


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Figure 11. Diagram of photocatalysis using GO/aptamer in visible-light irradiation: generation of reactive species by energy transformation and electron transfer (a), breakage of protein capsids and leak of nucleic acids (b), protein carbonylation, nucleic acid modification, and GO reduction (c).

can reduce the attachment of pollutants on graphene and increase graphene aggregation. The pH and salt strength may alter the surface changes and interaction forces between graphene and pollutants, thus leading to the dispersion or aggregation of graphene. Temperature could influence the thermodynamics of adsorption. Card et al.13 have reviewed the safety of food-related nanomaterials. Remarkably, the transfer of graphene via food chains could affect the ecosystem and human health, as presented in Figure 9. The safety and toxicity of food-related graphene are worth studying due to the possibility of graphene incorporation into the food matrix and ingredients. Unfortunately, risks of graphene in the food chains have not yet captured enough interest. 5.3. Unavailable Model Species

Aquatic organisms are important components of the ecosystem, and their responses to graphene are critical to the safety of the ecosystem. Knowledge of the aquatic toxicity of graphene has not been sufficiently understood. Standardized materials and rapid assays are two significant challenges to systematically evaluating nanomaterial ecotoxicity.245 Herein, three aquatic organisms as models are recommended for studying the aquatic toxicity of graphene. Daphnia magna is widely used as a laboratory animal for testing ecotoxicity, due to its relatively short life span, easy culture, and vigorous reproduction. The accumulation and elimination of multiwalled carbon nanotubes in D. magna has been explored.246 Danio rerio, commonly known as zebrafish, is proposed to be a quick, cheap, and easy model to conservatively assess the toxicity of nanomaterials in ecological risks.247 Furthermore, rainbow trout is a species of salmonid with a varied diet and serves as both food and medicine. A considerable number of researchers have used rainbow trout to test the ecotoxicity of nanomaterials.248 Rainbow trout can reveal risks of graphene in food chains. Given the numerous aquatic plants to be tested in aquatic

Figure 12. Interactions and challenges of graphene with plants.

To evaluate ecological risks, the reactive matrix should be consistent with the natural medium, such as pH, salt strength, temperature, or natural organic matter, which can influence the fate of graphene in the environment.73 Natural organic matter O


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toxicity, including emerged plants, submerged plants, or floating plants, a few sensitive aquatic plants should be screened out as priority species for graphene. For a terrestrial species, earthworms as an indicator of soil contamination are widely accepted. The detailed experimental procedure and assay parameters for earthworms have been discussed in our previous work.203,249 The identification of sensitive species and related bioresponses is the current urgent task in studying ecosystem risks.

previous work.72 GO immobilized with an aptamer was fairly polydispersed and selective in binding to viruses. The aptamer enhanced the photocatalytic activities of GO under the broad visible-light spectrum. The complex photocatalyst effectively inactivated the viruses and damaged the protein capsid and nucleic acids of the viruses in irradiation. Subsequently, the proteins underwent carbonylation, and the bases (especially for guanosine) of the nucleic acids suffered oxidized modification. GO formed reduced GO by loss of oxygen elements. The GO/ aptamer and viruses constitute a redox system. The mechanisms involve energy transfer (generation of singlet oxygen), electron transfer (generation of anion radicals), and water dissociation (generation of the precursor to the hydrated electron), which are described in Figure 11.

5.4. Bacterial Inactivation

Bacteria are ubiquitous in the ecosystem. Understanding the interaction of graphene with bacteria may facilitate the evaluation of environmental behaviors of graphene. The reductions of GO and antibacterial activity are two accepted results for the interaction of bacteria with graphene.52,250,251 The synergistic effects of graphene on other nanoparticles are obvious. The antibacterial activity of GO/Ag composites was investigated by Liu and colleagues252 and was remarkably enhanced compared with that of the original Ag nanoparticles. While the above graphene accumulated on the bacterial membrane and caused oxidative stress and apoptosis, the carboxyl- and amine-modified graphene pacified the hydrophobic interaction with bacteria. The carboxyl functionalization of graphene with small water contact angles induced lower toxicity to bacteria than unmodified graphene.251 Moreover, graphene functionalized with proteins and other amines specifically wrapped the bacteria,56,253 but did not damage the bacteria. In contrast, poly(N-vinylcarbazole) polymer-functionalized GO was more effective in preventing bacterial colonization relative to the unmodified GO.251 It is too early to draw a solid conclusion about the bacterial toxicity of graphene. Bacterial outer membranes maintain the morphology and protect the activity of bacteria. Therefore, the mechanisms of antibacterial activity of graphene are proposed as follows:52,254,255 graphene traps the bacteria; subsequently, the sharp edges of graphene damage the cytoplasmic membrane, interfere with the physiological activity of bacteria, and lead to bacterial inactivation. Compared with GO, RGO with limited reactive groups exhibits weak affinity for bacteria and induces slight toxicity.255 In addition, the generation of superoxide anions by graphene can disrupt bacterial membranes.54 So far, the mechanisms at a molecular level are not clear. GO and bacteria consist of a redox system via electron transfer and a mediator, where GO and bacteria act as an electron acceptor and donor, respectively.233,234 The proposed paths of electron transfer and electron mediator are conflicting. Salas et al.233 suggested that electrons flow from the inner-membrane quinine pool to the periplasmic MtrA protein without a route involving CymA. However, the report from Jiao et al.55 documented that CymA is critical to the redox system of graphene and bacteria. The electron transfer and mediator for bacteria with graphene require further analysis. The challenges of the interactions between graphene and bacteria are described in Figure 10.

5.6. Ignored Phytotoxicity

Plants are an essential component of the ecosystem. Understanding the interactions between nanomaterials and plants is crucial in comprehending the impact of nanotechnology on the ecosystem, but little progress has been made.258 Graphene significantly inhibits plant (cabbage, tomato, red spinach, and lettuce) growth and biomass compared to a control.259 The adverse effects include a dose-dependent manner, and the mechanisms of phytotoxicity involve oxidative stress necrosis.259 Generally, the plant nanotoxicology-related mechanisms subsume catalytic activities (oxidative stress or ROS generation), size-dependent interactions (damage to structures and clogging), and affinity-based interactions (covalent and noncovalent binding).260 It is unknown whether graphene possesses a similar phytotoxicity. Several issues should be addressed in future work: (i) the capability of graphene to penetrate the cell wall and cell membranes of intact plant cells; (ii) the uptake and translocation of graphene in plants are not clear, and the changes in the gene expression of plants are unknown; (iii) given that traditional phytotoxicity tests, such as germination and root elongation are not sensitive in evaluating nanoparticle toxicity,261 the sensitivity of the phytotoxicity test should be modified for graphene before drawing a conclusion; (iv) long-term exposure and the bioresponses of plant species merit further attention. The interactions and challenges of graphene with plants are displayed in Figure 12.

6. CONCLUSIONS Graphene-related research has grown at a spectacular pace in a wide range of disciplines, while more and more scientists are considering the health and ecosystem risks. The reported work and the emergent challenges have been reviewed from metals and small molecules to human health and the ecosystem. The current goal is to reduce the gaps between the expanding material applications and the related studies on human health and ecosystem risks through correct assay methods, valid administration procedures, long-term tests, and sufficient meaningful data.

5.5. Virus Photocatalysis

AUTHOR INFORMATION

Many epidemiological studies have indicated that viruses cause environmental contamination and outbreaks of water-borne diseases.256 Nanomaterials with excellent optical properties and electron transfer abilities have opened up a window of new applications in virus-contaminated water treatment.257 Bacteriophage MS2 as a model virus in the occurrence and purification of viruses was inactivated by GO/aptamer in our

Corresponding Author

*Phone: +86-22-23507800. Fax: +86-22-66229562. E-mail: [email protected]. Notes

The authors declare no competing financial interest. P


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ACKNOWLEDGMENTS This work was financially supported by the National Natural Science Foundation of China as key projects (Grants 21037002 and U1133006). REFERENCES (1) Brumfiel, G. Nature 2009, 458, 390. (2) Geim, A. K. Science 2009, 324, 1530. (3) Lee, Y. A.; Durandin, A.; Dedon, P. C.; Geacintov, N. E.; Sharirovich, V. J. Phys. Chem. B 2008, 112, 1834. (4) Choi, W.; Lahiri, I.; Seelaboyina, R.; Kang, Y. S. Crit. Rev. Solid State 2010, 35, 52−71. (5) Wei, Z.; Wang, D.; Kim, S.; Kim, S. Y.; Hu, Y.; Yakes, M. K.; Laracuente, A. R.; Dai, Z.; Marder, S. R.; Berger, C.; King, W. P.; de Heer, W. A.; Sheehan, P. E.; Riedo, E. Science 2010, 328, 1373. (6) Park, S.; Lee, K. S.; Bozoklu, G.; Cai, W.; Nguyen, S. T.; Ruoff, R. S. ACS Nano 2008, 2, 572. (7) Lee, S. H.; Kim, H. W.; Hwang, J. O.; Lee, W. J.; Kwon, J.; Bielawski, C. W.; Ruoff, R. S.; Kim, S. O. Angew. Chem., Int. Ed. 2010, 49, 10084. (8) Dreyer, D. R.; Park, S.; Bielawski, C. W.; Ruoff, R. S. Chem. Soc. Rev. 2010, 39, 228. (9) Loh, K. P.; Bao, Q.; Eda, G.; Chhowalla, M. Nat. Chem. 2010, 2, 1015. (10) De, M.; Chou, S. S.; Dravid, V. P. J. Am. Chem. Soc. 2011, 133, 17524. (11) Allen, M. J.; Tung, V. C.; Kaner, R. B. Chem. Rev. 2010, 110, 132. (12) Guo, C. X.; Yang, H. B.; Sheng, Z. M.; Lu, Z. S.; Song, Q. L.; Li, C. M. Angew. Chem., Int. Ed. 2010, 49, 3014. (13) Card, J. W.; Jonaitis, T. S.; Tafazoli, S.; Magnuson, B. A. Crit. Rev. Toxicol. 2011, 41, 22. (14) Paul, W.; Sharma, C. P. Trends Biomater. Artif. Organs 2011, 25, 91. (15) Sanchez, V. C.; Jachak, A.; Hurt, R. H.; Kane, A. B. Chem. Res. Toxicol. 2012, 25, 15. (16) Johnston, H. J.; Hutchison, G. R.; Christensen, F. M.; Aschberger, K.; Stone, V. Toxicol. Sci. 2009, 114, 162. (17) Choi, J. T.; Kim, D. H.; Ryu, K. S.; Lee, H.; Jeong, H. M.; Shin, C. M.; Kim, J. H.; Kim, B. K. Macromol. Res. 2011, 19, 809. (18) Nika, D. L.; Ghosh, S.; Pokatilov, E. P.; Balandin, A. A. Appl. Phys. Lett. 2009, 94, 203103. (19) Wang, Z.; Scharstein, R. W. Chem. Phys. Lett. 2010, 489, 229. (20) Luo, J.; Cote, L. J.; Tung, V. C.; Tan, A. T. L.; Goins, P. E.; Wu, J.; Huang, J. J. Am. Chem. Soc. 2010, 132, 17667. (21) Ciric, L.; Sienkiewicz, A.; Djokic, D. M.; Smajda, R.; Magrez, A.; Kaspar, T.; Nesper, R.; Forro, L. Phys. Status Solidi B 2010, 247, 2958. (22) Kim, H.; Lim, S. C.; Lee, Y. H. Phys. Lett. A 2011, 375, 2661. (23) Kwon, W.; Rhee, S. W. Chem. Commun. 2012, 48, 5256. (24) Zhou, K.; Zhu, Y.; Yang, X.; Jiang, X.; Li, C. New J. Chem. 2011, 35, 353. (25) Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M. L.; Stroevee, P.; Mahmoudi, M. Chem. Soc. Rev. 2012, 41, 2323. (26) Yang, K.; Wan, J.; Zhang, S.; Tian, B.; Zhang, Y.; Liu, Z. Biomaterials 2012, 33, 2206. (27) Ci, L.; Song, L.; Jariwala, D.; Laura Elias, A.; Gao, W.; Terrones, M.; Ajayan, P. M. Adv. Mater. 2009, 21, 4487. (28) Debgupta, J.; Shinde, D. B.; Pillai, V. K. Chem. Commun. 2012, 48, 3088. (29) Shao, G.; Lu, Y.; Wu, F.; Yang, C.; Zeng, F.; Wu, Q. J. Mater. Sci. 2012, 47, 4400. (30) Zhang, L.; Liang, J.; Huang, Y.; Ma, Y.; Wang, Y.; Chen, Y. Carbon 2009, 47, 3365. (31) Fasolino, A.; Los, J. H.; Katsnelson, M. I. Nat. Mater. 2007, 6, 858. (32) Dan, M.; Wu, P.; Grulke, E. A.; Graham, U. M.; Unrine, J. M.; Yokel, R. A. Nanomedicine (London, U. K.) 2012, 7, 95.

Xiangang Hu received his M.Sc. degree in 2009 and his Ph.D. degree in 2012 in Environmental Chemistry and Ecotoxicology from Nankai University, China. As a visiting scholar, he did research in the Chemistry Department at the University of Waterloo, Canada, from August 2010 to February 2012. He is currently a lecturer in the College of Environmental Science and Engineering at Nankai University, where he is an active faculty member in teaching and research. His scientific interests focus on application and risk evaluation of graphene in environmental health.

Qixing Zhou has been Professor of Ecotoxicological Chemistry and Environmental Remediation since 2005 in the College of Environmental Science and Engineering at Nankai University, China, before which he was selected as a Cheung Kong Scholar by the Education Ministry of China in 2004. He received his Ph.D. degree from the Chinese Academy of Sciences combined with Karlsruhe University, Germany, in 1992. Before 2005, Dr. Zhou worked as an Honorary Research Fellow and Advanced Research Fellow at Queen’s University Belfast and the University of London, United Kingdom, and served as Researching Professor and Chief Scientist at the Institute of Applied Ecology, Chinese Academy of Sciences. As the first author or the corresponding author, Dr. Zhou has published more than 400 papers in peer-reviewed journals, and his publications are frequently cited by his peers. His current research interests focus on ecotoxicological chemistry and environmental health, ecological remediation of contaminated soils and water, and environmental criteria/standards and ecological risk assessment. As early as 2002, Dr. Zhou received a grant from the National Science Fund for Distinguished Young Scholars in China. He has been invited to present his research results at many universities and institutions in China and other countries in the world. He is an editorial or advisory board member of several international journals. Q


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Review

(33) Jong, W. H. D.; Hagens, W. I.; Krystek, P.; Burger, M. C.; Sips, A. J. A. M.; Geertsma, R. E. Biomaterials 2008, 29, 1912. (34) Choi, H. S.; Liu, W.; Misra, P.; Tanaka, E.; Zimmer, J. P.; Itty Ipe, B.; Bawendi, M. G.; Frangioni, J. V. Nat. Biotechnol. 2007, 25, 1165. (35) Cho, M.; Cho, W. S.; Choi, M.; Kim, S. J.; Han, B. S.; Kim, S. H.; Kim, H. O.; Sheen, Y. Y.; Jeong, J. Toxicol. Lett. 2009, 189, 177. (36) Patlolla, R. R.; Vobalaboina, V. J. Pharm. Sci. 2005, 94, 437. (37) Jiang, W.; Kim, B. Y. S.; Rutka, J. T.; Chan, W. C. W. Nat. Nanotechnol. 2008, 3, 145. (38) Barnard, A. S.; Snook, I. K. J. Mater. Chem. 2010, 20, 10459. (39) Kalugin, N. G.; Kalichava, I.; Fallt, J.; Del Barga, C.; Cooper, C.; Duque, J. G.; Gonzales, E.; Doorn, S. K.; Shaner, E. A.; Gin, A. V. Carbon 2010, 48, 3405. (40) Georgantzinos, S. K.; Giannopoulos, G. I.; Katsareas, D. E.; Kakavas, P. A.; Anifantis, N. K. Comput. Mater. Sci. 2011, 50, 2057. (41) Zhou, Z.; Liu, Z.; Li, Z.; Huang, X.; Sun, C. J. Phys. Chem. C 2011, 115, 16282. (42) Verma, A.; Stellacci, F. Small 2010, 6, 12. (43) Al Faraj, A.; Bessaad, A.; Cieslar, K.; Lacroix, G.; Canet-Soulas, E.; Cremillieux, Y. Nanotechnology 2010, 21, 1. (44) Petkovic, J.; Zegura, B.; Stevanovic, M.; Drnovsek, N.; Uskokovic, D.; Novak, S.; Filipic, M. Nanotoxicology 2011, 5, 341. (45) Banhart, F.; Kotakoski, J.; Krasheninnikov, A. V. ACS Nano 2011, 5, 26. (46) Stadler, J.; Schmid, T.; Zenobi, R. ACS Nano 2011, 5, 8442. (47) Erickson, K.; Erni, R.; Lee, Z.; Alem, N.; Gannett, W.; Zettl, A. Adv. Mater. 2010, 22, 4467. (48) Geim, A. K.; Novoselov, K. S. Nat. Mater. 2007, 6, 183. (49) Mazzamuto, F.; Saint-Martin, J.; Valentin, A.; Chassat, C.; Dollfus, P. J. Appl. Phys. 2011, 109, 064516. (50) Yan, L.; Zheng, Y. B.; Zhao, F.; Li, S.; Gao, X.; Xu, B.; Weiss, P. S.; Zhao, Y. Chem. Soc. Rev. 2012, 41, 97. (51) Akhavan, O.; Ghaderi, E. ACS Nano 2010, 4, 5731. (52) Liu, S.; Zeng, T. H.; Hofmann, M.; Burcombe, E.; Wei, J.; Jiang, R.; Kong, J.; Chen, Y. ACS Nano 2011, 5, 6971. (53) Bagri, A.; Mattevi, C.; Acik, M.; Chabal, Y. J.; Chhowalla, M.; Shenoy, V. B. Nat. Chem. 2010, 2, 581. (54) Kotchey, G. P.; Allen, B. L.; Vedala, H.; Yanamala, N.; Kapralov, A. A.; Tyurina, Y. Y.; Klein-Seetharaman, J.; Kagan, V. E.; Star, A. ACS Nano 2011, 5, 2098. (55) Jiao, Y.; Qian, F.; Li, Y.; Wang, G.; Saltikov, C. W.; Gralnick, J. A. J. Bacteriol. 2011, 193, 3662. (56) Meyer, J. C.; Geim, A. K.; Katsnelson, M. I.; Novoselov, K. S.; Booth, T. J.; Roth, S. Nature 2007, 446, 60. (57) Mohanty, N.; Fahrenholtz, M.; Nagaraja, A.; Boyle, D.; Berry, V. Nano Lett. 2011, 11, 1270. (58) Koenig, S. P.; Boddeti, N. G.; Dunn, M. L.; Bunch, J. S. Nat. Nanotechnol. 2011, 6, 543. (59) Chra, V.; Park, J.; Chun, Y.; Lee, J. W.; Hwang, I.; Kim, K. S. ACS Nano 2010, 4, 3979. (60) Deng, X.; Lue, L.; Li, H.; Luo, F. J. Hazard. Mater. 2010, 183, 923. (61) Sreeprasad, T. S.; Maliyekkal, S. M.; Lisha, K. P.; Pradeep, T. J. Hazard. Mater. 2011, 186, 921. (62) Balapanuru, J.; Yang, J.; Xiao, S.; Bao, Q.; Jahan, M.; Polavarapu, L.; Wei, J.; Xu, Q.; Loh, K. P. Angew. Chem., Int. Ed. 2010, 49, 6549. (63) Wu, M.; Kempaiah, R.; Huang, P. J. J.; Maheshwari, V.; Liu, J. W. Langmuir 2011, 27, 2731. (64) Kim, S. N.; Kuang, Z.; Slocik, J. M.; Jones, S. E.; Cui, Y.; Farmer, B. L.; McAlpine, M. C.; Naik, R. R. J. Am. Chem. Soc. 2011, 133, 14480. (65) Pan, Y.; Bao, H.; Sahoo, N. G.; Wu, T.; Li, L. Adv. Funct. Mater. 2011, 21, 2754. (66) Salihoglu, O.; Balci, S.; Kocabas, C. Appl. Phys. Lett. 2012, 100, 213110. (67) Haque, A. M. J.; Park, H.; Sung, D.; Jon, S.; Choi, S. Y.; Kim, K. Anal. Chem. 2012, 84, 1871. (68) Chen, Y.; Vedala, H.; Kotchey, G. P.; Audfray, A.; Cecioni, S.; Imberty, A.; Vidal, S.; Star, A. ACS Nano 2012, 6, 760.

(69) Wang, L.; Pu, K. Y.; Li, J.; Qi, X.; Li, H.; Zhang, H.; Fan, C.; Liu, B. Adv. Mater. 2011, 23, 4386. (70) Park, S.; Mohanty, N.; Suk, J. W.; Nagaraja, A.; An, J.; Piner, R. D.; Cai, W.; Dreyer, D. R.; Berry, V.; Ruoff, R. S. Adv. Mater. 2010, 22, 1736. (71) Ruiz, O.; Ferno, K. A. S.; Wang, B.; Brown, N. A.; Luo, P. G.; McNamara, N. D.; Vangsness, M.; Sun, Y.; Bunker, C. E. ACS Nano 2011, 5, 8100. (72) Hu, X.; Mu, L.; Wen, J.; Zhou, Q. Carbon 2012, 50, 2772. (73) Hu, X.; Mu, L.; Wen, J.; Zhou, Q. J. Hazard. Mater. 2012, 213214, 387. (74) Li, Y.; Liu, Y.; Fu, Y.; Wei, T.; Le Guyader, L.; Gao, G.; Liu, R. S.; Chang, Y. Z.; Chen, C. Biomaterials 2012, 33, 402. (75) Lightcap, I. V.; Kosel, T. H.; Kamat, P. V. Nano Lett. 2010, 10, 577. (76) Matsumoto, Y.; Koinuma, M.; Ida, S.; Hayami, S.; Taniguchi, T.; Hatakeyama, K.; Tateishi, H.; Watanabe, Y.; Amano, S. J. Phys. Chem. C 2011, 115, 19280. (77) Ryu, S.; Han, M. Y.; Maultzsch, J.; Heinz, T. F.; Kim, P.; Steigerwald, M. L.; Brus, L. E. Nano Lett. 2008, 8, 4597. (78) Seo, J. T.; Green, A. A.; Antaris, A. L.; Hersam, M. C. J. Phys. Chem. Lett. 2011, 2, 1004. (79) Lee, D. Y.; Khatun, Z.; Lee, J.; Lee, Y.; In, I. Biomacromolecules 2011, 12, 336. (80) Bettinger, C. J.; Langer, R.; Borenstein, J. T. Angew. Chem., Int. Ed. 2009, 48, 5406. (81) Goodman, C. M.; McCusker, C. D.; Yilmaz, T.; Rotello, V. M. Bioconjugate Chem. 2004, 15, 897. (82) Arvizo, R. R.; Miranda, O. R.; Thompson, M. A.; Pabelick, C. M.; Bhattacharya, R.; Robertson, J. D.; Rotello, V. M.; Prakash, Y. S.; Mukherjee, P. Nano Lett. 2010, 10, 2543. (83) Liao, K. H.; Lin, Y. S.; Macosko, C. W.; Haynes, C. L. ACS Appl. Mater. Interfaces 2011, 3, 2607. (84) Singh, S. K.; Singh, M. K.; Nayak, M. K.; Kumari, S.; Shrivastava, S.; Gracio, J. J. A.; Dash, D. ACS Nano 2011, 5, 4987. (85) Singh, S. K.; Singh, M. K.; Kulkarni, P. P.; Sonkar, V. K.; Gracio, J. J. A.; Dash, D. ACS Nano 2012, 6, 2731. (86) Rafiee, J.; Rafiee, M. A.; Yu, Z.; Koratkar, N. Adv. Mater. 2010, 22, 2151. (87) Kim, J.; Cote, L. J.; Kim, F.; Yuan, W.; Shull, K. R.; Huang, J. J. Am. Chem. Soc. 2010, 132, 8180. (88) Maiorano, G.; Sabella, S.; Sorce, B.; Brunetti, V.; Malvindi, M. A.; Cingolani, R.; Pompa, P. P. ACS Nano 2010, 4, 7481. (89) Chang, Y.; Yang, S.; Liu, J.; Dong, E.; Wang, Y.; Cao, A.; Liu, Y.; Wang, H. Toxicol. Lett. 2011, 200, 201. (90) Zhang, J.; Zhang, F.; Yang, H.; Huang, X.; Liu, H.; Zhang, J.; Guo, S. Langmuir 2010, 26, 6083. (91) Zhao, G.; Ren, X.; Gao, X.; Tan, X.; Li, J.; Chen, C.; Huang, Y.; Wang, X. Dalton Trans. 2011, 40, 10945. (92) Ai, L.; Zhang, C.; Chen, Z. J. Hazard. Mater. 2011, 192, 1515. (93) Zhang, J.; Xiong, Z.; Zhao, X. S. J. Mater. Chem. 2011, 21, 3634. (94) Ponnusamy, V. K.; Jen, J. F. J. Chromatogr., A 2011, 1218, 6861. (95) Antony, J.; Grimme, S. Phys. Chem. Chem. Phys. 2008, 10, 2722. (96) Varghese, N.; Mogera, U.; Govindaraj, A.; Das, A.; Maiti, P. K.; Sood, A. K.; Rao, C. N. R. ChemPhysChem 2009, 10, 206. (97) Lu, C. H.; Li, J.; Liu, J. J.; Yang, H. H.; Chen, X.; Chen, G. N. Chem.Eur. J. 2010, 16, 4889. (98) Zhao, X. J. Phys. Chem. C 2011, 115, 6181. (99) Akca, S.; Foroughi, A.; Frochtzwajg, D.; Postma, H. W. C. PLoS One 2011, 6, e18442. (100) Spiwok, V.; Hobza, P.; Rezac, J. J. Phys. Chem. C 2011, 115, 19455. (101) Bonanni, A.; Pumera, M. ACS Nano 2011, 5, 2356. (102) Lei, H.; Mi, L.; Zhou, X.; Chen, J.; Hu, J.; Guo, S.; Zhang, Y. Nanoscale 2011, 3, 3888. (103) Liu, M.; Zhao, H.; Chen, S.; Yu, H.; Quan, X. J. Mater. Chem. 2011, 21, 15266. (104) Xu, Y.; Wu, Q.; Sun, Y.; Bai, H.; Shi, G. ACS Nano 2010, 4, 7358. R


Chemical Reviews

Review

(105) Lu, C.; Li, J.; Zhang, X.; Zheng, A.; Yang, H.; Chen, X.; Chen, G. Anal. Chem. 2011, 83, 7276. (106) Tang, L.; Wang, Y.; Liu, Y.; Li, J. ACS Nano 2011, 5, 3817. (107) Lu, C.; Zhu, C.; Li, J.; Liu, J.; Chen, X.; Yang, H. Chem. Commun. 2010, 46, 3116. (108) Cai, L.; Zhan, R.; Pu, K.; Qi, X.; Zhang, H.; Huang, W.; Liu, B. Anal. Chem. 2011, 83, 7849. (109) Zhao, X. J. Phys. Chem. C 2011, 115, 6181. (110) Ren, H.; Wang, C.; Zhang, J.; Zhou, X.; Xu, D.; Zheng, J.; Guo, S.; Zhang, J. ACS Nano 2010, 4, 7169. (111) Lv, W.; Guo, M.; Liang, M.; Jin, F.; Cui, L.; Zhi, L.; Yang, Q. J. Mater. Chem. 2010, 20, 6668. (112) Liu, J.; Li, Y.; Li, Y.; Li, J.; Deng, Z. J. Mater. Chem. 2010, 20, 10944. (113) Patil, A. J.; Vickery, J. L.; Scott, T. B.; Mann, S. Adv. Mater. 2009, 21, 3159. (114) Lin, J.; Teweldebrhan, D.; Ashraf, K.; Liu, G.; Jing, X.; Yan, Z.; Li, R.; Lake, R. K.; Ozkan, M.; Balin, A. A.; Ozkan, C. S. In Nanobiosystems: Processing, Characterization, and Applications II; Kobayashi, N., Ouchen, F., Rau, I., Eds.; SPIE: Bellingham, WA, 2009; Vol. 7403, pp 74030C−74030C-10. (115) Lin, L.; Liu, Y.; Zhao, X.; Li, J. Anal. Chem. 2011, 83, 8396. (116) Zhang, Q.; Qiao, Y.; Hao, F.; Zhang, L.; Wu, S.; Li, Y.; Li, J.; Song, X. Chem.Eur. J. 2010, 16, 8133. (117) He, Y.; Wang, Z.; Tang, H.; Pang, D. Biosens. Bioelectron. 2011, 29, 76. (118) Merchant, C. A.; Healy, K.; Wanunu, M.; Ray, V.; Peterman, N.; Bartel, J.; Fischbein, M. D.; Venta, K.; Luo, Z.; Johnson, A. T. C.; Drndic, M. Nano Lett. 2010, 10, 2915. (119) Schneider, G. F.; Kowalczyk, S. W.; Calado, V. E.; Praud, G.; Zbergen, H. W.; Versypen, L. M. K.; Dekker, C. Nano Lett. 2010, 10, 3163. (120) Postma, H. W. C. Nano Lett. 2010, 10, 420. (121) Prasongkit, J.; Grigoriev, A.; Pathak, B.; Ahuja, R.; Scheicher, R. H. Nano Lett. 2011, 11, 1941. (122) Min, S. K.; Kim, W. Y.; Cho, Y.; Kim, K. S. Nat. Nanotechnol. 2011, 6, 162. (123) Venkatesan, B. M.; Bashir, R. Nat. Nanotechnol. 2011, 6, 615. (124) Schultz, B. J.; Patridge, C. J.; Lee, V.; Jaye, C.; Lysaght, P. S.; Smith, C.; Barnett, J.; Fischer, D. A.; Prendergast, D.; Banerjee, S. Nat. Commun. 2011, 2, 372. (125) Han, T. H.; Lee, W. J.; Lee, D. H.; Kim, J. E.; Choi, E.; Kim, S. O. Adv. Mater. 2010, 22, 2060. (126) Hu, W.; Peng, C.; Lv, M.; Li, X.; Zhang, Y.; Chen, N.; Fan, C.; Huang, Q. ACS Nano 2011, 5, 3693. (127) Karmali, P. P.; Simberg, D. Expert Opin. Drug Delivery 2011, 8, 343. (128) Myung, S.; Solanki, A.; Kim, C.; Park, J.; Kim, K. S.; Lee, K. Adv. Mater. 2011, 23, 2221. (129) Qin, W.; Li, X.; Bian, W.; Fan, X.; Qi, J. Biomaterials 2010, 31, 1007. (130) Liu, J.; Fu, S.; Yuan, B.; Li, Y.; Deng, Z. J. Am. Chem. Soc. 2010, 132, 7279. (131) Balamurugan, K.; Singam, E. R. A.; Subramanian, V. J. Phys. Chem. C 2011, 115, 8886. (132) Willner, I.; Willner, B. Nano Lett. 2010, 10, 3805. (133) Zhang, M.; Yin, B.; Wang, X.; Ye, B. Chem. Commun. 2011, 47, 2399. (134) Feng, L.; Liu, Z. Nanomedicine (London, U. K.) 2011, 6, 317. (135) Zhang, W.; Guo, Z.; Huang, D.; Liu, Z.; Guo, X.; Zhong, H. Biomaterials 2011, 32, 8555. (136) Robinson, J. T.; Tabakman, S. M.; Liang, Y.; Wang, H.; Casalongue, H. S.; Daniel, V.; Dai, H. J. Am. Chem. Soc. 2011, 133, 6825. (137) Hong, H.; Yang, K.; Zhang, Y.; Engle, J. W.; Feng, L.; Yang, Y.; Nayak, T. R.; Goel, S.; Bean, J.; Theuer, C. P.; Barnhart, T. E.; Liu, Z.; Cai, W. ACS Nano 2012, 6, 2361. (138) Lu, C.; Yang, H.; Zhu, C.; Chen, X.; Chen, G. Angew. Chem., Int. Ed. 2009, 48, 4785.

(139) Mahmoudi, M.; Simchi, A.; Imani, M.; Shokrgozar, M. A.; Milani, A. S.; Hafeli, U. O.; Stroeve, P. Colloids Surf., B 2010, 75, 300. (140) Walczyk, D.; Bombelli, F. B.; Monopoli, M. P.; Lynch, I.; Dawson, K. A. J. Am. Chem. Soc. 2010, 132, 5761. (141) Mahmoudi, M.; Serpooshan, V. J. Phys. Chem. C 2011, 115, 18275. (142) Lynch, I.; Salvati, A.; Dawson, K. A. Nat. Nanotechnol. 2009, 4, 546. (143) Mahmoudi, M.; Lynch, I.; Ejtehadi, M. R.; Monopoli, M. P.; Bombelli, F. B.; Laurent, S. Chem. Rev. 2011, 111, 5610. (144) MacCuspie, R. I.; Allen, A. J.; Hackley, V. A. Nanotoxicology 2011, 5, 140. (145) Monopoli, M. P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Bombelli, F. B.; Dawson, K. A. J. Am. Chem. Soc. 2011, 133, 2525. (146) Jiang, X.; Weise, S.; Hafner, M.; Roecker, C.; Zhang, F.; Parak, W. J.; Nienhaus, G. U. J. R. Soc., Interface 2010, 7, S5. (147) Nel, A. E.; Maedler, L.; Velegol, D.; Xia, T.; Hoek, E. M. V.; Somasundaran, P.; Klaessig, F.; Castranova, V.; Thompson, M. Nat. Mater. 2009, 8, 543. (148) Linse, S.; Cabaliero-Lago, C.; Xue, W. F.; Lynch, I.; Lindman, S.; Thulin, E.; Radford, S. R.; Dawson, K. A. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 8691. (149) Xu, M.; Li, J.; Iwai, H.; Mei, Q.; Fujita, D.; Su, H.; Chen, H.; Hanagata, N. Sci. Rep. 2012, 2, 406. (150) Pazik, R.; Andersson, R.; Kepinski, L.; Nedelec, J.-M.; Kessler, V. G.; Seisenbaeva, G. A. J. Phys. Chem. C 2011, 115, 9850. (151) Ou, L.; Luo, Y.; Wei, G. J. Phys. Chem. B 2011, 115, 9813. (152) Zuo, X.; He, S.; Li, D.; Peng, C.; Huang, Q.; Song, S.; Fan, C. Langmuir 2010, 26, 1936. (153) Kodali, V. K.; Scrimgeour, J.; Kim, S.; Hankinson, J. H.; Carroll, K. M.; de Heer, W. A.; Berger, C.; Curtis, J. E. Langmuir 2011, 27, 863. (154) Ge, C.; Du, J.; Zhao, L.; Wang, L.; Liu, Y.; Li, D.; Yang, Y.; Zhou, R.; Zhao, Y.; Chai, Z.; Chen, C. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 16968. (155) Shan, C.; Yang, H.; Han, D.; Zhang, Q.; Ivaska, A.; Niu, L. Langmuir 2009, 25, 12030. (156) Alwarappan, S.; Liu, C.; Kumar, A.; Li, C. J. Phys. Chem. C 2010, 114, 12920. (157) Liu, X.; Sen, S.; Liu, J.; Kulaots, I.; Geohegan, D.; Kane, A.; Puretzky, A. A.; Rouleau, C. M.; More, K. L.; Palmore, G. T. R.; Hurt, R. H. Small 2011, 7, 2775. (158) Song, Y.; Qu, K.; Zhao, C.; Ren, J.; Qu, X. Adv. Mater. 2010, 22, 2206. (159) Song, Y.; Chen, Y.; Feng, L.; Ren, J.; Qu, X. Chem. Commun. 2011, 47, 4436. (160) Huang, C.; Bai, H.; Li, C.; Shi, G. Chem. Commun. 2011, 47, 4962. (161) Wang, Y.; Lu, J.; Tang, L.; Chang, H.; Li, J. Anal. Chem. 2009, 81, 9710. (162) Lim, B.; Xia, Y. Angew. Chem., Int. Ed. 2011, 50, 76. (163) Kempaiah, R.; Chung, A.; Maheshwari, V. ACS Nano 2011, 5, 6025. (164) Kempaiah, R.; Salgado, S.; Chung, W. L.; Maheshwari, V. Chem. Commun. 2011, 47, 11480. (165) Markovic, Z. M.; Harhaji-Trajkovic, L. M.; TodorovicMarkovic, B. M.; Kepic, D. P.; Arsikin, K. M.; Jovanovic, S. P.; Pantovic, A. C.; Dramicanin, M. D.; Trajkovic, V. S. Biomaterials 2011, 32, 1121. (166) Yuan, J.; Gao, H.; Ching, C. B. Toxicol. Lett. 2011, 207, 213. (167) Chen, H.; Mueller, M. B.; Gilmore, K. J.; Wallace, G. G.; Li, D. Adv. Mater. 2008, 20, 3557. (168) Fan, Z.; Yan, J.; Zhi, L.; Zhang, Q.; Wei, T.; Feng, J.; Zhang, M.; Qian, W.; Wei, F. Adv. Mater. 2010, 22, 3723. (169) Biris, A. R.; Mahmood, M.; Lazar, M. D.; Dervishi, E.; Watanabe, F.; Mustafa, T.; Baciut, G.; Baciut, M.; Bran, S.; Ali, S.; Biris, A. S. J. Phys. Chem. C 2011, 115, 18967. (170) Agarwal, S.; Zhou, X.; Ye, F.; He, Q.; Chen, G. C. K.; Soo, J.; Boey, F.; Zhang, H.; Chen, P. Langmuir 2010, 26, 2244. S


Chemical Reviews

Review

(205) Yan, L.; Zhao, F.; Li, S.; Hu, Z.; Zhao, Y. Nanoscale 2011, 3, 362. (206) Duarte, I. F. J. Controlled Release 2011, 153, 34. (207) Mahmoudi, M.; Simchi, A.; Imani, M.; Milani, A. S.; Stroeve, P. Nanotechnology 2009, 20, 225104. (208) Mahmoudi, M.; Simchi, A.; Imani, M.; Shokrgozard, M. A.; Milani, A. S.; Haefeli, U. O.; Stroeve, P. Colloids Surf., B 2010, 75, 300. (209) Krug, H. F.; Wick, P. Angew. Chem., Int. Ed. 2011, 50, 1260. (210) Lewinski, N.; Colvin, V.; Drezek, R. Small 2008, 4, 26. (211) Wang, Y.; Lai, S. K.; So, C.; Schneider, C.; Cone, R.; Hanes, J. PLoS One 2011, 6, e21547. (212) Laurent, S.; Burtea, C.; Thirifays, C.; Haefeli, U. O.; Mahmoudi, M. PLoS One 2012, 7, 306. (213) Mahmoudi, M.; Laurent, S.; Shokrgozar, M. A.; Hosseinkhani, M. ACS Nano 2011, 5, 7263. (214) Pogodin, S.; Slater, N. K. H.; Baulin, V. A. ACS Nano 2011, 5, 1141. (215) Titov, A. V.; Kral, P.; Pearson, R. ACS Nano 2010, 4, 229. (216) Johnston, H. J.; Hutchison, G. R.; Christensen, F. M.; Peters, S.; Hankin, S.; Aschberger, K.; Stone, V. Nanotoxicology 2010, 4, 207. (217) Xu, W.; Zhang, L.; Li, J.; Lu, Y.; Li, H.; Ma, Y.; Wang, W.; Yu, S. J. Mater. Chem. 2011, 21, 4593. (218) Murray, A. R.; Kisin, E.; Leonard, S. S.; Young, S. H.; Kommineni, C.; Kagan, V. E.; Castranova, V.; Shvedova, A. A. Toxicology 2009, 257, 161. (219) Alam, M. I.; Beg, S.; Samad, A.; Baboota, S.; Kohli, K.; Ali, J.; Ahuja, A.; Akbar, M. Eur. J. Pharm. Sci. 2010, 40, 385. (220) Keelan, J. A. Nat. Nanotechnol. 2011, 6, 263. (221) Kostarelos, K. Nat. Mater. 2010, 9, 793. (222) Mitchell, L. A.; Lauer, F. T.; Burchiel, S. W.; McDonald, J. D. Nat. Nanotechnol. 2009, 4, 451. (223) Al-Jamal, K. T.; Gherardini, L.; Bardi, G.; Nunes, A.; Guo, C.; Bussy, C.; Herrero, M. A.; Bianco, A.; Prato, M.; Kostarelos, K.; Pizzorusso, T. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 10952. (224) Kayat, J.; Gajbhiye, V.; Tekade, R. K.; Jain, N. K. Nanomedicine (New York, NY, U. S.) 2011, 7, 40. (225) Nayak, T. R.; Andersen, H.; Makam, V. S.; Khaw, C.; Bae, S.; Xu, P.; Ee, R.; Ahn, J.; Hong, B. H.; Pastorin, G.; Oezyilmaz, B. ACS Nano 2011, 5, 4670. (226) Park, S. Y.; Park, J.; Sim, S. H.; Sung, M. G.; Kim, K. S.; Hong, B. H.; Hong, S. Adv. Mater. 2011, 23, H263. (227) Liu, Y.; Yu, D.; Zeng, C.; Miao, Z.; Dai, L. Langmuir 2010, 26, 6158. (228) Vallabani, N. V.; Mittal, S.; Shukla, R. K.; Pey, A. K.; Dhakate, S. R.; Pasricha, R.; Dhawan, A. J. Biomed. Nanotechnol. 2011, 7, 106. (229) Kalbacova, M.; Broz, A.; Kong, J.; Kalbac, M. Carbon 2010, 48, 4323. (230) Liu, Z.; Robinson, J. T.; Tabakman, S. M.; Yang, K.; Dai, H. Mater. Today 2011, 14, 316. (231) Ishida, T.; Harashima, H.; Kiwada, H. Curr. Drug Metab. 2001, 2, 397. (232) Kagan, V. E.; Konduru, N. V.; Feng, W.; Allen, B. L.; Conroy, J.; Volkov, Y.; Vlasova, I. I.; Belikova, N. A.; Yanamala, N.; Kapralov, A.; Tyurina, Y. Y.; Shi, J.; Kisin, E. R.; Murray, A. R.; Franks, J.; Stolz, D.; Gou, P.; Klein-Seetharaman, J.; Fadeel, B.; Star, A.; Shvedova, A. A. Nat. Nanotechnol. 2010, 5, 354. (233) Salas, E. C.; Sun, Z.; Luttge, A.; Tour, J. M. AcS Nano 2010, 4, 4852. (234) Wang, G.; Qian, F.; Saltikov, C. W.; Jiao, Y.; Li, Y. Nano Res. 2011, 4, 563. (235) Bai, Y.; Zhang, Y.; Zhang, J.; Mu, Q.; Zhang, W.; Butch, E. R.; Snyder, S. E.; Yan, B. Nat. Nanotechnol. 2010, 5, 683. (236) Singh, V.; Joung, D.; Zhai, L.; Das, S.; Khondaker, S. I.; Seal, S. Prog. Mater. Sci. 2011, 56, 1178. (237) Rallo, R.; France, B.; Liu, R.; Nair, S.; George, S.; Damoiseaux, R.; Giralt, F.; Nel, A.; Bradley, K.; Cohen, Y. Environ. Sci. Technol. 2011, 45, 1695. (238) Hernez-Alonso, M. D.; Fresno, F.; Suarez, S.; Coronado, J. M. Energy Environ. Sci. 2009, 2, 1231.

(171) Chen, G. Y.; Pang, D. W. P.; Hwang, S. M.; Tuan, H. Y.; Hu, Y. C. Biomaterials 2012, 33, 418. (172) Guo, C. X.; Zheng, X. T.; Lu, Z. S.; Lou, X. W.; Li, C. M. Adv. Mater. 2010, 22, 5164. (173) Chen, W.; Yi, P.; Zhang, Y.; Zhang, L.; Deng, Z.; Zhang, Z. ACS Appl. Mater. Interfaces 2011, 3, 4085. (174) Kim, S.; Ku, S. H.; Lim, S. Y.; Kim, J. H.; Park, C. B. Adv. Mater. 2011, 23, 2009. (175) Chen, M.; Liu, J.; Hu, B.; Chen, M.; Wang, J. Analyst 2011, 136, 4277. (176) Wang, Y.; Li, Z.; Hu, D.; Lin, C. T.; Li, J.; Lin, Y. J. Am. Chem. Soc. 2010, 132, 9274. (177) Wang, K.; Ruan, J.; Song, H.; Zhang, J.; Wo, Y.; Guo, S.; Cui, D. Nanoscale Res. Lett. 2011, 6, 1. (178) Hu, Z.; Huang, Y.; Sun, S.; Guan, W.; Yao, Y.; Tang, P.; Li, C. Carbon 2012, 50, 994. (179) Oh, S.; Brammer, K. S.; Li, Y. S. J.; Teng, D.; Engler, A. J.; Chien, S.; Jin, S. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 2130. (180) Zhang, S.; Yang, K.; Feng, L.; Liu, Z. Carbon 2011, 49, 4040. (181) Zhang, X.; Yin, J.; Peng, C.; Hu, W.; Zhu, Z.; Li, W.; Fan, C.; Huang, Q. Carbon 2011, 49, 986. (182) Zhang, Y.; Xu, Y.; Li, Z.; Chen, T.; Lantz, S. M.; Howard, P. C.; Paule, M. G.; Slikker, W.; Watanabe, F.; Mustafa, T.; Biris, A. S.; Ali, S. F. ACS Nano 2011, 5, 7020. (183) Peng, C.; Hu, W.; Zhou, Y.; Fan, C.; Huang, Q. Small 2010, 6, 1686. (184) Liu, K.; Zhang, J.; Cheng, F.; Zheng, T.; Wang, C.; Zhu, J. J. Mater. Chem. 2011, 21, 12034. (185) Lee, W. C.; Lim, C. H. Y. X.; Shi, H.; Tang, L. A. L.; Wang, Y.; Lim, C. T.; Loh, K. P. ACS Nano 2011, 5, 7334. (186) Li, N.; Zhang, X.; Song, Q.; Su, R.; Zhang, Q.; Kong, T.; Liu, L.; Jin, G.; Tang, M.; Cheng, G. Biomaterials 2011, 32, 9374. (187) Ryoo, S.; Kim, Y.; Kim, M.; Min, D. ACS Nano 2010, 4, 6587. (188) Jones, C. F.; Grainger, D. W. Adv. Drug Delivery Rev. 2009, 61, 438. (189) Li, N.; Xia, T.; Nel, A. E. Free Radical Biol. Med. 2008, 44, 1698. (190) Burello, E.; Worth, A. Nat. Nanotechnol. 2011, 6, 138. (191) Summers, H. D.; Rees, P.; Holton, M. D.; Rowan Brown, M.; Chappell, S. C.; Smith, P. J.; Errington, R. J. Nat. Nanotechnol. 2011, 6, 170. (192) Gousset, K.; Schiff, E.; Langevin, C.; Marijanovic, Z.; Caputo, A.; Browman, D. T.; Chenouard, N.; de Chaumont, F.; Martino, A.; Enninga, J.; Olivo-Marin, J. C.; Männel, D.; Zurzolo, C. Nat. Cell Biol. 2009, 11, 328. (193) Wang, X.; Veruki, M. L.; Bukoreshtliev, N. V.; Hartveit, E.; Gerdes, H. H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 17194. (194) Li, Z.; Geng, Y.; Zhang, X.; Qi, W.; Fan, Q.; Li, Y.; Jiao, Z.; Wang, J.; Tang, Y.; Duan, X.; Wu, W. J. Nanopart. Res. 2011, 13, 2939. (195) Yang, K.; Wan, J.; Zhang, S.; Zhang, Y.; Lee, S. T.; Liu, Z. ACS Nano 2011, 5, 516. (196) Duch, M. Nano Lett. 2011, 11, 5120. (197) Haniu, H.; Matsuda, Y.; Usui, Y.; Aoki, K.; Shimizu, M.; Ogihara, N.; Hara, K.; Okamoto, M.; Takanashi, S.; Ishigaki, N.; Nakamura, K.; Kato, H.; Saito, N. J. Proteomics 2011, 74, 2703. (198) Murdock, R. C.; Braydich-Stolle, L.; Schr, A. M.; Schlager, J. J.; Hussain, S. M. Toxicol. Sci. 2008, 101, 239. (199) Maynard, A. D.; Warheit, D. B.; Philbert, M. A. Toxicol. Sci. 2011, 120, S109. (200) Fischer, H. C.; Chan, W. C. Curr. Opin. Biotechnol. 2007, 18, 565. (201) Ren, H. X.; Chen, X.; Liu, J. H.; Gu, N.; Huang, X. J. Mater. Today 2010, 13, 6. (202) Yu, Z.; Zhou, Q. J. Hazard. Mater. 2009, 167, 38. (203) Liu, S.; Zhou, Q.; Wang, Y. Chemosphere 2011, 83, 1080. (204) Galluzzi, L.; Chiarantini, L.; Pantucci, E.; Curci, R.; Merikhi, J.; Hummel, H.; Bachmann, P. K.; Manuali, E.; Pezzotti, G.; Magnani, M. Nanomedicine (London, U. K.) 2012, 7, 393. T


Chemical Reviews

Review

(239) Thuy-Duong, N.; Viet Hung, P.; Shin, E. W.; Hai-Dinh, P.; Kim, S.; Chung, J. S.; Kim, E. J.; Hur, S. H. Chem. Eng. J. 2011, 170, 226. (240) Liu, X.; Vinson, D.; Abt, D.; Hurt, R. H.; Rand, D. M. Environ. Sci. Technol. 2009, 43, 6357. (241) Thomas, C. R.; George, S.; Horst, A. M.; Ji, Z.; Miller, R. J.; Peralta-Videa, J. R.; Xia, T.; Pokhrel, S.; Mädler, L.; Gardea-Torresdey, J. L.; Holden, P. A.; Keller, A. A.; Lenihan, H. S.; Nel, A. E.; Zink, J. I. ACS Nano 2011, 5, 13. (242) Chra, V.; Yu, S. U.; Kim, S. H.; Yoon, Y. S.; Kim, D. Y.; Kwon, A. H.; Meyyappan, M.; Kim, K. S. Chem. Commun. 2012, 48, 735. (243) Farhangi, N.; Chowdhury, R. R.; Medina-Gonzalez, Y.; Ray, M. B.; Charpentier, P. A. Appl. Catal. B 2011, 110, 25. (244) Jiang, G.; Lin, Z.; Chen, C.; Zhu, L.; Chang, Q.; Wang, N.; Wei, W.; Tang, H. Carbon 2011, 49, 2693. (245) Harper, S. L.; Carriere, J. L.; Miller, J. M.; Hutchison, J. E.; Maddux, B. L. S.; Tanguay, R. L. ACS Nano 2011, 5, 4688. (246) Petersen, E. J.; Pinto, R. A.; Mai, D. J.; Lrum, P. F.; Weber, W. J., Jr. Environ. Sci. Technol. 2011, 45, 1133. (247) Fako, V. E.; Furgeson, D. Y. Adv. Drug Delivery Rev. 2009, 61, 478. (248) Scown, T.; M. Santos, E. M.; Johnston, B. D.; Gaiser, B.; Baalousha, M.; Mitov, S.; Lead, J. R.; Stone, V.; Fernes, T. F.; Jepson, M.; van Aerle, R.; Tyler, C. R. Toxicol. Sci. 2010, 115, 521. (249) Chen, C.; Zhou, Q.; Liu, S.; Xiu, Z. Chemosphere 2011, 83, 1147. (250) Santos, P. S. M.; Otero, M.; Filipe, O. M. S.; Santos, E. B. H.; Duarte, A. C. Talanta 2010, 83, 505. (251) Santos, C. M.; Tria, M. C. R.; Vergara, R. A. M. V.; Ahmed, F.; Advincula, R. C.; Rodrigues, D. F. Chem. Commun. 2011, 47, 8892. (252) Liu, L.; Liu, J.; Wang, Y.; Yan, X.; Sun, D. D. New J. Chem. 2011, 35, 1418. (253) Mohanty, N.; Berry, V. Nano Lett. 2008, 8, 4469. (254) Cai, X.; Tan, S.; Lin, M.; Xie, A.; Mai, W.; Zhang, X.; Lin, Z.; Wu, T.; Liu, Y. Langmuir 2011, 27, 7828. (255) Hu, W.; Peng, C.; Luo, W.; Lv, M.; Li, X.; Li, D.; Huang, Q.; Fan, C. ACS Nano 2010, 4, 4317. (256) Sinclair, R. G.; Jones, E. L.; Gerba, C. P. J. Appl. Microbiol. 2009, 107, 1769. (257) Liga, M. V.; Bryant, E. L.; Colvin, V. L.; Li, Q. Water Res. 2011, 45, 535. (258) Khodakovskaya, M. V.; De Silva, K.; Nedosekin, D. A.; Dervishi, E.; Biris, A. S.; Shashkov, E. V.; Galanzha, E. I.; Zharov, V. P. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 1028. (259) Begurn, P.; Ikhtiari, R.; Fugetsu, B. Carbon 2011, 49, 3907. (260) Dietz, K. J.; Herth, S. Trends Plant Sci. 2011, 16, 582. (261) Stampoulis, D.; Sinha, S. K.; White, J. C. Environ. Sci. Technol. 2009, 43, 9473.

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