Interaction of Amino Acids and Graphene Oxide: Trends in

Publication Date (Web): December 21, 2016. Copyright ... This trend and origin of interaction is further confirmed by zeta potential (for cationic ami...
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Interaction of Amino Acids and Graphene Oxide: Trends in Thermodynamic Properties Subrata Pandit, and Mrinmoy De J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.6b11571 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on January 3, 2017

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Interaction of Amino Acids and Graphene Oxide: Trends in Thermodynamic Properties Subrata Pandit and Mrinmoy De* Department of Organic Chemistry, Indian Institute of Science, Bangalore-560094, India Email: [email protected]

Abstract: Recent studies on interaction of graphene oxide with proteins and peptides attracts lot of attentions in biomedical applications. Hence the fundamental and experimental estimation of binding thermodynamics between various amino acids and graphene oxide is highly significant for many aspects. In this study, the interaction of graphene oxide (GO) with amino acids bearing variable charge, hydrophobicity and aromatic moieties are studied by using isothermal titration calorimetry (ITC). To explore the effect of lateral size and degree of oxidation in GO we employ two different sizes of GO. The result shows, the interactions of GO with amino acids are mainly governed by electrostatic and π-π interaction with variable enthalpy and entropy values. The highest complex stability is observed in case of tryptophan and arginine followed by other amino acids containing either positive charge or aromatic moieties. Amino acids bearing other functional groups either exhibit very weak interaction or did not show any detectable binding. This trend and origin of interaction is further confirmed by zeta potential (for cationic amino acids) and fluorescence titration assay (for aromatic amino acids). Furthermore, there is a significant enthalpy entropy correlation between GO and amino acids that is observed with near unit slope (α = 0.87) and positive intercept (T∆S0 = 18.65). This indicates flexible nature of GO as a receptor against small molecules. In future, this investigation will help in designing the peptide based receptors for GO and understanding the nature interaction of proteins and peptides with GO based receptors.

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Introduction Interactions of graphene oxide (GO) with peptides and proteins attract great attention that has been applied in a variety of applications ranging from modulation of enzymatic activity to delivery and sensing by specific and nonspecific binding.1-2 In particular GO has been considered as an attractive artificial receptor due to its highest surface to volume ratio, structural flexibility, biomimetic multi surface functionality and possibility of diverse surface modification.3-4 Due to these advantages significant improvement in various biological applications have been demonstrated. For example, GO can inhibit the enzymatic activity of α-chymotrypsin most efficiently in compare to any other reported artificial inhibitor.5 Jung et.al showed that GOaptamer noncovalent conjugate can be used in aptasensing microarrays for the detection of human thrombin at picomolar (pM) concentration.6 Even in nose/tongue based non-specific sensing GO-protein conjugates had demonstrated superior performance in array based protein sensing compare to the state of art functionalized gold nanoparticles.7 Not only the improvement in sensing or enzymatic inhibition, GO-protein/peptide interaction also had used for several other applications.8 In all above applications noncovalent interactions play major role in GO-protein/peptide complexation and the different amino acids are the key elements in those interactions. In proteins and peptides; amino acids provide the source of the variable noncovalent interaction such as, electrostatic, hydrophobic, aromatic and hydrogen bonding which plays the pivotal role in protein’s secondary structure, protein-protein interaction, protein-peptide complexation and development of artificial receptors. So to understand and develop the GO based artificial receptor for aforementioned biological applications the study on binding thermodynamic of GO and amino acid is highly significant and necessary.

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Earlier reports on interaction of graphene and carbon nanotube with amino acids are studied both experimentally and theoretically.9-10 In those reports it was established that these interactions are hydrophobic in nature and entropically controlled. But unlike graphene, GO consisted with other functional groups such as epoxide and hydroxyl groups on the basal plane along with carbonyl and carboxyl groups at the edges, which are suitable for electrostatic and hydrogen bonding interactions along with hydrophobic interaction (figure 1). Hence the estimation and understanding of GO-amino acid interaction is more complicated and may not show the similar results and trends between theoretical and experimental finding. For instance, recently the interactions of nucleobases with GO was reported both experimentally and theoretically in two different reports.11-13 According to the theoretical study the trend of relative binding strength is cytosine (C) > guanine (G) > adenine (A) > thymine (T).12 Whereas the experimental result appears in different trend which is G > A >C~T.11 While the theoretical results are based on several assumptions and predetermine structure of GO, experimental results are more realistic in practical application and receptor structure designing. The theoretical study on interaction of aromatic amino acids with GO was performed by Vovusha et al. and according to their report the trend was altered from one method to another method. According to M05-2X method it is histidine (His) > tryptophan (Trp) > tyrosine (Tyr) > phenylalanine (Phe), while using M06-2X the trend is His>Tyr>Trp>Phe.11 So the experimental study is vital in designing the interactive peptide sequence and understanding the GO-protein/peptide complexation. Not only are the aromatic amino acids, other hydrophobic, cationic and anionic amino acids also very important in the development of artificial receptors. To the best of our knowledge only qualitative assessment has been reported so far by Zhang et. al. According to that report., the ratio of amino acid concentration was measured after and before incubation with GO and based

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on that they had reported the relative affinity of different amino acids with GO.14 According to their observation they have found the following trends, Arg>His>Lys>Trp>Tyr>Phe. But quantitative estimation and comparison of all thermodynamic parameters have not been reported which are highly imperative in fundamental understanding for interactions between GO and protein/peptide.

Figure 1: Structural features of GO and amino acids and possible way of GO-amino acid complexation.

In this context we have considered series of natural L-amino acids with possibility of diverse noncovalent interactions with GO. To estimate the nature of interaction we have used isothermal titration calorimetry (ITC) study, which is an ultrasensitive tool to determine the thermodynamic properties in molecular interaction. To support the ITC analysis we have also estimated and compared the binding constant of GO with amino acids by fluorescence quenching study for aromatic amino acids and measured the change of zeta potential of complexes for charged amino acids. We have also explored the effect of lateral size of GO in thermodynamic

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measurements. Hence we have used GO with average lateral diameter of 600-800 nm (GO) and 25-40 nm (nano-GO). Followed by thermodynamic properties estimation, entropy-enthalpy compensation analysis indicates a linear correlation similar to protein small molecular interaction which demonstrates the possible flexibility initiated from GO surface. Experimental Section Materials: Graphite (Asbury carbon) and carbon nanofiber (Sigma Aldrich) were used to prepare GO and nano-GO respectively. Phosphorus pentoxide (P2O5), Potassium persulfate (K2S2O8) and Potassium permanganate (KMnO4) was purchased from SD fine chemicals and used as received. All of the GO-amino acid interaction experiments were performed in sodium phosphate buffer (5 mM, pH 7.4). Synthesis of GO and nGO: GO was prepared using modified Hummer’s method from graphite. In a typical reaction K2S2O8 (1 gm) and P2O5 (1 gm) were added to 15 ml of conc. H2SO4 and stirred at 80 oC temperature until the all reagents are fully dissolved. Then graphite powder (1 gm) was added to the solution and kept at 80 oC for 3 hr. After that the reaction mixture was cooled, diluted with deionised water (100 ml) and filtered using filter paper grade 3. The filtrate was further rinsed with additional 1.2 liter of distilled water to remove all residual salt and dried in air to get the pretreated graphite. The pretreated graphite powder was collected and transferred into 500 ml round bottom flask with 45 ml conc. H2SO4 at 0 oC using an ice bath. While stirring, KMnO4 (6 gm) was slowly added to the mixture and kept the temperature below 10 oC. Then the flask was moved to 42 oC water bath and left for 2.5 hr. To that solution 100 ml water was added at 0 oC. After dilution, very slowly 4 ml of a 30% H2O2 solution was added. The colour of the solution turned bright yellow. This was filtered and rinsed with 3.4% HCL solution to remove residual salts. The filtrate was re-disperse in water and dialyzed against working buffer solution.

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Nano-GO was prepared similar way form carbon nanofiber. Weight concentration was determined with lypholyzed aliquots. Isothermal titration calorimetry (ITC) experiment: We have carried out the ITC (nano ITC, TA instruments) experiment at 25 oC in 5 mM sodium phosphate buffer, pH 7.4. For this study, we used GO (1 mg/ml) and nano-GO (0.75 mg/ml) in cell and 20 mM amino acids in syringe during titration. Titration was carried out by 22 time successively injecting 10 µl of amino acid solution to 1 ml of GO or nano-GO solution. For the control experiment (i.e. the heat of dilution of the amino acid solutions when added to the buffer solution in the absence of GO) was determined in each run, using the same experimental parameters. The control titration enthalpies were subtracted from the above titration experiment and used in plotting the final titration curve. The curve was fitted by using NanoAnalyzer program supplied by TA instruments to determine the binding constants (KS), enthalpy changes (∆H) and binding ratio (n). Fluorescence quenching titration: fluorescence spectra were measured on a photon counting steady state using Varian fluorescence spectrophotometer instrument. The sample amino acids (20 mM) were excited of their corresponding excitation wavelength (280 nm for Tyr, Trp, His and 260 nm for Phe) and was measured the emission spectra from 280 nm to 380 nm. GO (1 mg/ml) or nano-GO (0.75 mg/ml) was added in 5 µL aliquots for 20 times to quench the complete fluorescence. All conditions (concentration, buffer, temperature etc.) were kept same as those in ITC study. Zeta potential: Zeta potential of the GO and nano-GO with various amino acids in buffer solution were measured on a MALVERN Zetasizer Nano ZS. Three rounds of assays have been performed, and the average values were reported.

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Result and Discussion Preparation and Characterization of GO and nano-GO: Several studies were reported on size dependent properties and applications of GO, where lateral size and oxidation state were played a very crucial role.15-16 Hence in our study on the interaction between GO and amino acids, first we prepared the two types of GO solution with variable size and oxidation state. As expected bigger GO layers are more hydrophobic in nature due the presence of less oxygenated functionality, hence they interact stronger with hydrophobic molecules such as drug molecules and exhibit less colloidal stability.17-18 Alternatively small GO sheets have higher charge density with stronger electrostatic interaction.19-20 As amino acids demonstrate variable types and degree of noncovalent interactions so we have decided to synthesize small as well as large lateral sized GO solutions. GO is typically made by oxidation of graphite powder using strong oxidizing agent such as KMnO4.21 During the preparation and reaction the graphene sheets are oxidized with various oxygen containing group and exfoliated to single layers. By this method polydisperse GO solution is produced which can be used for size separation by centrifugation.22 In our experiment we have used graphite powder as the raw material for larger GO. We follow the conventional modified Hummer’s method to exfoliate the graphite powder followed by size separation by centrifugation.23 Alternatively for smaller lateral sized GO, we chemically exfoliated carbon nanofiber with 100 nm diameter using reported procedure.20 Both GO and nano-GO are characterized by dynamic light scattering (DLS), atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). For GO, DLS measurements suggest the average hydrodynamic diameter 720 nm (figure S1) with broad peak which indicates the presence of some polydispersity. Similar result was also observed in case AFM characterization which

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revealed the 1.5 nm thick single layered GO with lateral size distribution of 600-800 nm (figure 2a, c and S2a).

Figure 2. AFM micrograph of (a) GO and (b) nano-GO. (c,d) The thickness and lateral diameter of the exfoliated materials. (e) XPS spectra of GO and nano-GO, indicates the higher quantity of oxygen in nano-GO. (f,g) C1s peak analysis of GO and nano-GO indicates the presence of more oxygenated C in nano-GO.

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For nano-GO the observed diameter is 25-40 nm by AFM (figure 2b, d and S2b) and 290 nm by DLS (figure S1), which indicates much less polydispersity. Observed zeta potential for GO and nano-GO in 5 mM sodium phosphate buffer, pH 7.4 are -52.1 mV and -58.8 mV respectively indicates high colloidal stability. XPS analysis confirmed that the oxygen content in nano-GO (45.4%) is much higher than GO (38.2%) which indicates higher hydrophilic functionality in nano-GO (figure 2e). Also the C1s peak analysis suggests the presence of oxygenated carbon in higher percentage in nano-GO compare to GO (figure 2f, g and table S1 in supplementary information).

Binding Thermodynamics of Amino Acid-GO Interaction: Until now only fluorescence titration24 and concentration differential studies14 have been used to estimate the GO-amino acid interaction but these techniques provide only minimal information in molecular binding and no information about binding thermodynamics. In this respect ITC has been extensively used to explore the nature of molecular interaction quantitatively ranging from small molecule to macromolecules.25-28 Calorimetric titration directly provides the binding constant (Ks), hence the free energy (∆G) and enthalpy (∆H) of association, and the entropy (∆S) from the former two values by using the following standard thermodynamic equations, ∆G = -RTlnKs and ∆G = ∆H T∆S. To investigate the GO-amino acid interactions by ITC we considered various set of amino acids depending on their inherent physical properties such as cationic amino acids (Arg, Lys) which leads to the electrostatic interaction, aromatic amino acids (Phe, Tyr, Trp) which are responsible for π-πinteraction, polar amino acids (Ser, Asn) to explore the possibility of hydrophilic interaction, aliphatic (Ala, Leu) and anionic (Asp, Glu) amino acids (figure 1).

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Figure 3: ITC analysis for the interaction of (a-c) GO with Arg, Trp, Asp and (d-f) nano-GO with Arg, Trp, Asp in 5 mM sodium phosphate buffer pH 7.4, at 25 oC. Cationic (Arg) and aromatic (Trp) amino acid show strong interaction whereas no binding was observed in case of anionic amino acid (Asp). The independent binding curve fit analysis gives the thermodynamic parameters, stability constants (Ks) and enthalpy changes (∆H).

We carried out the ITC experiment at 25 oC by titrating 20 mM amino acid solution in to the GO and nano-GO solution in 5 mM sodium phosphate buffer, pH 7.4. As can be seen from the

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titration experiment different amino acids interact in different extent even they are in similar subgroup (figure 3 and figure S3, S4 in supplementary information). For example the complexation with cationic amino acid, Arg is very enthalpically similar with aromatic amino acid Trp but their binding constants are different (table 1). Similarly Arg (pKa 12.10) and Lys (pKa 10.67) are both positively charged amino acids but their binding parameters are very diverse (table 1). To compare the binding nature from one amino acid to other amino acid and the effect of the size of GO we have summarized the obtained thermodynamic parameters in table 1. Table 1. Binding constants (KS), Gibbs free energy changes (∆G), enthalpy changes (∆H) and entropy changes (T∆S) for the complexation of various amino acids with GO and Nano-GO (5 mM sodium phosphate, pH 7.4) at 25 oC. * Interaction is very week to perform the curve fitting analysis. ND: Nondetectable

Amino Acids

Cationic

Aromatic

Polar Nonpolar Anionic Control

Arg Lys His Phe Tyr Trp Ser Asn Ala Leu Asp Gly

KS / M-1 of AA 2.36×103±3.3 1.13×103±8.3 3.29×103±6.2 1.92×103±15 2.28×103±17 4.55×103±9.1 4.25×102±21 2.74×102±55

-∆G / kJ mol-1 19.24 17.42 20.06 18.73 19.16 20.87 14.99 13.91

GO -∆H / kJ mol-1 9.77±0.03 6.97±0.01 2.16±0.05 1.23±0.16 2.61±0.01 10.94±0.03 4.88±0.17 1.07±0.21 -ND - ND - ND - ND -

T∆S / kJ mol-1 9.48 10.43 17.90 17.51 16.55 9.93 10.12 12.84

n mol / mg ml-1 0.75 0.51 1.09 0.22 0.26 0.34 0.32 0.48

KS / M-1 of AA 3.82×103±6.2 2.18×103±9.2 3.98×103±11 1.41×103±3.2 2.26×103±9.8 5.98×103±2.9

Nano-GO -∆G / -∆H / kJ mol-1 kJ mol-1 20.44 14.35±0.05 19.05 9.82±0.03 20.54 3.96±0.14 17.97 1.28±0.06 19.13 1.99±0.08 21.55 14.75±0.12 -ND*-ND*-ND - ND - ND - ND -

n T∆S / kJ mol-1 mol / mg ml-1 6.09 0.55 9.23 0.46 16.58 1.01 16.69 0.45 17.14 0.41 6.80 0.49

As we can see from table 1, only two groups of amino acids, cationic and aromatic amino acids are interacting stronger than others. Week interaction with polar amino acids was also observed but ITC experiment did not able to detect any significant interaction with anionic and nonpolar amino acids including reference amino acid, glycine. This suggests that electrostatic and π−π interactions are key contributor in GO-amino acid interaction through oxygenated functionality

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and aromatic back bone of GO respectively. In all detectable cases, GO-amino acid complexation happen through favorable enthalpic contribution (∆H0). The complexation behavior of cationic amino acids, mainly for Arg and Lys involves with large positive enthalpy changes (-9.77 to -6.97 kJ mol-1 of amino acids for GO and -14.35 to -9.82 kJ mole-1 of amino acids for nano-GO) compare to amino acids with aromatic functionality (-2.61 to -1.07 kJ mol-1 of amino acids for GO and -3.96 to -1.28 kJ mol-1 of amino acids for nano-GO) except Trp. From the thermodynamic point of view, noncovalent complexation is exothermic (∆H 0 kJ mol-1). Thus by using the slope (α) and intercept (T∆S0) we can quantitatively measures the changes in conformation and complex stabilization of a host-guest system, which was applied many chemical and biological supramolecular systems.40-41 Here we have extended this correlation approach to the GO and small molecular (amino acid) system by plotting the entropy changes (Τ∆S) against corresponding enthalpy changes (∆H) as listed in table 1. As shown in figure 6a, a good linear relationship is observed for the GO-amino acid system with a correlation coefficient of 0.94. The compensation plot yields near unit slope (α = 0.87) and positive intercept (T∆S0 = 18.65). Using correlation analysis, it has been reported that rigid receptors give the smaller slope values, whereas the flexible receptors produce larger and near unit slope value. As we can see from figure 6b (adapted and modified from ref 28), rigid small receptors such as cryptand, metalloporphyrin etc. show smaller slope values (α = 0.44 and 0.63 respectively) in compare to flexible large receptors such as substituted cyclodextrin (α = 1.02), protein (α = 0.96) etc. The α value for GO-amino acid interaction is close to protein ligand interaction, indicates the favorable conformational change of GO during complexation which is good accordance with previous reports.5,

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Similarly the T∆S0 value falls near the other large host and small guest molecule

systems, indicates GO as a suitable receptor for amino acids and other related small molecular derivatives. This enthalpy-entropy correlation plot also can be used to determine the nature of complexation for specific amino acid. As can be seen from figure 6a, that the aromatic amino acids are behaving similarly and the complexation is entropically controlled. Alternatively the interaction of cationic amino acids are enthalpically controlled except Trp.

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Figure 6. (a) Correlation plot of entropy (T∆S) versus enthalpy (∆H) for the interaction of GO and nanoGO with cationic and aromatic amino acids. In the plot nGO indicates nano-GO. (b) Comparison plot of slope (α) and intercept (T∆S0) values of various host-guest system with GO-amino acids (GO-AA).

Conclusion: In this report we have estimated the thermodynamic properties for interaction of graphene oxides with various amino acids by using ITC. We have observed that electrostatic and π−π interactions either independently (for Lys, Arg, His, Phe and Tyr) or cooperatively (Trp) control the GO-amino acid complexation and playing the major role in interaction. ITC studies reveled that interaction of cationic amino acids and Trp are controlled by enthalpy changes whereas the complexation with other aromatic amino acids are entropy driven. While considering the effect of lateral size of GO, we did not observed any effect on aromatic amino

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acids but enhancement in binding with cationic amino acids was detected. In support of the electrostatic and π−π interaction we performed zeta potential measurement and fluorescence titration respectively. Zeta potential of the GO-amino acid complexes suggest that Arg and Lys are binding through electrostatic interaction and even though Trp exhibits similar thermodynamic parameters to Arg, but the electrostatic interaction does not play the major role in complexation. Fluorescence titration analysis follows the same trend as we observed in ITC experiments. Using the obtained thermodynamic parameters, an excellent linear relationship between ∆H and T∆S was observed. This indicates the enthalpy-entropy compensation correlation in GO-amino acid system as observed in several other host guest systems. Near unit slope and positive intercept in enthalpy-entropy compensation analysis suggest the suitability of GO as a biological receptor. These kind of strategic studies also can be used for further optimization of GO with surface functionalizations as well as can be applied for other analytes such as proteins and peptides. Associated Content: Supporting Information includes DLS, size distribution of GO and nano-GO, ITC of other amino acids, zeta potential for nano-GO and other fluorescence titration. Author Information: Corresponding Author: Mrinmoy De Email: [email protected] The authors declare no competing financial interest. Acknowledgements: The authors would like to thank DST-SERB (SB/FT/CS-139/2014) for financial support. SP thanks IISc and CSIR for doctoral fellowship. The authors also thank Prof. S. Ramakrishnan for allowing access to the ITC instrument.

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References: 1. Feng, L. Y.; Wu, L.; Qu, X. G. New Horizons for Diagnostics and Therapeutic Applications of Graphene and Graphene Oxide. Adv. Mater. 2013, 25, 168-186. 2. Biju, V. Chemical Modifications and Bioconjugate Reactions of Nanomaterials for Sensing, Imaging, Drug Delivery and Therapy. Chem. Soc. Rev. 2014, 43, 744-764. 3. Dreyer, D. R.; Todd, A. D.; Bielawski, C. W. Harnessing the Chemistry of Graphene Oxide. Chem. Soc. Rev. 2014, 43, 5288-5301. 4. Zhu, Y. W.; Murali, S.; Cai, W. W.; Li, X. S.; Suk, J. W.; Potts, J. R.; Ruoff, R. S. Graphene and Graphene Oxide: Synthesis, Properties, and Applications. Adv. Mater. 2010, 22, 3906-3924. 5. De, M.; Chou, S. S.; Dravid, V. P. Graphene Oxide as an Enzyme Inhibitor: Modulation of Activity of Alpha-Chymotrypsin. J. Am. Chem. Soc. 2011, 133, 17524-17527. 6. Jung, Y. K.; Lee, T.; Shin, E.; Kim, B. S. Highly Tunable Aptasensing Microarrays with Graphene Oxide Multilayers. Sci. Rep. 2013, 3. 7. Chou, S. S.; De, M.; Luo, J. Y.; Rotello, V. M.; Huang, J. X.; Dravid, V. P. Nanoscale Graphene Oxide (nGO) as Artificial Receptors: Implications for Biomolecular Interactions and Sensing. J. Am. Chem. Soc. 2012, 134, 16725-16733. 8. Loh, K. P.; Bao, Q. L.; Eda, G.; Chhowalla, M. Graphene Oxide as a Chemically Tunable Platform for Optical Applications. Nat. Chem. 2010, 2, 1015-1024. 9. Rajesh, C.; Majumder, C.; Mizuseki, H.; Kawazoe, Y. A Theoretical Study on the Interaction of Aromatic Amino Acids with Graphene and Single Walled Carbon Nanotube. J. Chem. Phy. 2009, 130, 124911. 10. Piao, L. Y.; Liu, Q. R.; Li, Y. D. Interaction of Amino Acids and Single-Wall Carbon Nanotubes. J. Phy. Chem. C 2012, 116, 1724-1731. 11. Varghese, N.; Mogera, U.; Govindaraj, A.; Das, A.; Maiti, P. K.; Sood, A. K.; Rao, C. N. R. Binding of DNA Nucleobases and Nucleosides with Graphene. Chemphyschem 2009, 10, 206210. 12. Vovusha, H.; Sanyal, S.; Sanyal, B. Interaction of Nucleobases and Aromatic Amino Acids with Graphene Oxide and Graphene Flakes. J. Phy. Chem. Lett. 2013, 4, 3710-3718. 13. Larijani, H. T.; Ganji, M. D.; Jahanshahi, M. Trends of Amino Acid Adsorption onto Graphene and Graphene Oxide Surfaces: A Dispersion Corrected Dft Study. RSC Adv. 2015, 5, 92843-92857. 14. Zhang, M.; Yin, B. C.; Wang, X. F.; Ye, B. C. Interaction of Peptides with Graphene Oxide and Its Application for Real-Time Monitoring of Protease Activity. Chem. Comm. 2011, 47, 2399-2401.

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15. Perreault, F.; de Faria, A. F.; Nejati, S.; Elimelech, M. Antimicrobial Properties of Graphene Oxide Nanosheets: Why Size Matters. ACS Nano 2015, 9, 7226-7236. 16. Frost, R.; Svedhem, S.; Langhammer, C.; Kasemo, B. Graphene Oxide and Lipid Membranes: Size-Dependent Interactions. Langmuir 2016, 32, 2708-2717. 17. Compton, O. C.; Nguyen, S. T. Graphene Oxide, Highly Reduced Graphene Oxide, and Graphene: Versatile Building Blocks for Carbon-Based Materials. Small 2010, 6, 711-723. 18. Liu, J. Q.; Cui, L.; Losic, D. Graphene and Graphene Oxide as New Nanocarriers for Drug Delivery Applications. Acta Biomater. 2013, 9, 9243-9257. 19. Sun, X. M.; Liu, Z.; Welsher, K.; Robinson, J. T.; Goodwin, A.; Zaric, S.; Dai, H. J. NanoGraphene Oxide for Cellular Imaging and Drug Delivery. Nano Res. 2008, 1, 203-212. 20. Luo, J. Y.; Cote, L. J.; Tung, V. C.; Tan, A. T. L.; Goins, P. E.; Wu, J. S.; Huang, J. X. Graphene Oxide Nanocolloids. J. Am. Chem. Soc. 2010, 132, 17667-17669. 21. Hummers, W. S.; Offeman, R. E. Preparation of Graphitic Oxide. J. Am. Chem. Soc. 1958, 80, 1339-1339. 22. Sun, X. M.; Luo, D. C.; Liu, J. F.; Evans, D. G. Monodisperse Chemically Modified Graphene Obtained by Density Gradient Ultracentrifugal Rate Separation. ACS Nano 2010, 4, 3381-3389. 23. Cote, L. J.; Kim, F.; Huang, J. X. Langmuir-Blodgett Assembly of Graphite Oxide Single Layers. J. Am. Chem. Soc. 2009, 131, 1043-1049. 24. Li, S. H.; Aphale, A. N.; Macwan, I. G.; Patra, P. K.; Gonzalez, W. G.; Miksovska, J.; Leblanc, R. M. Graphene Oxide as a Quencher for Fluorescent Assay of Amino Acids, Peptides, and Proteins. ACS Appl. Mater. Interfaces 2012, 4, 7069-7075. 25. Joshi, H.; Shirude, P. S.; Bansal, V.; Ganesh, K. N.; Sastry, M. Isothermal Titration Calorimetry Studies on the Binding of Amino Acids to Gold Nanoparticles. J. Phy. Chem. B 2004, 108, 11535-11540. 26. Kabiri, M.; Unsworth, L. D. Application of Isothermal Titration Calorimetry for Characterizing Thermodynamic Parameters of Biomolecular Interactions: Peptide Self-Assembly and Protein Adsorption Case Studies. Biomacromolecules 2014, 15, 3463-3473. 27. Ghai, R.; Falconer, R. J.; Collins, B. M. Applications of Isothermal Titration Calorimetry in Pure and Applied Researchusurvey of the Literature from 2010. J. Mol. Recogn. 2012, 25, 32-52. 28. Choudhary, S.; Talele, P.; Kishore, N. Thermodynamic Insights into Drug-Surfactant Interactions: Study of the Interactions of Naporxen, Diclofenac Sodium, Neomycin, and Lincomycin with Hexadecytrimethylammonium Bromide by Using Isothermal Titration Calorimetry. Colloids Surf. B 2015, 132, 313-321.

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