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Monitoring bound HA1(H1N1) and HA1(H5N1) on freely suspended graphene over plasmonic platforms with infrared spectroscopy
Chemical Physics Letters xxx (2013) xxx–xxx
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Monitoring bound HA1(H1N1) and HA1(H5N1) on freely suspended graphene over plasmonic platforms with infrared spectroscopy Amrita Banerjee a, Sumit Chakraborty b, Nihal Altan-Bonnet c,1, Haim Grebel a,⇑ a
The Electronic Imaging Center at NJIT, Newark, NJ 07102, United States Department of Microbiology & Immunology, Weill Cornell Medical College, New York City, NY 10025, United States c Department of Biological Sciences, Rutgers University, Newark, NJ 07102, United States b
a r t i c l e
i n f o
Article history: Received 22 May 2013 In final form 5 July 2013 Available online xxxx
a b s t r a c t Infrared (IR) spectroscopy provides fingerprinting of the energy and orientation of molecular bonds. The IR signals are generally weak and require amplification. Here we present a new plasmonic platform, made of freely suspended graphene, which was coating periodic metal structures. Only monolayer thick films were needed for a fast signal recording. We demonstrated unique IR absorption signals of bound proteins: these were the hemagglutinin area (HA1) of swine influenza (H1N1) and the avian influenza (H5N1) viruses bound to their respective tri-saccharides ligand receptors. The simplicity and sensitivity of such approach may find applications in fast monitoring of binding events. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Infrared (IR) spectroscopy is an important spectroscopic tool to detect molecular absorption in the 1–100 lm wavelength range [1]. The signals are relatively weak and techniques have been devised to amplify it. For example, in Attenuation Total Reflection (ATR) the sample is depositing a transparent slab. A long interaction length between the interrogating electromagnetic wave and molecules is achieved through multiple internal reflections. However, upon each reflection a different set of molecules is assessed along the slab. Here we propose another amplification approach while keeping the thickness of the molecular film to monolayer dimensions. In our approach, the IR beam irradiates the sample depositing a graphene-coated metal mesh structure. The periodic metal structure scatters the resultant surface modes back and forth, thus increasing the interaction between the IR beam and the same set of molecules. The platform fits existing Fourier Transform IR (FTIR) spectrometers in transmission mode without modification. The technique is demonstrated with bound hemagglutin of influenza viruses to their respective tri-saccharides ligand receptors. Binding the hemagglutinin area of influenza virus (HA) to sialylated glycans is an important part of virus infection studies [2–6]. Besides interests in its devastating effects on birds and fowls, avian flu has been also researched for potential risks to humans [7–13]. Crystal structures of HA from H1 (swine) and H5 (avian) and their ⇑ Corresponding author. E-mail address: [email protected] (H. Grebel). Current address: Laboratory of Host-Pathogen Dynamics, Cell Biology and Physiology Center, National Heart Lung and Blood Institute, NIH, Bethesda MD, United, States. 1
complexes with oligosaccharides provided useful information [14–16]. Real-Time PCR protocol has been issued by the Center of Decease Control, expediting the detection of swine flu [17]. Yet, despite advances in key micro-array technologies [18], and facing global pandemic prospects, a need arises for a simple detection method, augmented by drug development and implemented in an environment closely mimicking the virus expression location. IR spectroscopy [19] is a detection method widely applied to the study of proteins. Proteins share many IR absorption lines thus, obscuring their identification in complex environments. Yet, protein binding involve hydrogen bonds, thus in principle, we may follow the binding mechanism instead. Our signal amplifying platforms enabled us to use very small concentration levels, e.g., the starting molar concentration of HA1 in our stock solution was 1 lM spread over an area, larger than 1 cm2. Cellular plasma membranes are essential structural elements of eukaryotic cells, which define the cells’ outer surface. The primary structural elements of a plasma membrane are amphiphilic phospholipid molecules, self-assembled into sheet-like structures, which we deposited onto bio-compatible, micron-size, graphenecoated periodic hole-array. In the experiments, tri-saccharide ligand receptors were incorporated with lipid membranes via mixing. The ligand receptors have a hydrophobic linker which could aid its attachment to the hydrophobic part of the membrane. Saccharides ligands stabilize the lipid bilayers by dispersing water molecules around their hydrophylic heads [20,21]. By incorporating sialosaccharides with the membrane we are not only immobilizing the receptors but also attempting to mimic the natural binding process between protein and ligand. Graphite is hydrophobic while the hydrophobicity of graphene is unclear. Thus, the stability of the membrane-ligand receptor complex on graphene-
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coated substrates is of interest. Detection and understanding of the interactions between analytes and lipid membranes is an essential task for drug targeting. Specifically, one of the fundamental properties of a drug or drug-like molecule is its binding affinity to a target. Strong H-bond mediated interactions are, therefore a very desirable property (see for example, Lipinski et al., in Adv. Drug Delivery Rev. 46 (2001) 3–26). The methodology described here outlines a simple approach to monitor H-bond formation between a small molecule and a large biomolecule. Figure 1. Schematics of the platform layout. The binding between receptor and protein is at the lipid membrane surface but the receptor is most probably anchored to the hydrophobic tail of the phospholipid.
2. Experimental results Experimental methods are described at the end of the SI section. The platform is presented in Figure 1. Binding of HA1 proteins to the respective ligand receptors are simulated in Figure 2. The
Figure 2. (a) HA1(H5N1) bound to tr40: key interaction distances between ligand and protein are highlighted blue (left). Bound receptor (right). (b) HA1(H1N1) bound to tr43: key interaction distances between ligand and protein are highlighted blue (left). Bound receptor (right). (see Fig. S5 for the entire data). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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A. Banerjee et al. / Chemical Physics Letters xxx (2013) xxx–xxx
hydrophobic (or lack of) nature of the suspended graphene layer(s) is assessed by its contact angle [22] with di-ionized (DI) water and buffer solutions (Figure 3). Several fitting methods differed somewhat in their final results but provided with similar trend: contact angles with buffer solution droplets on graphite and nickel plates were substantially smaller (20°) than those with DI water (>80°). Contact angles for highly ordered pyrolitic graphite (HOPG) substrates were 5° larger than graphene coated screens. DMPC in buffer solution exhibited larger contact angles than the buffer solution alone (by approximately 15°): this was true for all samples including HOPG substrates for which an angle of 59° was assessed. The range of contact angles obtained with a buffer solution alludes to the hydrophilic nature of our graphene coated surfaces. Fluorescence recovery after photobleaching (FRAP) experiments were conducted [23] to determine the diffusion time of dye within lipid bilayers on glass and on our suspended graphene layers. The recovery life-time appeared to be similar for both graphene-coated screens and reference glass substrates (Figure 4). IR absorption spectra of bound ligand receptors exhibited new IR peaks. In Figure 5a we show the IR absorption spectra of HA1 of H1N1 and H5N1 on the avian receptor tr40. Clearly, the HA1(H5N1) interacts with the tr40 while the HA1(H1N1) is not: newly formed IR absorption bands at 1400–1650 and 3000– 3800 cm 1 appear for HA1(H5N1). IR peaks appear only upon coupling between HA1(H1N1) and the human receptor tr43 as shown in Figure 2b. We also note a small but clear affinity of the HA1(H5N1) to the human receptor tr43 indicating a weak binding after washing the sample with a buffer solution. Relative affinity for Avian to 3’SL over 6’SLN is 70-fold. Apparent association constants (Ka, lM 1) for Avian (Duck/Bavaria/1/77) are 70 and 1 for 3’SL and 6’SLN, respectively. Relative affinity for classical swine (to 6‘SLN over 3‘SL is >3-fold. Apparent association constants (Ka,
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lM 1) for swine (Turkey/MO/1/81) are 8 and 25 for 3’SL and 6’SLN, respectively [24]. Infrared peak fitting is shown in the SI section and is provided below. The absorption was referenced to a platform with only the receptor imbedded lipid bilayer. That is why the DMPC signature [25] is missing from the data. IR data were obtained for partially wet samples. Infrared peak analysis: we fitted the various peaks by GAUSSIAN distributions: Tr40-HA1(H5N1): there exist three peaks at 1500 cm 1: 1442 (CH3), 1635 and 1673 cm 1 (bent vibrations of H2O and C@O). The broad peak at 3400 cm 1 may be fitted with five peaks: 3078 (C H), 3292, 3450 and 3585 (H O H) and 3797 cm 1 (N H). The region between these peaks may be fitted with small and broad two peaks at 1921 (@C) 2454 cm 1 (O H). One small peak may also be identified at 1162 cm 1 (CH2). Tr43-HA1(H1N1): there exist two peaks at 1500 cm 1: 1438 (CH3) and 1641 cm 1 (mostly bent vibrations of H2O). The broad peak at 3400 cm 1 may be fitted with six peaks: 3085 (C H), 3255, 3344, 3467 and 3608 (H O H) and 3856 cm 1 (N H). The region between these peaks may be fitted with small and broad two peaks at 2090 (@C) and 2422 cm 1 (O H). One small peak may also be identified at 1064 cm 1 (CH2). After one hour of drying in air, the bound tr40-HA1(H5N1) and tr43-HA1(H1N1) exhibited somewhat reduced peaks’ amplitude, yet all peak characteristics were intact. Other notable features for both viruses are the down-shift of the 1640 peak by 15 cm 1 which indicates the vibrations of other than just water in the sample (e.g., COO ). The two small peaks at 2848 and 2918 cm 1 after dehydration are attributed to the DMPC and the fact that the reference signal was initially obtained for wet (as opposed to the dehydrated) sample.
Figure 3. (a) DI water droplet on graphite (CA = contact angle) (CAleft = 85°; CAright = 81°). (b) DI water droplet on nickel plate (CAleft = 83°; CAright = 84°). (c) Buffer droplet on nickel plate (CAleft = 22°; CAright = 21°). (d) DI water droplet on graphene-coated nickel screen (CAleft = 56°; CAright = 56°). (e) Buffer solution droplet on graphenecoated nickel screen (CAleft = 39°; CAright = 43°).
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Figure 4. Fluorescence pictures of DMPC films: 0.5% dye-conjugated DMPC was mixed in non-conjugated DMPC. (a) On glass slide. (b) On a few layer graphene, coating a nickel screen. (c) Typical FRAP results for DMPC on glass and (d) for DMPC on screen. The recovery time was 39 s for films on glass substrates and 42.4 s for films on screens. After 80 s, the film on glass recovered to 65% whereas the coated screen recovered to 35% of its respective initial FL values. While the data was collected from the entire yellow marked area of the screen, no significant FL signal change was observed from the graphene-covered metal areas (the dark cross-shape regions in (b)). The area covered by the suspended graphene was 1/3 of the total screen area thus explaining the lower FL intensity value from films on screens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Figure 5. IR absorption spectra of HA1 from either H1N1 or H5N1 on bilayers situated on graphenated screens. The data is expressed as the negative logarithm of transmission, or, log(transmission) and is referenced to the absorption of the receptor imbedded lipid bilayers: (a) Bilayers with tr40(a2–3) receptor. (b) Bilayers with tr43 (a2–6) receptor. Note the weak affinity of tr43 to HA1(avian).
While we are interested in the formation of H-bonds, one could argue that the IR peak are the result of conformal changes in the HA itself [26]. If so, these peaks will be pH dependent. The effect of pH on the IR spectra with pH 5 through pH 7 is shown in Figure 6 (pH 6 data were omitted for clarity; it resembled the other two). Several experiments were conducted and at least two complete sets (on same date by same person) were carried out. The figure
is based on one of these sets. A constant was added to the pH 7 data to better compare the two data. The two major bands at 1640 and 3500 cm 1 imply absorption by bonds between protein and ligand receptors. Anchoring of ligand receptors required the lipid membrane. The HA protein was administered to tri-saccharides, deposited directly on the graphene without the lipid membrane. The complex of
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A. Banerjee et al. / Chemical Physics Letters xxx (2013) xxx–xxx
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ligand and protein was then washed with a buffer solution. The IR signal disappeared after the wash stage (see SI section). We note that the protein cannot attach itself to either the graphene nor to the lipid membrane: binding of the protein is enabled only through ligand receptors, which anchored itself to the lipid membrane through a hydrophobic linker. Further indication to the role of water (or lack of it) was made by replacing the buffer solution with deuterated water, D2O as shown in Figure 7. Every component was prepared with D2O with the exception of HA1 which arrived in a buffer solution. The latter was diluted 25 times with D2O. The bands at 3400 and 1600 cm 1 remained with slight modification; it is expected that the bent vibration of H2O at 1640 cm 1 will be affected by the presence of deuterated water as indeed happened. The additional band at 2500 cm 1 is attributed to D2O. In addition, and unlike HA1(H5N1), two additional peaks appear at 1434 (CH3) and 1199 cm 1 (CH2), respectively for HA1(H1N1). 3. Discussion Signal enhancing platforms allowed us to use low concentration levels. In fact, thick layers (including water) would mask the IR features. The binding process was associated with mainly two IR bands: the Amid I group at 1645 cm 1 and the H–O–H and N–H
in the 3400 cm 1 range. The arrangement accentuated the IR absorption of newly formed H-bonds between protein and ligand near the substrate’s surface without being masked by the rich IR spectra of the protein. In a bit of irony, such approach also masks membrane imperfections since only newly formed bonds, which are close to the substrate’s surface, will be accentuated. (a) The characteristic bands at various pH: Figure 6 present spectra of our bound proteins at pH 5 through pH 7 environments. Molecular structure of HA1 may not be as stable if not combined with HA2 and in principle the HA1 could have gone conformational change. We note that we do not monitor the protein but its bonding to a ligand receptor: the IR spectra as a function of pH remains the same for the three pH cases. (b) The 3000–3800 cm 1 region: The large peak in the 3000– 3800 cm 1 is of interest [27]. All other, water-dissolved films exhibited weak peaks at the 3400 cm 1 region in contrast to very strong signals exhibited when HA1 binds to its related ligand receptor. Upon drying, the IR peaks fainted but did not disappear. This means that membrane disintegration eventually led to signal deterioration. Washing the sample after 3 h of drying in air eliminated the IR signal completely. Destabilization of the HA through drying (and thereby, releasing fusion peptide) cannot account for the data (at least during the first hour after deposition) since one would expect similar IR data for the proteins regardless of the presence of ligand receptors. The fact that unbound proteins did not exhibit any IR signature and the fact that signals were detected for hydrated samples also suggest that the HA has not been destabilized (since the fusion is expected to be triggered by the drying process). The possibility that some or all of the IR peaks are associated with water molecules could be tested by replacing the buffer solution with D2O. The band at 3000–3800 cm 1 decreased a bit and shifted towards the larger frequencies regime for both viruses (Figure 7). The band at 1645 cm 1 substantially decreased for HA1(H1N1) but not for HA1(H5N1): this means that the signal for HA1(H1N1) line had a large contribution from bent vibrations of OH molecules with only a small contribution from Amide I while the reverse is true for HA1(H5N1). The intensity of IR peaks for bound receptors in H2O was substantially larger than for peaks obtained with free H2O, free D2O molecules, a film of only buffer solution, saliva, saccharide only on graphene, or samples treated with D2O. This means that the enhanced IR absorption may be attributed to (a) only water-like molecules trapped between ligand and protein or (b) relatively strong
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Figure 7. (a) Comparison between tr40-HA1(H5N1) in water (buffer solution) and in D2O. The peak at 2500 cm 1 is attributed to D2O. The curve for HA1(H5N1) in D2O was up-shifted for convenience. (b) Comparison between tr43-HA1(H1N1) in water (buffer solution) and in D2O. The peak at 1450 cm 1 has increased in consistence with the effect of D2O. The peaks at 1200 and 2500 cm 1 are attributed to D2O. For D2O at 3000–3800 cm 1, note the small shift to larger frequencies for both samples.
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hydrogen bonds [28]. Hypothesis (a) is not consistent with docking simulations: bound receptors were associated with negative binding energy (for tr43-HA1(H1N1) it was 2.34 kcal/mol and for tr40-HA1(H5N1) it was 1.87 kcal/mol), and very short distanced H-bonds, below 2 A; such short distance cannot support trapped water molecules. Structural studies of hemagglutinin-sugar complexes (pdb id: 3HTO, 3HTP, 3HTQ, and 3HTT, 3LZG, 3 AL4 [29,30]) provide strong evidence to the absence of trapped free water molecules (not involved in hydrogen bonding) and strong H-bonds which are associated with very short distances. Thus, docking simulations provide good qualitative and predictive picture. Based on the strength of the absorption and the response to D2O we conclude that the peaks ought to be attributed to mostly Hbonds. The short-distanced interaction between the ligand and protein and the fact that the ligand receptor did not bind to graphene, effectively places the binding sites of the saccharide at the membrane surface. Note that a bilayer of phospholipid is typically 50 A thick compared with a bond length of less than 3 A. (c) Are our graphene layer(s) hydrophobic? Graphite is hydrophobic. The hydrophobicity of graphene is affected by its substrate [31]. Our graphene layer is depositing copper or nickel mesh (known to be hydrophobic) and is freely suspended over the mesh opening, yet, Figure 6 indicates that it is hydrophilic to the buffer solution used. Our preparation method does not exclude intercalated water molecules within the few layered graphene and the interaction between defect states and the OH group; defect states and carboxylic group advance functionalization of carbon nanotubes (which are but rolled graphene) [32,33]. In addition, our deposition method of lipid bilayers involves vesicles fusion unlike mono lipid layers on graphite [34,35]. These, the FRAP experiments and the lack of adhesion to the suspended graphene by tri-saccharides imply hydrophylic graphene capable of attaching lipid bilayer membranes. (d) Simulations: Docking simulations (see SI section) suggested interaction distance, on the order of 3 A, between unbound protein and receptor. The positive free energy values (+354.72 kcal/ mol for swine flu-avian ligand receptor and +4.93 kcal/mol for avian flu-swine ligand receptor may indicate unstable binding. Yet, we point to the smaller distances between the avian HA1(H5N1) flu and the human receptor tr43 than the corresponding distances between the swine HA1(H1N1) flu and the avian receptor, tr40. This may indicate a weak interaction between the HA1(H5N1) to the human receptor, as alluded to by the very weak IR absorption peaks in Figure 5b. We should caution that docking simulations do not include water molecules and could be justified where the distances between protein and the ligand receptor are very short. Therefore, the issue of water mediated interaction between HA1(H5N1) and the human ligand receptor remains open. The specificity of these receptors has been assessed by others [13]. The anchoring of the receptor to the lipid membrane could, in principle be maintained through the hydrophilic (polar) membrane heads [20,21]. However, in a wet environment, such interaction may be weak since it competes with the surrounding water molecules. We note that the tri-saccharides used have a hydrophobic linker (CH2–CH2–azide tail), originally aimed at anchoring glycans to micro-arrays [18]; therefore, we propose that during the deposition stage, the hydrophobic receptor tail protrudes the surface of the lipid membrane and chemically attaches itself to the hydrophobic tail of the phospholipid. This may explain the robustness of the receptor/membrane against repeated washing. Vigorous washing the substrate with a buffer solution effectively limits the overall coverage of the DMPC to basically two layers. A reference experiment with biotinylated lipid bilayers and streptavidin on SiO2/Si and on graphene demonstrated the
adhesion of the lipid bilayers to both SiO2 and graphene and exhibited IR peaks that matched almost one to one for these two substrates [36]. We may, therefore conclude that lipid bilayers are similarly grown on (hydrophilic) suspended graphene and glass substrates. 4. Conclusion We monitored selective binding between the hemagglutinin area of influenza viruses and their respective receptors. Such extremely thin-film methodology could be employed in detailing the characteristics of hydrogen bonds involved and in developing a database for unique signatures of known strains, therefore, facilitating global surveillance of the viruses. Acknowledgements We wish to acknowledge the Consortium for Functional Glycomics Grant number GM62116 for providing us with the tri-saccharides. We thank David F. Smith, Emory University, School of Medicine and James H. Prestegard, Complex Carbohydrate Research Center, University of Georgia for useful discussions. We also thank Samarth Trivedi of NJIT for taking the SEM pictures. NAB was funded by NIH 5R01AI091985-03. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cplett.2013. 07.010. References [1] H. Barbara, Infrared Spectroscopy: Fundamentals and Applications, Wiley, 2004. [2] Y. Ha, D.J. Stevens, J.J. Skehel, D.C. Wiley, Proc. Natl. Acad. Sci. USA 98 (2001) 11181. [3] J. Stevens et al., Science 312 (2006) 404. [4] R.J. Russell, D.J. Stevens, L.F. Haire, S.J. Gamblin, J.J. Skehel, Glycoconj. J. 23 (2006) 85. [5] J.J. Skehel, D.C. Wiley, Annu. Rev. Biochem. 69 (2000) 531. [6] J. Stevens et al., J. Mol. Biol. 355 (2006) 1143. [7] H.L. Yen et al., J. Virol. 81 (2007) 6890. [8] T.R. Maines et al., Proc. Natl. Acad. Sci. USA 103 (2006) 12121. [9] T. Kuiken et al., Science 312 (2006) 394. [10] K. Shinya et al., Nature 440 (2006) 435. [11] D. van Riel et al., Science 312 (2006) 399. [12] J.M. Nicholls, A.J. Bourne, H. Chen, Y. Guan, J.S. Peiris, Respir. Res. 8 (2007) 73. [13] Aarthi Chandrasekaran et al., Nat. Biotechnol. 26 (2008) 107. [14] M.B. Eisen, S. Sabesan, J.J. Skehel, D.C. Wiley, Virology 232 (1997) 19. [15] S.J. Gamblin et al., Science 303 (2004) (1918) 1838. [16] T. Ito et al., J. Virol. 72 (1998) 7367. [17] CDCrealtimeRTPCRprotocol_20090428. [18] J. Stevens, O. Blixt, J.C. Paulson, I.A. Wilson, Nat. Rev. Microbiol. 4 (2006) 857. [19] Andreas Barth, Biochim. Biophys. Acta 1767 (2007) 1073–1101. [20] John H. Crowe, Lois M. Crowe, John F. Carpenter, Christina Aurell Wistrom, Biochem. J. 242 (1987) 1. [21] Sukit Leekumjorn, Amadeu K. Sum, J. Phys. Chem. B 112 (2008) 10732. [22] A.F. Stalder, G. Kulik, D. Sage, L. Barbieri, P. Hoffmann, Colloids Surf. A 286 (2006) 92. [23] D. Axelrod, D. Koppel, J. Schlessinger, E. Elson, W. Webb, Mobility measurement by analysis of fluorescence photobleaching recovery kinetics, Biophys. J . 16 (1976) 1055, http://dx.doi.org/10.1016/S0006-3495 (76)85755-4. [24] Mikhail Matrosovich et al., J. Virol. 74 (2000) 8502. [25] Andreas O. Hohner, Maria Pamela C. David, Joachim O. Rädler, Biointerphases 5 (2010) 1. [26] Lukas K. Tamm, Suren A. Tatulian, Q. Rev. Biophys. 30 (1997) 365. [27] Teresa L. Tarbuck, Stephanie T. Ota, Geraldine L. Richmond, J. Am. Chem. Soc. 128 (2006) 14519. [28] Shinya Yamada et al., Nature 444 (2006) 378, http://dx.doi.org/10.1038/ nature05264. [29] Tianwei Lin et al., Virology 392 (1) (2009) 73. [30] Rui Xu, Damian C. Ekiert, Jens C. Krause, James E. Crowe Jr., Ian A. Wilson, Science 328 (2010) (2009) 357.
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Monitoring bound HA1(H1N1) and HA1(H5N1) on freely suspended graphene over plasmonic platforms with infrared spectroscopy Amrita Banerjee a, Sumit Chakraborty b, Nihal Altan-Bonnet c,1, Haim Grebel a,⇑ a
The Electronic Imaging Center at NJIT, Newark, NJ 07102, United States Department of Microbiology & Immunology, Weill Cornell Medical College, New York City, NY 10025, United States c Department of Biological Sciences, Rutgers University, Newark, NJ 07102, United States b
a r t i c l e
i n f o
Article history: Received 22 May 2013 In final form 5 July 2013 Available online xxxx
a b s t r a c t Infrared (IR) spectroscopy provides fingerprinting of the energy and orientation of molecular bonds. The IR signals are generally weak and require amplification. Here we present a new plasmonic platform, made of freely suspended graphene, which was coating periodic metal structures. Only monolayer thick films were needed for a fast signal recording. We demonstrated unique IR absorption signals of bound proteins: these were the hemagglutinin area (HA1) of swine influenza (H1N1) and the avian influenza (H5N1) viruses bound to their respective tri-saccharides ligand receptors. The simplicity and sensitivity of such approach may find applications in fast monitoring of binding events. Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction Infrared (IR) spectroscopy is an important spectroscopic tool to detect molecular absorption in the 1–100 lm wavelength range [1]. The signals are relatively weak and techniques have been devised to amplify it. For example, in Attenuation Total Reflection (ATR) the sample is depositing a transparent slab. A long interaction length between the interrogating electromagnetic wave and molecules is achieved through multiple internal reflections. However, upon each reflection a different set of molecules is assessed along the slab. Here we propose another amplification approach while keeping the thickness of the molecular film to monolayer dimensions. In our approach, the IR beam irradiates the sample depositing a graphene-coated metal mesh structure. The periodic metal structure scatters the resultant surface modes back and forth, thus increasing the interaction between the IR beam and the same set of molecules. The platform fits existing Fourier Transform IR (FTIR) spectrometers in transmission mode without modification. The technique is demonstrated with bound hemagglutin of influenza viruses to their respective tri-saccharides ligand receptors. Binding the hemagglutinin area of influenza virus (HA) to sialylated glycans is an important part of virus infection studies [2–6]. Besides interests in its devastating effects on birds and fowls, avian flu has been also researched for potential risks to humans [7–13]. Crystal structures of HA from H1 (swine) and H5 (avian) and their ⇑ Corresponding author. E-mail address: [email protected] (H. Grebel). Current address: Laboratory of Host-Pathogen Dynamics, Cell Biology and Physiology Center, National Heart Lung and Blood Institute, NIH, Bethesda MD, United, States. 1
complexes with oligosaccharides provided useful information [14–16]. Real-Time PCR protocol has been issued by the Center of Decease Control, expediting the detection of swine flu [17]. Yet, despite advances in key micro-array technologies [18], and facing global pandemic prospects, a need arises for a simple detection method, augmented by drug development and implemented in an environment closely mimicking the virus expression location. IR spectroscopy [19] is a detection method widely applied to the study of proteins. Proteins share many IR absorption lines thus, obscuring their identification in complex environments. Yet, protein binding involve hydrogen bonds, thus in principle, we may follow the binding mechanism instead. Our signal amplifying platforms enabled us to use very small concentration levels, e.g., the starting molar concentration of HA1 in our stock solution was 1 lM spread over an area, larger than 1 cm2. Cellular plasma membranes are essential structural elements of eukaryotic cells, which define the cells’ outer surface. The primary structural elements of a plasma membrane are amphiphilic phospholipid molecules, self-assembled into sheet-like structures, which we deposited onto bio-compatible, micron-size, graphenecoated periodic hole-array. In the experiments, tri-saccharide ligand receptors were incorporated with lipid membranes via mixing. The ligand receptors have a hydrophobic linker which could aid its attachment to the hydrophobic part of the membrane. Saccharides ligands stabilize the lipid bilayers by dispersing water molecules around their hydrophylic heads [20,21]. By incorporating sialosaccharides with the membrane we are not only immobilizing the receptors but also attempting to mimic the natural binding process between protein and ligand. Graphite is hydrophobic while the hydrophobicity of graphene is unclear. Thus, the stability of the membrane-ligand receptor complex on graphene-
0009-2614/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.cplett.2013.07.010
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coated substrates is of interest. Detection and understanding of the interactions between analytes and lipid membranes is an essential task for drug targeting. Specifically, one of the fundamental properties of a drug or drug-like molecule is its binding affinity to a target. Strong H-bond mediated interactions are, therefore a very desirable property (see for example, Lipinski et al., in Adv. Drug Delivery Rev. 46 (2001) 3–26). The methodology described here outlines a simple approach to monitor H-bond formation between a small molecule and a large biomolecule. Figure 1. Schematics of the platform layout. The binding between receptor and protein is at the lipid membrane surface but the receptor is most probably anchored to the hydrophobic tail of the phospholipid.
2. Experimental results Experimental methods are described at the end of the SI section. The platform is presented in Figure 1. Binding of HA1 proteins to the respective ligand receptors are simulated in Figure 2. The
Figure 2. (a) HA1(H5N1) bound to tr40: key interaction distances between ligand and protein are highlighted blue (left). Bound receptor (right). (b) HA1(H1N1) bound to tr43: key interaction distances between ligand and protein are highlighted blue (left). Bound receptor (right). (see Fig. S5 for the entire data). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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hydrophobic (or lack of) nature of the suspended graphene layer(s) is assessed by its contact angle [22] with di-ionized (DI) water and buffer solutions (Figure 3). Several fitting methods differed somewhat in their final results but provided with similar trend: contact angles with buffer solution droplets on graphite and nickel plates were substantially smaller (20°) than those with DI water (>80°). Contact angles for highly ordered pyrolitic graphite (HOPG) substrates were 5° larger than graphene coated screens. DMPC in buffer solution exhibited larger contact angles than the buffer solution alone (by approximately 15°): this was true for all samples including HOPG substrates for which an angle of 59° was assessed. The range of contact angles obtained with a buffer solution alludes to the hydrophilic nature of our graphene coated surfaces. Fluorescence recovery after photobleaching (FRAP) experiments were conducted [23] to determine the diffusion time of dye within lipid bilayers on glass and on our suspended graphene layers. The recovery life-time appeared to be similar for both graphene-coated screens and reference glass substrates (Figure 4). IR absorption spectra of bound ligand receptors exhibited new IR peaks. In Figure 5a we show the IR absorption spectra of HA1 of H1N1 and H5N1 on the avian receptor tr40. Clearly, the HA1(H5N1) interacts with the tr40 while the HA1(H1N1) is not: newly formed IR absorption bands at 1400–1650 and 3000– 3800 cm 1 appear for HA1(H5N1). IR peaks appear only upon coupling between HA1(H1N1) and the human receptor tr43 as shown in Figure 2b. We also note a small but clear affinity of the HA1(H5N1) to the human receptor tr43 indicating a weak binding after washing the sample with a buffer solution. Relative affinity for Avian to 3’SL over 6’SLN is 70-fold. Apparent association constants (Ka, lM 1) for Avian (Duck/Bavaria/1/77) are 70 and 1 for 3’SL and 6’SLN, respectively. Relative affinity for classical swine (to 6‘SLN over 3‘SL is >3-fold. Apparent association constants (Ka,
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lM 1) for swine (Turkey/MO/1/81) are 8 and 25 for 3’SL and 6’SLN, respectively [24]. Infrared peak fitting is shown in the SI section and is provided below. The absorption was referenced to a platform with only the receptor imbedded lipid bilayer. That is why the DMPC signature [25] is missing from the data. IR data were obtained for partially wet samples. Infrared peak analysis: we fitted the various peaks by GAUSSIAN distributions: Tr40-HA1(H5N1): there exist three peaks at 1500 cm 1: 1442 (CH3), 1635 and 1673 cm 1 (bent vibrations of H2O and C@O). The broad peak at 3400 cm 1 may be fitted with five peaks: 3078 (C H), 3292, 3450 and 3585 (H O H) and 3797 cm 1 (N H). The region between these peaks may be fitted with small and broad two peaks at 1921 (@C) 2454 cm 1 (O H). One small peak may also be identified at 1162 cm 1 (CH2). Tr43-HA1(H1N1): there exist two peaks at 1500 cm 1: 1438 (CH3) and 1641 cm 1 (mostly bent vibrations of H2O). The broad peak at 3400 cm 1 may be fitted with six peaks: 3085 (C H), 3255, 3344, 3467 and 3608 (H O H) and 3856 cm 1 (N H). The region between these peaks may be fitted with small and broad two peaks at 2090 (@C) and 2422 cm 1 (O H). One small peak may also be identified at 1064 cm 1 (CH2). After one hour of drying in air, the bound tr40-HA1(H5N1) and tr43-HA1(H1N1) exhibited somewhat reduced peaks’ amplitude, yet all peak characteristics were intact. Other notable features for both viruses are the down-shift of the 1640 peak by 15 cm 1 which indicates the vibrations of other than just water in the sample (e.g., COO ). The two small peaks at 2848 and 2918 cm 1 after dehydration are attributed to the DMPC and the fact that the reference signal was initially obtained for wet (as opposed to the dehydrated) sample.
Figure 3. (a) DI water droplet on graphite (CA = contact angle) (CAleft = 85°; CAright = 81°). (b) DI water droplet on nickel plate (CAleft = 83°; CAright = 84°). (c) Buffer droplet on nickel plate (CAleft = 22°; CAright = 21°). (d) DI water droplet on graphene-coated nickel screen (CAleft = 56°; CAright = 56°). (e) Buffer solution droplet on graphenecoated nickel screen (CAleft = 39°; CAright = 43°).
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Figure 4. Fluorescence pictures of DMPC films: 0.5% dye-conjugated DMPC was mixed in non-conjugated DMPC. (a) On glass slide. (b) On a few layer graphene, coating a nickel screen. (c) Typical FRAP results for DMPC on glass and (d) for DMPC on screen. The recovery time was 39 s for films on glass substrates and 42.4 s for films on screens. After 80 s, the film on glass recovered to 65% whereas the coated screen recovered to 35% of its respective initial FL values. While the data was collected from the entire yellow marked area of the screen, no significant FL signal change was observed from the graphene-covered metal areas (the dark cross-shape regions in (b)). The area covered by the suspended graphene was 1/3 of the total screen area thus explaining the lower FL intensity value from films on screens. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Figure 5. IR absorption spectra of HA1 from either H1N1 or H5N1 on bilayers situated on graphenated screens. The data is expressed as the negative logarithm of transmission, or, log(transmission) and is referenced to the absorption of the receptor imbedded lipid bilayers: (a) Bilayers with tr40(a2–3) receptor. (b) Bilayers with tr43 (a2–6) receptor. Note the weak affinity of tr43 to HA1(avian).
While we are interested in the formation of H-bonds, one could argue that the IR peak are the result of conformal changes in the HA itself [26]. If so, these peaks will be pH dependent. The effect of pH on the IR spectra with pH 5 through pH 7 is shown in Figure 6 (pH 6 data were omitted for clarity; it resembled the other two). Several experiments were conducted and at least two complete sets (on same date by same person) were carried out. The figure
is based on one of these sets. A constant was added to the pH 7 data to better compare the two data. The two major bands at 1640 and 3500 cm 1 imply absorption by bonds between protein and ligand receptors. Anchoring of ligand receptors required the lipid membrane. The HA protein was administered to tri-saccharides, deposited directly on the graphene without the lipid membrane. The complex of
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ligand and protein was then washed with a buffer solution. The IR signal disappeared after the wash stage (see SI section). We note that the protein cannot attach itself to either the graphene nor to the lipid membrane: binding of the protein is enabled only through ligand receptors, which anchored itself to the lipid membrane through a hydrophobic linker. Further indication to the role of water (or lack of it) was made by replacing the buffer solution with deuterated water, D2O as shown in Figure 7. Every component was prepared with D2O with the exception of HA1 which arrived in a buffer solution. The latter was diluted 25 times with D2O. The bands at 3400 and 1600 cm 1 remained with slight modification; it is expected that the bent vibration of H2O at 1640 cm 1 will be affected by the presence of deuterated water as indeed happened. The additional band at 2500 cm 1 is attributed to D2O. In addition, and unlike HA1(H5N1), two additional peaks appear at 1434 (CH3) and 1199 cm 1 (CH2), respectively for HA1(H1N1). 3. Discussion Signal enhancing platforms allowed us to use low concentration levels. In fact, thick layers (including water) would mask the IR features. The binding process was associated with mainly two IR bands: the Amid I group at 1645 cm 1 and the H–O–H and N–H
in the 3400 cm 1 range. The arrangement accentuated the IR absorption of newly formed H-bonds between protein and ligand near the substrate’s surface without being masked by the rich IR spectra of the protein. In a bit of irony, such approach also masks membrane imperfections since only newly formed bonds, which are close to the substrate’s surface, will be accentuated. (a) The characteristic bands at various pH: Figure 6 present spectra of our bound proteins at pH 5 through pH 7 environments. Molecular structure of HA1 may not be as stable if not combined with HA2 and in principle the HA1 could have gone conformational change. We note that we do not monitor the protein but its bonding to a ligand receptor: the IR spectra as a function of pH remains the same for the three pH cases. (b) The 3000–3800 cm 1 region: The large peak in the 3000– 3800 cm 1 is of interest [27]. All other, water-dissolved films exhibited weak peaks at the 3400 cm 1 region in contrast to very strong signals exhibited when HA1 binds to its related ligand receptor. Upon drying, the IR peaks fainted but did not disappear. This means that membrane disintegration eventually led to signal deterioration. Washing the sample after 3 h of drying in air eliminated the IR signal completely. Destabilization of the HA through drying (and thereby, releasing fusion peptide) cannot account for the data (at least during the first hour after deposition) since one would expect similar IR data for the proteins regardless of the presence of ligand receptors. The fact that unbound proteins did not exhibit any IR signature and the fact that signals were detected for hydrated samples also suggest that the HA has not been destabilized (since the fusion is expected to be triggered by the drying process). The possibility that some or all of the IR peaks are associated with water molecules could be tested by replacing the buffer solution with D2O. The band at 3000–3800 cm 1 decreased a bit and shifted towards the larger frequencies regime for both viruses (Figure 7). The band at 1645 cm 1 substantially decreased for HA1(H1N1) but not for HA1(H5N1): this means that the signal for HA1(H1N1) line had a large contribution from bent vibrations of OH molecules with only a small contribution from Amide I while the reverse is true for HA1(H5N1). The intensity of IR peaks for bound receptors in H2O was substantially larger than for peaks obtained with free H2O, free D2O molecules, a film of only buffer solution, saliva, saccharide only on graphene, or samples treated with D2O. This means that the enhanced IR absorption may be attributed to (a) only water-like molecules trapped between ligand and protein or (b) relatively strong
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Figure 7. (a) Comparison between tr40-HA1(H5N1) in water (buffer solution) and in D2O. The peak at 2500 cm 1 is attributed to D2O. The curve for HA1(H5N1) in D2O was up-shifted for convenience. (b) Comparison between tr43-HA1(H1N1) in water (buffer solution) and in D2O. The peak at 1450 cm 1 has increased in consistence with the effect of D2O. The peaks at 1200 and 2500 cm 1 are attributed to D2O. For D2O at 3000–3800 cm 1, note the small shift to larger frequencies for both samples.
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hydrogen bonds [28]. Hypothesis (a) is not consistent with docking simulations: bound receptors were associated with negative binding energy (for tr43-HA1(H1N1) it was 2.34 kcal/mol and for tr40-HA1(H5N1) it was 1.87 kcal/mol), and very short distanced H-bonds, below 2 A; such short distance cannot support trapped water molecules. Structural studies of hemagglutinin-sugar complexes (pdb id: 3HTO, 3HTP, 3HTQ, and 3HTT, 3LZG, 3 AL4 [29,30]) provide strong evidence to the absence of trapped free water molecules (not involved in hydrogen bonding) and strong H-bonds which are associated with very short distances. Thus, docking simulations provide good qualitative and predictive picture. Based on the strength of the absorption and the response to D2O we conclude that the peaks ought to be attributed to mostly Hbonds. The short-distanced interaction between the ligand and protein and the fact that the ligand receptor did not bind to graphene, effectively places the binding sites of the saccharide at the membrane surface. Note that a bilayer of phospholipid is typically 50 A thick compared with a bond length of less than 3 A. (c) Are our graphene layer(s) hydrophobic? Graphite is hydrophobic. The hydrophobicity of graphene is affected by its substrate [31]. Our graphene layer is depositing copper or nickel mesh (known to be hydrophobic) and is freely suspended over the mesh opening, yet, Figure 6 indicates that it is hydrophilic to the buffer solution used. Our preparation method does not exclude intercalated water molecules within the few layered graphene and the interaction between defect states and the OH group; defect states and carboxylic group advance functionalization of carbon nanotubes (which are but rolled graphene) [32,33]. In addition, our deposition method of lipid bilayers involves vesicles fusion unlike mono lipid layers on graphite [34,35]. These, the FRAP experiments and the lack of adhesion to the suspended graphene by tri-saccharides imply hydrophylic graphene capable of attaching lipid bilayer membranes. (d) Simulations: Docking simulations (see SI section) suggested interaction distance, on the order of 3 A, between unbound protein and receptor. The positive free energy values (+354.72 kcal/ mol for swine flu-avian ligand receptor and +4.93 kcal/mol for avian flu-swine ligand receptor may indicate unstable binding. Yet, we point to the smaller distances between the avian HA1(H5N1) flu and the human receptor tr43 than the corresponding distances between the swine HA1(H1N1) flu and the avian receptor, tr40. This may indicate a weak interaction between the HA1(H5N1) to the human receptor, as alluded to by the very weak IR absorption peaks in Figure 5b. We should caution that docking simulations do not include water molecules and could be justified where the distances between protein and the ligand receptor are very short. Therefore, the issue of water mediated interaction between HA1(H5N1) and the human ligand receptor remains open. The specificity of these receptors has been assessed by others [13]. The anchoring of the receptor to the lipid membrane could, in principle be maintained through the hydrophilic (polar) membrane heads [20,21]. However, in a wet environment, such interaction may be weak since it competes with the surrounding water molecules. We note that the tri-saccharides used have a hydrophobic linker (CH2–CH2–azide tail), originally aimed at anchoring glycans to micro-arrays [18]; therefore, we propose that during the deposition stage, the hydrophobic receptor tail protrudes the surface of the lipid membrane and chemically attaches itself to the hydrophobic tail of the phospholipid. This may explain the robustness of the receptor/membrane against repeated washing. Vigorous washing the substrate with a buffer solution effectively limits the overall coverage of the DMPC to basically two layers. A reference experiment with biotinylated lipid bilayers and streptavidin on SiO2/Si and on graphene demonstrated the
adhesion of the lipid bilayers to both SiO2 and graphene and exhibited IR peaks that matched almost one to one for these two substrates [36]. We may, therefore conclude that lipid bilayers are similarly grown on (hydrophilic) suspended graphene and glass substrates. 4. Conclusion We monitored selective binding between the hemagglutinin area of influenza viruses and their respective receptors. Such extremely thin-film methodology could be employed in detailing the characteristics of hydrogen bonds involved and in developing a database for unique signatures of known strains, therefore, facilitating global surveillance of the viruses. Acknowledgements We wish to acknowledge the Consortium for Functional Glycomics Grant number GM62116 for providing us with the tri-saccharides. We thank David F. Smith, Emory University, School of Medicine and James H. Prestegard, Complex Carbohydrate Research Center, University of Georgia for useful discussions. We also thank Samarth Trivedi of NJIT for taking the SEM pictures. NAB was funded by NIH 5R01AI091985-03. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.cplett.2013. 07.010. References [1] H. Barbara, Infrared Spectroscopy: Fundamentals and Applications, Wiley, 2004. [2] Y. Ha, D.J. Stevens, J.J. Skehel, D.C. Wiley, Proc. Natl. Acad. Sci. USA 98 (2001) 11181. [3] J. Stevens et al., Science 312 (2006) 404. [4] R.J. Russell, D.J. Stevens, L.F. Haire, S.J. Gamblin, J.J. Skehel, Glycoconj. J. 23 (2006) 85. [5] J.J. Skehel, D.C. Wiley, Annu. Rev. Biochem. 69 (2000) 531. [6] J. Stevens et al., J. Mol. Biol. 355 (2006) 1143. [7] H.L. Yen et al., J. Virol. 81 (2007) 6890. [8] T.R. Maines et al., Proc. Natl. Acad. Sci. USA 103 (2006) 12121. [9] T. Kuiken et al., Science 312 (2006) 394. [10] K. Shinya et al., Nature 440 (2006) 435. [11] D. van Riel et al., Science 312 (2006) 399. [12] J.M. Nicholls, A.J. Bourne, H. Chen, Y. Guan, J.S. Peiris, Respir. Res. 8 (2007) 73. [13] Aarthi Chandrasekaran et al., Nat. Biotechnol. 26 (2008) 107. [14] M.B. Eisen, S. Sabesan, J.J. Skehel, D.C. Wiley, Virology 232 (1997) 19. [15] S.J. Gamblin et al., Science 303 (2004) (1918) 1838. [16] T. Ito et al., J. Virol. 72 (1998) 7367. [17] CDCrealtimeRTPCRprotocol_20090428. [18] J. Stevens, O. Blixt, J.C. Paulson, I.A. Wilson, Nat. Rev. Microbiol. 4 (2006) 857. [19] Andreas Barth, Biochim. Biophys. Acta 1767 (2007) 1073–1101. [20] John H. Crowe, Lois M. Crowe, John F. Carpenter, Christina Aurell Wistrom, Biochem. J. 242 (1987) 1. [21] Sukit Leekumjorn, Amadeu K. Sum, J. Phys. Chem. B 112 (2008) 10732. [22] A.F. Stalder, G. Kulik, D. Sage, L. Barbieri, P. Hoffmann, Colloids Surf. A 286 (2006) 92. [23] D. Axelrod, D. Koppel, J. Schlessinger, E. Elson, W. Webb, Mobility measurement by analysis of fluorescence photobleaching recovery kinetics, Biophys. J . 16 (1976) 1055, http://dx.doi.org/10.1016/S0006-3495 (76)85755-4. [24] Mikhail Matrosovich et al., J. Virol. 74 (2000) 8502. [25] Andreas O. Hohner, Maria Pamela C. David, Joachim O. Rädler, Biointerphases 5 (2010) 1. [26] Lukas K. Tamm, Suren A. Tatulian, Q. Rev. Biophys. 30 (1997) 365. [27] Teresa L. Tarbuck, Stephanie T. Ota, Geraldine L. Richmond, J. Am. Chem. Soc. 128 (2006) 14519. [28] Shinya Yamada et al., Nature 444 (2006) 378, http://dx.doi.org/10.1038/ nature05264. [29] Tianwei Lin et al., Virology 392 (1) (2009) 73. [30] Rui Xu, Damian C. Ekiert, Jens C. Krause, James E. Crowe Jr., Ian A. Wilson, Science 328 (2010) (2009) 357.
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