Interactions between Graphene Oxide and Biomolecules from Surface

Chapter 3

Downloaded by UNIV LAVAL on December 16, 2015 | http://pubs.acs.org Publication Date (Web): December 8, 2015 | doi: 10.1021/bk-2015-1215.ch003

Interactions between Graphene Oxide and Biomolecules from Surface Chemistry and Spectroscopy Shanghao Li,1 Zhili Peng,1 Xu Han,1 and Roger M. Leblanc*,1 1Department

of Chemistry, University of Miami, 1301 Memorial Drive, Coral Gables, Florida 33146, United States *E-mail: [email protected]. Tel.: +1-305-284-2194. Fax: +1-305-284-6367.

Graphene oxide (GO), an oxidized form of graphene, holds a similar single-atom-thick structure to graphene, but possesses plenty of oxygen−containing functional groups, such as carboxyls on the edges and hydroxyls and epoxies on the basal plane. GO possesses extraordinary properties, including low cost manufacturing, rich colloidal properties, high adsorption, and strong fluorescence quenching. GO has also recently been demonstrated to have advantageous applications in the biomedical field and in biosensing. Nevertheless, one critical question needs to be addressed before any actual applications can be discussed: how does GO interact with biomolecules? In this chapter, we will approach this question and briefly summarize the recent progress from the views of surface chemistry and spectroscopy methodology.

Introduction Graphene oxide (GO) is a two−dimensional, atomically thin carbon nanomaterial with functional oxygen−containing groups, such as carboxyl groups at the edges, hydroxyl and epoxide groups mainly at the basal plane, and some C=C sp2 domains (1, 2). Compared with other nanomaterials, the extremely large surface area on both sides, one−atom thickness (1.1 ± 0.2 nm), abundant functional groups, and good dispersion in water render GO as an ideal solid substrate to bind biomolecules through both covalent and non−covalent interactions (3, 4). The history of GO related research can be extended back to © 2015 American Chemical Society In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


about 150 years ago on some of the earliest studies involving the chemistry of graphite oxide (5). The recent decades have witnessed great research progress of GO in biological applications, such as biosensing (6–8), controlled drug delivery (including peptides, proteins, nucleic acids and anticancer drugs) (9–11), cellular uptake (12, 13), microscopic imaging (14–16), and photothermal treatment for cancers and Alzheimer’s disease (17–19). Although great progress has been achieved in research for the potential applications of GO in the biological systems, one significant question needs to be considered before any actual applications: How does GO interact with biomolecules, i.e. amino acids, peptides, proteins, nucleic acids, and lipids? Studies of the interactions between these biomolecules and GO can provide further supports and insights for the potential applications of GO. Unfortunately, there is very limited information available to answer such a fundamental and important question. Hence, the purpose of this chapter is intended to summarize the recent progress of the interacting studies between biomolecules and GO from a viewpoint of surface chemistry and spectroscopy.

Structure and Characterization of GO Although tremendous efforts have been made in the research of GO, the exact atomic structure of GO currently remains largely unknown and under debate (5, 20, 21). Usually, the nature of the functional groups in GO strongly depends on the reaction conditions, such as starting materials, preparation time, and reaction temperatures. In fact, GO may be considered as a family of carbon containing nanomaterials, rather than a single compound, due to its complex structure. We will briefly discuss the structure and characterization of GO from the most commonly used methods, such as spectroscopy and microscopy. The GO sheet consists of a carbon network with both sp2−hybridized carbon atoms in hexagonal rings (i.e. graphene−like C=C) and carbon atoms bearing oxygen functional groups (i.e. C–OH, C-O-C, C=O, and -COOH) (5, 22). The sp2−hybridized graphene−like domains are often found in the sheets of GO, while the oxygen−containing functional groups are mainly dispersed at the basal plane or the defects of the GO sheet (Figure 1A). Various spectroscopic and microscopic techniques have been applied to characterize the structure and morphology of GO. Figure 1B shows a typical UV-vis absorption spectrum of GO aqueous dispersion. It displays a maximum absorption at 229 nm due to the π−π* transition of aromatic C=C bonds and a shoulder around 300 nm due to the n−π* transition of C=O bonds (23–25). As displayed in Figure 1C, the Fourier transform infrared spectroscopy (FTIR) of GO synthesized by the strong acid oxidation method shows in the structures of GO the groups of hydroxyl (C–OH, 3000–3600 cm-1), ketone (C=O, ~1750–1850 cm-1), carboxyl (COOH) (~1600–1750 cm-1), sp2-hybridized C=C (in-plane stretching, ~1500–1600 cm-1), and epoxide (C–O–C, ~1280–1330 and 800–900 cm-1) (26, 27). GO has a couple of bands in the Raman spectrum (Figure 1D), i.e. the in-phase vibration of sp2–hybridized carbon networks (G band) at ~1590 cm-1 and a disorder band caused by the oxygenated groups at the edges (D band) at ~1350 cm-1 (28). The 44 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


integrated intensity ratio of the D- and G-bands (ID/IG) is often used to indicate the oxidation degree in the structure of GO. The morphology of GO has been widely studied using atomic force microscopy (AFM), transmission electron microscopy (TEM) and scanning electron microscopy (SEM) (Figure 2). Among these techniques, AFM is likely the most widely used tool in the determination of the surface topography of GO with lateral dimension and precise height profile. While the size distribution of GO sheet obtained from microscopic studies is quite diverse, the height of single layer GO is usually around ~1 nm (18, 24, 29–31).

Figure 1. (A) Proposed chemical structure; (B) UV-vis absorption; (C) FTIR spectroscopy; and (D) Raman spectroscopy of graphene oxide. Figure 1B is reproduced with permission from reference (23). Copyright 2012 American Chemical Society; Figure 1C is reproduced with permission from reference (26). Copyright 2013 Institute of Physics.

Interaction between GO and Amino Acids Interaction between amino acids with GO has attracted significant attention in research because of the ubiquitousness of the amino acids and the potential biomedical applications of GO in biological systems. α-Amino acids are biologically essential organic compounds composed of amine (–NH2) and carboxylic acid (–COOH) functional groups at the α position, along with a side-chain. They are the basic building-blocks of peptides and proteins. Depending on the properties of the side-chain group (i.e. polar, non-polar, acidic, 45 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


basic, and aromatic), the interaction between amino acids and GO is driven by various forces, such as electrostatic, hydrophobic, hydrogen bond and/or van der Waals interactions (32).

Figure 2. (a) A tapping mode AFM image of graphene oxide (GO) sheets on mica surface, (b) the height profile of the AFM image, (c) TEM image of the GO, and (d) SEM image of the GO. Reproduced with permission from reference (31). Copyright 2011 Ivyspring International Publisher.

Adsorption Study Zhang et al. investigated experimentally the possible adsorption of 20 amino acids to the surface of GO (Table 1) (32). The adsorption was determined by the concentration changes before and after incubation with GO. It was found that the interaction strength between GO and the amino acids in phosphate buffered saline (PBS) at pH 7.4 followed the order Arg > His > Lys > Trp > Tyr > Phe (Table 1) (32). Their experiments showed that the other 14 amino acids have little adsorption on the surface of GO. As expected, the amino acids Arg, His and Lys with positive charge have strong electrostatic interactions as the main driving forces with negatively charged GO surface. Compared with Arg and Lys, His has an imidazole (C3H4N2) ring substituent, which has been demonstrated from molecular modeling studies that the imidazole ring has strong π–π interaction (33). 46 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Therefore, besides electrostatic interactions, π–π interaction also contributes to the adsorption of His to the surface of GO. Theoretical studies also display that the aromatic rings of the amino acids (e.g. Trp, Tyr and Phe) prefer to orient in parallel with respect to the plane of graphene via the π–π interactions (33). The π–π binding strength with graphene surface follows the trend: Trp > Tyr > Phe > His depending on the polarizability of the amino acid. As mentioned above on its structure, GO has some intact graphene–like islands which facilitate the adsorption between the GO nanosheet and the aromatic amino acids (Trp, Tyr, Phe and His) via the π–π interaction.

Table 1. The Concentrations of the Tested Amino Acids (nmol/mL) before and after Incubation of GO in PBS at pH 7.4 and the Corresponding Concentration Ratios (Revised from Reference (32)) Amino Acid

Before incubation (nmol/mL)

After incubation (nmol/mL)

Ratio

Arg

87.80

55.52

0.6323

His

77.30

55.32

0.7157

Lys

93.89

69.91

0.7446

Trp

125.02

95.17

0.7612

Tyr

92.81

81.05

0.8733

Phe

94.89

89.34

0.9415

Other 14 amino acids

--

--

0.9722~1.0262

Fluorescence Quenching of Trp or Tyr by GO Fluorescence spectroscopy and quenching are widely used to investigate the interaction of substances with molecules due to its sensitivity, low cost and ease for operation. These techniques can sensitively detect the changes of the local environment of the fluorophore by simply measuring the fluorescence signal (34). It has been known that GO can quench the emission of fluorescent molecules or nanoparticles, such as organic dye molecules (35, 36), fluorescent labels (29, 37), and quantum dots (QD) (24, 38), through Förster resonance energy transfer (FRET) from the fluorescent part to GO. The fluorescent dyes or labels usually contain aromatic rings and the quenching is via non−covalent interactions, such as electrostatic interactions, hydrogen bonding, hydrophobic and π−π interactions between GO and the dye molecules or fluorescent labels (29, 39, 40). Based on the structure and component of fluorescent amino acids (Trp or Tyr), there should be a non−covalent interaction between them and GO, quenching the fluorescent assay. Indeed, the experiments showed the strong quenching effect of GO. 47 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Experimentally, the amino acid Phe is also fluorescent, but it is not suitable for the quenching study due to its low quantum yield (41). Therefore, Phe will not be mentioned in this part. When mixed with GO, the fluorescence intensity of Trp was strongly quenched without shifting the emission peak (Figure 3A). Similar fluorescence quenching phenomenon was also observed in Tyr assay.

Figure 3. Fluorescence quenching of Trp by GO. (A) The fluorescence quenching of Trp (10-6 M) by mixing with different concentrations of GO. (B) The quenching of Trp (F0/F, black) and fluorescence lifetime ratio (τ0/τ, red) as a function of GO concentrations. Reproduced with permission from reference (23). Copyright 2012 American Chemical Society. After the correction of the “inner filter effect” (23, 42, 43), Stern−Volmer plot of F0/F against the concentration of GO is showed in Figure 3B (black color), where F0 and F are the fluorescence intensity at the maxima in the absence and in the presence of GO, respectively. If the quenching mechanism is static only, the fluorescence lifetime should remain the same, as this process does not affect the excitation state of the fluorophore (43). Hence, the static quenching can be described by the classical Stern−Volmer plot with linear fit. However, the fluorescence lifetime of Trp increases slightly with linearity when GO is added (red line, Figure 3B), and the plot of F0/F against GO has an upward curvature (black line, Figure 3B). Similar observation was also found for Tyr. These observations indicate that the quenching of Trp and Tyr by GO is mainly static quenching, slightly combined with dynamic quenching.

Hydrophobic Interaction Study between Trp or Tyr and GO Since both Trp and Tyr have a hydrophobic moiety, they may have hydrophobic interaction with the hydrophobic part of GO in the process of quenching. Triblock copolymer Pluronic F127 (PF127) was utilized in the experiment with a hope to block this interaction due to the fact that PF127 was previously shown to have strong hydrophobic interaction with GO (44, 45). PF127 consists of a central hydrophobic block of polypropylene glycol flanked by two hydrophilic blocks of polyethylene glycol. The hydrophobic segments 48 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


have previously been shown to interact with the hydrophobic part of GO while the hydrophilic chains extend to the aqueous solution (44, 45). When mixed GO with PF127, the hydrophobic moiety of GO is proposed to be covered by the hydrophobic part of PF127, screening the hydrophobic interaction between GO and Trp. As shown in Figure 4A, the mixture of GO:PF127 (1:1, w/w) has a lower quenching efficiency than the corresponding GO concentration in the absence of PF127. Since PF127 itself does not affect the fluorescence of Trp, the observation of reduced quenching supports the assumption that the added PF127 blocks the hydrophobic interaction between GO and Trp. Lower quenching effect of the mixture of GO and PF127 is also observed for Tyr.

Figure 4. Sterm–Volmer plot of Trp under different conditions. (A) 10-6 M Trp against the concentration of GO alone, and the mixture of GO:PF127 (1:1, w/w). (B) 10-6 M Trp against the concentration of GO at pH 5.6 and 9. Reproduced with permission from reference (23). Copyright 2012 American Chemical Society.

Electrostatic Interactions between Trp or Tyr and GO Electrostatic interactions can also exist between GO and Trp during quenching. The carboxylic groups of GO are readily deprotonated with negative charge when GO is dispersed in pure water at pH 5.6. If electrostatic interactions are important for the quenching, decreased quenching efficiency between GO and Trp at basic pH is expected, as Trp is also negatively charged (the isoelectric point for Trp is 5.9). As expected, the value of F0/F is lower at pH 9 compared with that at pH 5.6 (Figure 4B), indicating the existence of electrostatic interactions in the quenching process. Similar to Trp, the quenching efficiency between GO and Tyr at pH 9 is also lower than that at pH 5.6.

Interaction between Peptides or Proteins and GO As GO has recently been exploited for drug delivery, biomolecule detection, and near−infrared photothermal treatment for cancers and Alzheimer’s disease (17–19), the understanding of the interaction between GO and peptides or proteins 49 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


is fundamentally essential, especially for those drug− or disease−related peptides or proteins. Similar to the studies mentioned above, fluorescence spectroscopy is a simple method to investigate the interaction between peptide/proteins and GO. The quenching property of GO can also be used for protein detection by measuring the fluorescence signal with or without target proteins. Furthermore, the unique nanostructure of GO with large specific surface area makes this material an ideal platform for protein immobilization through non-covalent binding. In this part, we will focus on the interaction between GO and peptides or proteins through the measurement of their intrinsic fluorescence, fluorescence quenching and immobilization.

Intrinsic Fluorescence of Peptides and Proteins The intrinsic fluorescence of a peptide or protein is a mixture of the fluorescence from individual fluorescent amino acid residues (i.e. Trp, Tyr and Phe). Among them, most of the intrinsic fluorescence emissions of a folded protein are due to excitation of Trp residues, with some weak emissions due to Tyr and even weaker emission from Phe (46). Typically, the emission peak of Trp depends on the polarity of the local environment around it and can be used as a probe of the conformational state of a protein (46, 47). Furthermore, Trp is a relatively rare residue found in proteins; many proteins contain only one or a few Trp residues. Therefore, Trp fluorescence can be used as an intrinsic measurement for the conformational and environmental change of the residues. Aβ40 is the most abundant form of Aβ peptides, and its fibril formation is associated with the development of Alzheimer’s disease. Human islet amyloid polypeptide (hIAPP) is a 37 amino acid residue peptide and its amyloid deposits are the major source of fibrils found in the islets of Langerhans of type 2 diabetic patients (48, 49). Both have only one fluorophore, the Tyr residue at position 10 for Aβ40 and position 37 for hIAPP. When mixed with GO, the intrinsic fluorescence of Tyr in the amyloid beta 1-40 (Aβ40) and human islet amyloid polypeptide (hIAPP) is quenched (Figure 5A) (23). While the emission peaks of both peptides are not shifted, the quenching indicates that there is binding interaction between GO and Aβ40 or hIAPP. The higher quenching of Aβ40 should not be due to the charge differences between Aβ40 and hIAPP, because the slightly negatively charged Aβ40 (isoelectric point 5.4) in principle should have weaker electrostatic interactions with GO than the positively charged hIAPP (isoelectric point 8.8). Instead, the structural differences between Aβ40 and hIAPP are likely the reason for the observed difference between their quenching efficiency. The single Tyr in Aβ40 has six aromatic amino acid residues in its vicinity (three phenylalanines and three histidines). Strong π−π and/or hydrophobic interactions may thus exist between these residues and GO. However, the Tyr residue in hIAPP is located at the negatively charged C−terminus, and there are no aromatic residues in this domain for the interaction with GO. Therefore, the quenching efficiency of hIAPP is lower than that of Aβ40. 50 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 5. The Stern-Volmer plot of different proteins aqueous solution quenched by GO at pH 5.6. (A) 10-5 M hIAPP and 10-5 M Aβ40 against the concentration of GO; (B) 10-6 M BSA and 10-6 M HSA against GO. Reproduced with permission from reference (23). Copyright 2012 American Chemical Society. GO also strongly quenches the fluorescence of bovine serum albumin (BSA) and human serum albumin (HSA), with a slightly higher quenching efficiency for HSA, as shown in Figure 5B. The quenching difference between these two proteins is likely related to their respective number of Trp residues. BSA has two Trp (Trp135 and Trp214) and HSA only one (Trp214) (50). It could also be due to protein conformational changes as a result of their interaction with GO, leading to a decrease of their fluorescence intensity. We also studied the fluorescence of other proteins, such as lysozyme and pepsin, and quenching was always observed in our experiments. Therefore, it is possible that GO is a universal quencher for intrinsic fluorescence of proteins.

Protein Detection Using the Fluorescence Quenching of GO If the peptide or protein detection system using GO is based on the fluorescence signal, the process in most cases follows the following pattern: (1) fluorescence quenching of the probe due to the strong non-covalent binding between the fluorescent moiety and GO; (2) target recognition by the probe detaching the fluorescent moiety from GO surfaces; (3) fluorescence recovery of the probe. In principle, by substituting the corresponding probe for the target, the same strategy can be easily extended to detect different peptides or proteins. In fact, this process in literature has been widely used in the field of analytical chemistry to detect proteins. Dong et al. designed a platform for effective sensing of thrombin by FRET from quantum dots (QDs) as donors to GO as an acceptor (24). The QDs were first conjugated to a molecular beacon which can recognize thrombin. The strong interaction between the molecular beacon and GO resulted in the fluorescence quenching of QDs. Once the molecular beacon was bound to thrombin, the distance between the QDs and GO became larger, weakening the interaction between GO and QDs. Therefore, the fluorescence of QDs was recovered. 51 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


To increase the sensitivity of protein detection using peptide-protein binding, Wang et al. utilized the fluorescence quenching property of GO to selectively detect cyclin A2, a prognostic protein indicator in many early-stage cancers (51). The fluorescence of the dye-labeled peptide probe, FITC-HAKRRLIF, is efficiently quenched when mixed with GO. Similar to what we have mentioned above about the interaction between aromatic amino acids and GO, the authors also proposed that non-covalent binding (i.e., electrostatic interactions, hydrophobic interaction, and π-π interaction) contributes to the efficient quenching of the dye by GO. Once the target protein, cyclin A2, is added, the fluorescence of dye was recovered.

Protein Adsorption on the Surface of GO GO used as a matrix for enzyme immobilization was first reported by Zhang et al. (3) Horseradish peroxidase and lysozyme molecules were immobilized onto the surface of GO via non-covalent bonding without any cross-linking reagents by simply mixing these components. The immobilized enzyme can be observed clearly by AFM imaging. Apparently, protein immobilization onto the surface of GO results from the synergic effect of different types of interactions. However, this process is dominated by the electrostatic interactions between negatively charged GO sheets and enzyme molecules. Lysozyme (LYZ) content is relatively high in biological fluid samples, such as tears, milk, saliva, urine and blood serum (52–54). Lysozyme is a small globular enzymatic protein with 129 residues linked by four disulfide bridges. This enzyme is part of the innate immune system, damaging the bacterial cell walls by hydrolyzing the peptidoglycan (55). GO has been previously used for the purpose of analyte detection and quantification from the biological fluid. To detect tetracyclines from milk samples, Liu et al. applied GO to enrich the analyte and then detect the target by MALDI−TOF mass spectroscopy (56). In another study, Song et al. constructed a system of GO-fluorescein isothiocyanate-labeled peptide for the detection of matrix metalloproteinase 2 in complex serum samples (57). Yan et al. studied both in vitro and in vivo biocompatibility and cytotoxicity of GO when it was intravitreally injected into rabbit eyes (58). Their preliminary results showed that GO has good intraocular biocompatibility with little cytotoxicity on cell viability, cell morphology and membrane integrity. Due to the high abundance of lysozyme in these biological fluid samples, one has to consider the possible interaction and adsorption between lysozyme and GO in the experiments. Our group recently investigated the interaction between GO and lysozyme and the possible applications of this interaction in separation and selective adsorption of lysozyme (59). Surprisingly, compared to BSA and HSA (Figure 5B), a more dramatic quenching of lysozyme is observed under the same concentration with the increase in the content of GO (Figure 6A). In the presence of 10 µg/mL GO, the value F0/F of lysozyme increases to proximately 26.5. The fast reduction of fluorescence intensity indicates a much stronger interaction between GO and 52 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


lysozyme. To determine the nature of interaction between GO and lysozyme, the quenching has been investigated at pH 5.6, 10 and 12. Considering the isoelectric point of lysozyme is about 11, its charge strongly depends on the pH of the solution (60). Thus lysozyme is more positively charged at pH 5.6 than pH 10. From Figure 6A, lysozyme at lower pH has higher quenching effect by the negatively charged GO, indicating that the strong quenching of lysozyme by GO is predominantly due to attractions between lysozyme and GO. Other methods were used to further characterize the interaction between GO and lysozyme, i.e. zeta potential, dynamic light scattering and AFM imaging. At pH 5.6, the zeta potential of 5 µg/mL GO is -38.85 mV, however, the value increases to -9.05 mV in the presence of 14.3 µg/mL lysozyme. In order to find out the trend of zeta potential change in the GO/lysozyme mixture, a titration experiment of zeta potential was performed with a constant GO concentration at 5 µg/mL (Figure 6B). As more lysozyme is present, the zeta potential value of GO/lysozyme mixture shifts toward the more positive. When lysozyme is added to 20 µg/mL, the zeta potential reaches about 0 mV, probably corresponding to the maximum loading of lysozyme on the surface of GO. As shown in Figure 6C, in the GO/lysozyme mixture, the hydrodynamic diameter distribution depends on the pH and the concentration of GO. The size of GO/lysozyme decreases as the pH increases from 5.6 to 12 at each corresponding concentration of GO, indicating a weaker interaction at higher pH. In fact, the size of the mixture at pH 12 is almost the same as that of pure GO (data not shown). This may be due to the electrostatic repulsion between lysozyme and GO at pH 12 which are then both negatively charged. AFM was used to directly observe the morphology of GO/lysozyme at pH 5.6 on a freshly cleaved mica surface. It seems that lysozyme “glues” GO sheets together with uneven height from 3.5 nm to more than 20 nm (Figure 6D). We have so far demonstrated that strong interaction between GO and lysozyme results from the electrostatic interactions as described above, but it is worth noticing that some weak interactions may also exist, such as π-π interaction, hydrophobic interaction, and hydrogen bonding.

Adsorption and Desorption of Lysozyme on GO We demonstrated above that the strong interaction between GO and lysozyme is predominately resulting from electrostatic interactions. In our experiment, this interaction was so strong that we could use GO to remove and separate lysozyme from aqueous solution (59). Briefly, GO is added into a lysozyme aqueous solution to form a GO/lysozyme assembly, and then NaCl is added to precipitate GO/lysozyme. After centrifugation, the fluorescence (Figure 7A) and UV-vis absorbance (Figure 7B) of lysozyme in the supernatant almost completely disappears. Therefore, GO could be an excellent adsorbent material to remove lysozyme from aqueous solutions. In the pellet, the adsorbed lysozyme can be released by adding NaOH and CaCl2, which separate lysozyme from the surface of GO (59). 53 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Figure 6. (A) F0/F of 10-6 M lysozyme against the concentration of GO at pH 5.6, 10 and 12. (B) Zeta potential of GO/LYZ aqueous solution against LYZ concentration at pH 5.6. The concentration of GO was fixed at 5 µg/mL. (C) Mean hydrodynamic diameter of GO/LYZ mixture. (D) AFM image of GO/LYZ mixture. The scale bar at the bottom right is 1 µm. Reproduced with permission from reference (59). Copyright 2014 American Chemical Society.

Figure 7. (A) Fluorescence spectra and (B) UV−vis absorption spectra of lysozyme before and after adsorption by GO. Reproduced with permission from reference (59). Copyright 2014 American Chemical Society. 54 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Selective Adsorption of Lysozyme One may use the strong interaction between lysozyme and GO to selectively adsorb lysozyme from a mixture of proteins on the basis of their electrostatic interactions. If two proteins are positively charged and have similar isoelectric points (pI), both will be adsorbed by GO without selectivity. Fortunately, very few natural proteins or enzymes bear as many positive charge as lysozyme. To study the selective adsorption of lysozyme by GO, we mixed lysozyme (LYZ, pI 11, 14.3 kDa) in 0.1 M phosphate buffer at pH 7 with ovalbumin (OVA, pI 4.9, 43 kDa), bovine serum albumin (BSA, pI 5.3, 68 kDa), or human serum albumin (HSA, pI 4.7, 66.5 kDa) (59). After GO and sodium chloride are added subsequently, the mixture was centrifuged to obtain the supernatant. In a binary protein mixture (i.e. LYZ/BSA, LYZ/HSA, or LYZ/OVA), the supernatant after centrifugation was characterized by spectroscopy and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS−PAGE). The obtained fluorescence and UV−vis absorption spectra of the mixture after adsorption by GO were very similar to those of the control experiments using BSA, HSA or OVA alone (59). Experiments from the SDS-PAGE showed that the band of lysozyme at ~15 kDa disappeared upon addition of GO. Similarly, in a ternary of protein mixture, as shown in Figure 8, the SDS-PAGE displays that the band of lysozyme disappeared upon addition of GO (see Lane 2 and 4). These observations confirm that lysozyme was selectively adsorbed by GO, leaving other proteins in the solution (i.e. BSA, HSA, and OVA).

Interactions between Graphene Oxide and Oligonucleotides DNA and RNA based oligonucleotide nanotechnology plays an important role in biological research, and have attracted attention for decades (61, 62). The information encoded within the well-known Watson-Crick structure of DNA and RNA is the key to a variety of biological phenomena. However, there still remain huge challenges regarding their biological applications, such as detection limit, cost efficiency, and biocompatibility. Due to the remarkable chemical and physical properties of GO, the hybridized nanomaterials consisting of DNA or RNA with GO display a high potential for new generations of biosensors, drug delivery systems, molecular machines, and devices in many other fields (63–65). In an effort to investigate the role of interactions between oligonucleotides and GO, covalent and non-covalent binding strategies are generally employed to attach DNA or RNA to the GO, as illustrated in Figure 9. The fluorescence quenching of the probe by GO is due to the strong binding between the fluorophore and GO. When the target recognition happens, the fluorescent probe will be detached from GO surfaces, resulting in the recovery of the fluorescence of the probe. For example, Berry’s group reported biological applications of graphene based device in 2008 (66). In this study, the terminal amine group of single stranded DNA (ssDNA) covalently bonded to the carboxylic group from GO, which was confirmed by hybridizing it with its conjugated fluorescent labeled DNA probe. The preference of DNA tethering on thicker layers and on wrinkles 55 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


of GO were observed. In addition to covalent binding approach, non-covalent binding methods using van der Waals, π-π stacking and electrostatic interactions are more widely utilized for DNA detection (67–69).

Figure 8. Electrophoresis of a ternary mixture of LYZ/OVA/HSA and LYZ/OVA/BSA separated by 12 % SDS−PAGE. Protein marker (Lane M), LYZ/OVA/HSA control (Lane 1), LYZ/OVA/HSA adsorbed by GO (Lane 2), LYZ/OVA/BSA control (Lane 3), LYZ/OVA/BSA adsorbed by GO (Lane 4). Reproduced with permission from reference (59). Copyright 2014 American Chemical Society.

Figure 9. i) Fluorophore attached DNA was absorbed onto GO, and its fluorescence was quenched because of FRET. ii) Specific sequence ssDNA was added to the surface, which leads to the fluorescence restoration. 56 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


Studies have shown that nucleobases, nucleosides, DNA and RNA can be absorbed to graphene based materials. Rao’s group studied the interaction of DNA base pairs with graphene by isothermal titration calotrimetry (ITC) (70). Their data have shown experimental binding energies of Guanine (G) > adenine (A) > cytosine (C) ≈ thymine (T). Moreover, Lu et al. immobilized dye labeled ssDNA onto the GO via the ionic interaction (11). The fluorescence of the conjugated organic dye (fluorescein-based dye) was quenched after adsorption onto the surface of GO (Figure 9A). When the target DNA was added (Figure 9B), the fluorescent dye would be detached from GO surface due to the formation of the complementary DNA between ssDNA and the target DNA, resulting in the reappearance of the fluorescence (Figure 9C). Recently, thiolated DNA was employed to assemble gold nanoparticles into two dimensional materials on top of the GO (71). DNA was coated onto the basal plane of GO by electrostatic interactions. This hybrid nanostructure showed good water solubility and exhibited a high potential for applications in catalysis, nanoelectronics, and biosensing platforms. Similarly, Huang’s group modified metal nanoparticles with DNA, followed by depositing it onto the GO via π-π stacking (16).

Interaction between GO and Lipids GO has been extensively studied for various biomedical applications (4, 72), and one novel direction of these studies is the development of new nanocomposite materials of GO with lipid membranes. However, detailed studies regarding the interactions between GO and cell membranes or model membrane systems is rather limited. In this part, we will focus on the latest studies on the interaction between GO and lipids.

Orientation of GO in the Presence of Lipid Monolayers at the Air−Water Interface Due to the unique structure of GO, which has an extremely thin layer (~1 nm) with large surface area and irregular shape, it is important to know how GO orientates itself when interacting with cell membranes. Our group recently investigated the interaction between GO and model membranes using the Langmuir monolayer technique that was applied at the air-water/aqueous interface and revealed the possible nature and orientations of GO when interacting with different lipids (45). Langmuir monolayer at the air−water interface is a typical two−dimensional (2–D) surface chemistry approach, widely applied for the structure and property studies of proteins and lipids at the air−water interface (73–75). Because of the deprotonation of carboxyl groups of GO sheets (76, 77), electrostatic interactions are expected to occur between negatively charged GO and charged headgroups of lipids. The air−water interface is an ideal platform for the study of interaction between lipids and GO, as the lipid molecules have readily oriented themselves at the interface with polar/charged groups merged 57 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


in the aqueous phase while hydrophobic moiety facing toward the air phase. Furthermore, Langmuir monolayer of lipid is a well-accepted in vitro model to mimic biological membranes (78), which can be considered as two weakly assembled Langmuir monolayers. Five different lipids were used in this study: positively charged dioctadecyldimethylammonium bromide (DODAB), 1,2-distearoyl-snglycero-3-ethylphosphocholine chloride salt (DSEPC); zwitterionic 1,2distearoyl-sn-glycero-3-phosphocholine (DSPC), and negatively charged 1,2-distearoyl-sn-glycero-3-phosphate sodium salt (DSPA) and stearic acid (SA). The same 18-carbon chain alkyl groups in the nonpolar tail with different charged head groups are purposely chosen to rationalize the possible interactions. The study revealed that the interactions are governed by electrostatic forces between the polar head groups of the lipids and GO, evidenced by the fact that GO successfully incorporated into the monolayer of DODAB and DSEPC (positive charged), but not DSPC, DSPA and SA (neutral/negative charged). It was further elucidated that the shielding effect from the substructure of the head group is also an important fact when considering the interactions between GO and lipids. Collectively considering the possible orientations of GO and experimental data, we proposed that an “edge-in” fashion is more likely to occur where GO penetrated into the DODAB lipid monolayer (Figure 10a and 10b). However, “face-in” fashion to penetrate the monolayer is not likely to happen for DSEPC monolayer (Figure 10C and 10D), probably due to the possible electrostatic shielding from sn-glycero-3-ethylphospho groups.

Interactions Between GO and Lipid Bilayers Frost et al. investigated the interactions between GO and liposomes (79). They prepared supported lipid bilayers with different charges on SiO2 surfaces by using extruded liposomes with a mean diameter of 80-90 nm. The model negatively charged liposomes, POPC/POPS (3:1), were prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS); and positively charged liposomes, POPC/POEPC (3:1), were prepared from POPC and 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC). The model membranes were then exposed to an aqueous suspension of GO, and then monitored by the quartz crystal microbalance with dissipation monitoring technique (QCM-D). It was found that when the negatively charged POPC/POPS (3:1) membrane surface is exposed to GO, there is no sign of adsorption of GO based on the QCM-D signal, which could be attributed to the electrostatic repulsion between the lipid membrane head groups and GO. In contrast, when the positively charged POPC/POEPC (3:1) membrane was used, there is a clear signal from the QCM-D indicating the flat adsorption of the GO flakes on the membrane and covers most of the surface. The finding once again demonstrates the importance of electrostatic forces between GO and lipid head groups when studying their interactions. Further 58 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


experiments found that after the adsorption of GO onto the positively charged POPC/POEPC (3:1) lipid membrane, an interesting multilayered structure could be obtained with further sequential additions of POPC/POEPC (3:1) liposomes and GO to the surface (Figure 11).

Figure 10. Schematic diagrams of the possible orientations of GO when interacting with different types of lipids monolayer at the air-water interface. Reproduced with permission from reference (45). Copyright 2013 American Chemical Society.

Antibacterial Activity of GO Toxicity of synthetic carbon nano-materials, such as fullerenes and carbon nanotubes have been extensively studied while only a few toxicity investigations on GO related materials are available. It is believed that the physical properties of GO such as solubility, dispersion and size would strongly influence their antibacterial activities. Recent investigations have showed that GO could exhibit strong antibacterial activities (80–83) The physical damages on the bacterial cell membranes by the sharp edges of GO nanosheets are believed to be responsible for the antibacterial activity observed in these studies. The interaction between GO and the bacterial membrane could result in destruction of membrane structure and the leakage of RNA from the cell (membrane stress) (83). Another mechanism was proposed that graphene-based materials might induce oxidative stress on 59 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.


bacterial cells (80). A three-step antibacterial activity mechanism was also proposed (81). The process begins with the bacterial deposition onto GO resulting in direct contact between bacteria and GO, which is followed by a disruptive interaction with bacterial, inducing membrane stress. The last step would involve the disruption of a specific bacterial process by oxidizing a vital cellular structure or component, inducing oxidation stress. These studies demonstrated that GO could be used as potential antibacterial materials in daily life to protect the public health.

Figure 11. Schematic representation of the formation of a POPC/ POEPC (3:1) lipid membrane and subsequent adsorption of GO. Further addition of POPC/POEPC (3:1) liposomes and GO results in a multilayered structure. The figure is not drawn to scale. Reproduced with permission from reference (79). Copyright 2012 American Chemical Society.

Conclusion Graphene oxide (GO) holds promising applications in biological and biomedicine fields due to its unique physical and chemical properties as a novel 2-dimensional nanomaterial. It is fundamentally important to investigate the interactions between GO and biomolecules for any practical applications. From the views of surface chemistry and spectroscopy, we briefly summarized the recent progress of the interaction studies between GO and biomolecules, including amino acids, peptides, proteins, oligonucleotides and lipids. Because of its unique one-atom-thick structure with plenty oxygen-containing groups and graphene-like domains, GO surface has high adsorbility of biomolecules via non-covalent binding, particularly electrostatic and π−π interactions. When the biomolecules are adsorbed onto the surface of GO by these interactions, the fluorescence of the intrinsic fluorophores or the dye attached will be strongly quenched. When these biomolecules are detached from the GO surface by recognition or competition, the fluorescence will be restored. Many new biosensors are reported and fabricated on the base of this mechanism for the detections of peptides, proteins, DNA and RNA. The interaction between lipid model membranes and GO is governed primarily by electrostatic interactions, as the experiments showed that positively charged lipids have a strong binding effect on GO. Recent 60 In Recent Progress in Colloid and Surface Chemistry with Biological Applications; Wang, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2015.

investigations demonstrated that GO can exhibit strong antibacterial activities, which may be due to the physical damage or oxidation stress of the bacterial cell membranes caused by GO.

Acknowledgments


R.M.L. gratefully acknowledges the support of the National Science Foundation under Grant 1355317. All authors gratefully acknowledge the reviewers for the valuable and constructive suggestions and comments on this chapter.

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Interactions between Graphene Oxide and Biomolecules from Surface

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