Ionic Strength, Surface Charge, and Packing Density Effects on the

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Ionic strength, surface charge and packing density effects on the properties of peptide self-assembled monolayers Norman Robert Leo, Juan Liu, Ian Archbold, Yongan Tang, and Xiangqun Zeng Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b04038 • Publication Date (Web): 30 Jan 2017 Downloaded from http://pubs.acs.org on February 10, 2017

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Leo et. al 1

Ionic strength, surface charge and packing density effects on the properties of peptide self-assembled monolayers Norman Leo, Juan Liu, Ian Archbold, Yongan Tang, Xiangqun Zeng* Department of Chemistry, Oakland University, Rochester, MI 48309

* To whom correspondence should be addressed.

Tel: 248-370-2881 Fax: 248-370-2321 e-mail: [email protected]

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Leo et. al 2 Abstract The various environmental parameters of packing density, ionic strength, and solution charge were examined for their effects on the properties of the immobilized peptide mimotope CH19 (CGSGSGSQLGPYELWELSH) that binds with the therapeutic antibody Trastuzumab (Herceptin®) on a gold substrate. The immobilization of CH19 onto gold was examined with Quartz Crystal Microbalance (QCM). The QCM data showed the presence of intermolecular interactions resulting in the increase of viscoelastic properties of the peptide self-assembled monolayer (SAM). The CH19 SAM was diluted with CS7 (CGSGSGS) to decrease the packing density as CH19/CS7.The packing density and ionic strength parameters were evaluated by atomic force microscopy (AFM), ellipsometry, and QCM. AFM and ellipsometry showed a distinct conformational difference between CH19 and CH19/CS7, indicating a relationship between packing density and conformational state of the immobilized peptide. The CH19 SAM thickness was 40 Angstrom with a rough topology, while the CH19/CS7 SAM thickness was 20 Angstrom with a smooth topology. The affinity studies showed that the affinity of CH19 and CH19/CS7 to Trastuzumab were both on the order of 107 M-1 in undiluted PBS buffer, while the dilution of the buffer by 1000x increased both SAMs affinities to Trastuzumab to the order of 1015 M-2 and changed the binding behavior from non-cooperative to cooperative binding. This indicated that ionic strength had a more pronounced effect on binding properties of the CH19 SAM than packing density. Electrochemical impedance spectroscopy (EIS) was conducted on the CH19/CS7 SAM, which showed an increase in impedance after each EIS measurement cycle. Cyclic voltammetry on the CH19/CS7 SAM decreased impedance to near initial values. The impact of the packing density, buffer ionic strength and local charge perturbation of the peptide SAM properties were interpreted based on the titratable sites in CH19 that could participate in the proton transfer and water equilibrium.


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Leo et. al 3 INTRODUCTION In recent decades, biosensor technology has advanced significantly. However, few of the current biosensors can satisfy the requirements of clinical applications in terms of accuracy, cost, portability, and accessibility. Peptide based biosensors which utilize antibody mimic peptides as recognition elements to detect minute changes, as they bind with analytes, have shown great potential in clinical detection technology. In comparison with widely used biorecognition elements such as antibody, aptamer, enzyme, nucleic acid, protein receptor etc., peptides have many advantages making them ideal candidates for biosensor development. Peptides can provide excellent affinity and specificity of an antibody. They are small, significantly decreasing their susceptibility to proteases and non-specific binding/trapping of antigen. They can be massproduced through standard solid-phase synthesis protocols at low cost. Of the class of peptides exist mimetic peptides that emulate the active sites of proteins which are called mimotopes.1 Mimotopes can be as little as 3 amino acids long, but can exhibit very high affinity constants. Mimotopes have high application value in peptide libraries for determination of structural motifs, and drug development. Mimotopes can be synthesized in lieu of monoclonal antibodies purified from cells which allows for the development of low cost, high affinity protein analogs. In most biosensor applications, the mimotope is immobilized onto a surface such as in enzyme-linked immunosorbent assay (ELISA)2, or techniques that utilize SAMs3. However, there is a current lack of principles to rationally design mimotopes that retain their biological activity when immobilized. When a mimotope is immobilized on a solid surface, its affinity to a target protein may change due to the loss of freedom caused by the peptidesurface interaction. Also, the surface adsorbed mimotopes likely experience new interactions with neighboring peptides that are not normally observed in solvated conditions, leading to


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Leo et. al 4 different peptide conformations. In summary, the three major factors influencing peptide properties on a surface include: (1) peptide-surface interactions (related to the substrate surface property and sequences of mimotope and peptide linker), (2) peptide-peptide interactions, and (3) environment (e.g., ionic strength of the buffer). All these factors can affect peptide structure on the surface, which can affect the binding affinity of the peptide to its target molecule Due to the near-infinite combinations of amino acids and the high concentration of solvents compared to peptide, the structure and conformation of the immobilized small peptides, especially those of mimotopes, have been difficult to understand. Computational simulations of self-assembled monolayers (SAM) have been used to study immobilized peptides.4 Our lab has provided an initial experimental framework for understanding immobilized peptide monolayers through a titratable site perspective. The amino acid residues of peptides that are capable in varying in protonation states are called titratable sites5. Titratable sites are very important to proteins as they help define shape and function.6-8 Latour et al. has demonstrated using a semi-empirical approach that the charge of the titratable residues can affect binding behavior of proteins to the peptide SAM.9 Furthermore, peptide interactions can be systematically influenced by varying protonation states of amino acids of the peptide chain depending on environmental.10 Two peptide mimotope modified gold Quartz Crystal Microbalance (QCM) sensors were characterized for the detection of Tratuzumab11 and Rituximab12 respectively. These investigations utilized peptide mimotope SAMs on a gold surface of a QCM electrode using a cysteine containing peptide linker. In our Trastuzumab studies, we utilized mimotope sequences of QLGPYELWELSH and GPYELWELSH with linkers of GSGSGS, R, and RGRGRG derived from the work of Shou et al’s with phage display libraries.13 The sequence of CGSGSGSQLGPYELWELSH, which we designated as CH19 was


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Leo et. al 5 found to have the highest affinity to Trastuzumab. Using CH19, we developed a biosensor for Trastuzumab detection in human serum that was comparable to existing ELISA Trastuzumab detection methods.14 Reflecting on our Trastuzumab and CH19 work, we postulated that the mimotope SAMs could be manipulated by altering the pKa’s of the titratable sites through the addition of electron donating/withdrawing groups. The different linkers used here were distinct in their shape and charge. Our GSGSGS was an aliphatic linker whereas our RGRGRG was a positively charged linker. The mimotope sequence contained three titratable sites which were capable of being influenced by the presence of different linkers. In this study, we hypothesized that we may be able to influence titratable sites by environmental parameters, which span from the substrates to solution properties. We focused on the latter. The solution parameters that influence the protonation of the titratable sites include pH, ionic strength, charge, and packing density. pH is an obvious parameter, but most biological systems occur under a buffered pH and a change in pH would have deleterious effects on the protein as well. The ionic strength represented as buffer concentration has been shown to have a pronounce effect on proper functioning of proteins.15 Electric fields have been shown to greatly influence the active site and catalytic properties of proteins.16, 17 Packing density, or how many mimotopes are packed together on a surface, may also have an effect on titratable sites.18 To understand these relationships, our CH19 peptide mimotope was revisited. In this study, the environmental parameters of ionic strength, packing density, and surface charge were altered to gauge the effects on titratable sites and peptide affinity to Trastuzumab such as in Scheme 1. The packing density of the CH19 SAM was altered by diluting the surface with the linker component of CH19, i.e. CS7, which consists of the sequence CGSGSGS. The resulting SAMs were characterized by surface techniques. In addition, an Electrochemical Impedance


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Leo et. al 6 Spectroscopy (EIS) analysis was performed on the SAM of CH19/CS7 to see how charge perturbation influences SAMs. This combined approach of QCM, surface characterization, and EIS could reveal fundamental insight on peptide dynamics.


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Leo et. al 7 MATERIALS AND METHODS Chemicals and Materials: Peptides, designated as CH19 (sequence: CGSGSGSQLGPYELWELSH, purity >95.84%, MW 2007.14) and CS7 (sequence: CGSGSGS, purity 95.99%, MW 553.54)), were chemically synthesized by Bio. Basic, Inc. (Ontario, Canada) and received in a lyophilized condition. The sequence and quality of both peptides were confirmed and assessed by matrixassisted laser desorption/ionization (MALDI) mass spectrometry analysis. The purity was determined by high performance liquid chromatography (HPLC). Therapeutic mAbs Trastuzumab was provided by Beaumont Hospital, Royal Oak, Michigan. Gold wire (0.025mm diameter, annealed, 99.95%), silver conductive adhesive paste and E-120HP hysol epoxy adhesive were obtained from VWR (Radnor, PA). Corning 8161 patch clamp glass capillaries (LB16, 1.50 mm outer diameter, 1.10 mm inner diameter, wall thickness 0.20mm, length 100mm) and tungsten rods (0.010 × 3.000 in.) were acquired from Warner Instrument (Hamden, CT) and AM Systems, Inc. (Sequim, WA) respectively. Microcut/Carbimet discs (240 grit, 600 grit and 1200 grit), microcloth, 1.00 µm and 0.05 µm alumina micropolish powder for polishing electrodes were purchased from Buehler (Lake Bluff, IL). Phosphate Buffer Saline (PBS, 1X, pH 7.4) was purchased from Thermo Fisher Scientific (Waltham, MA). All other chemicals are used as received.

Sensor Fabrication and Interrogation 25 µm gold electrodes (details in the Supporting Information (1)) were cleaned by hand polishing with 1.00 and 0.05 µm alumina slurry on microcloth for 2 min. The electrodes were sonicated in ethanol and then in H2O. The gold electrodes were further cleaned by cycling the


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Leo et. al 8 potential from -0.35V to 1.5V in 0.05M sulfuric acid. The cleaned gold electrodes were incubated with 1mM CH19 peptide solution overnight (~26h) at 4oC to form the CH19 SAM. The CH19/CS7 SAM was formed by immersing the CH19 functionalized electrode into 1.8 mM CS7 solution overnight (~15h) at 4oC. Cyclic voltammetry in 1 mM 1:1 K3Fe(CN)6: K4Fe(CN)6 before and after peptide surface modification was used to characterize surface functionalization. Ferri-ferro cyanides were only used for the characterization of the peptide SAMs and the experimental time was about 1-2 minutes. The SAMs were subsequently rinsed off with DI water to remove these redox residues. The impedance of the fabricated sensor in PBS (diluted and undiluted) was monitored using a three-electrode system, which utilized a 25µm gold electrode as the working electrode (WE), platinum wire as the counter electrode (CE) and Ag/AgCl wire as the reference electrode (RE). All experiments were carried out using Gamry Multichannel Potentiostat (Gamry Instruments). EIS was conducted on the peptide SAM at 0.2Hz, 0V DC, 5mV AC until a steady state signal was reached.

Gold quartz crystal electrode and QCM measurement The sensor was fabricated using an AT cut (35˚ cut from the Z-axis) gold QCM electrode with 1000-Å of gold onto ∼0.23 cm2 geometric area (International Crystal Co.). Before surface functionalization, the gold QCM electrode was cleaned thrice with 1:1 (v/v) mixture of concentrated nitric acid and sulfuric acid, rinsed with copious amounts of ethanol and water in series, then dried in nitrogen. The cleaned gold QCM electrode was mounted into a Kel-F cell sealed by two viton O-rings. After surface functionalization, the obtained gold coated quartz wafer electrode was washed with PBS three times. The QCM device was filled with 1.2mL PBS and placed into a Faraday cage to reduce noise from environment. Network/Spectrum/Impedance


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Leo et. al 9 Analyzer (Agilent 4395A) was used for monitoring the frequency change and the damping resistance change due to the binding of the analyte (Trastuzumab) with the peptide SAMs.

Atomic Force Microscopy (AFM) and ellipsometry Atomic Force Microscopy (AFM) and ellipsometry were used to characterize the CH19 and CH19/CS7 SAMs. AFM images using the AC mode were obtained from a PicoPlus Atomic Force Microscope (Agilent Technologies, CA) by using Au coated silicon probes of resonant frequency 75 kHz and spring constant 3.5 N/m. The Au surface was subjected to electrochemical polishing followed by flame annealing and cooling in nitrogen to obtain a clean surface. A Gaertner Single Angle Stokes Ellipsometer was used to obtain the thicknesses of the CH19 and CH19/CS7 monolayer films.

RESULTS Self-assembled Monolayers (SAM) formation SAMs of peptide and alkanethiols have some similarities.19 While the structure of alkanethiol SAMs is primarily formed and stabilized by covalent bonds, van der Waals forces, and hydrophobic interactions; the molecular interactions in peptide SAMs also include salt bridging, pi-cation stacking, hydrophobicity, and hydrophilicity allowing to stabilize more complex structures.20 The interplay among intramolecular, intermolecular, and peptide-protein interactions provides the potential for a wide range of structures, which makes peptide SAMs a potentially useful platform to study how titratable sites influence binding affinities. Thus, we first characterized the CH19 peptide SAM immobilization process. Immobilization ensures desolvation of the peptide, peptide-peptide interactions, and peptide folding. By immobilization


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Leo et. al 10 into a SAM via the thiol functional group in cysteine of CH19, we can use the available alkanethiol literature as a fundamental discussion point of peptide folding and connect titratable sites to peptide folding when environmental parameters are altered. The formation of a peptide SAM is thought to be like that of alkanethiols where there exists extensive research on the properties and the mechanisms of alkanethiol SAM formation. Based on the kinetics of alkanethiol SAMs, it is speculated that there are three phases to peptide SAM formation: nucleation, growth, and folding. Peptides offer more complicated interactions than that of alkanethiols with the presence of different functional groups yielding to salt bridging, hydrophilic, hydrophobic, and van der Waals both between peptides and amongst themselves. According to Figure 1, peptide immobilization parallels alkanethiol SAM formation. When the peptide approaches the gold QCM electrode, it randomly adsorbs to the surface. Many nucleation points occur randomly and grow outward until the peptide covers most of the surface. This causes a large initial increase in change of frequency (|∆F|) and damping resistance (R).21 The adsorption slows, as the peptide predominately covers all the exposed gold binding sites. Like alkanethiols, the peptides align as the nuclei grows. The initial adsorbed peptides do not form strong interactions with neighboring peptides which causes the change in damping resistance, an indication of the surface viscoelastic effects. However, as the monolayer rigidity increases, the damping resistance will not change and Sauerbrey’s equation can be used to relate the change of the mass with the decrease of QCM frequency |∆F|.22 The change in damping resistance R is more straight-forward as it is reflective of the viscoelastic properties of the SAM.23 Nucleation causes a large change in resistance due to the peptide binding to the surface. The alignment phase aligns the peptides of the SAM to a common


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Leo et. al 11 orientation to interact with neighboring peptides. These interactions make the SAM more rigid, decreasing resistance. Additionally, alignment creates new sites for more peptide to nucleate which increase the mass as shown in the change of frequency (|∆F|). It is clear from Figure 1 that as the SAM matures, interactions between peptides make the SAM a rigid monolayer. These interactions are believed to be dependent on the titratable sites present in the peptide mimotope. They influence the viscoelastic properties of the SAM by establishing hydrogen bonding networks and salt bridging. This is not readily observable during the self-assembling process. To isolate the titratable sites involved with the folding behavior of CH19 on gold, characterization of the peptide mimotope and conformational shape of the peptide mimotope need to be understood.

SAM Characterization The QCM monitoring of the immobilization of CH19 showed interactions within the SAM that may have resulted from the effects of packing density, ionic strength, or surface charge. The characterization of the CH19 and CH19/CS7 SAMs by ellipsometry and atomic force microscopy will further define the role of the titratable sites during the peptide SAM formation processes. The difference in morphology of the monolayers from thickness (ellipsometry) and topology (AFM) properties by varying the packing density of the CH19 peptide mimotope and ionic strength of solution will help determine how titratable sites interact within the SAM and influence binding behavior of CH19 to Trastuzumab. Table 1 represents the data obtained from ellipsometry on different conditions of the SAMs of CH19. Note the wet and dry conditions yield the same thickness and the addition of Trastuzumab produced an approximate 40Å increase in thickness for both SAMs. The CH19 and


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Leo et. al 12 CH19/CS7 SAM differed by approximately 20 Å. Dilution of the buffer did not have a large impact on the thickness of both SAMs. However, the packing density does show a large effect on the thickness of the CH19 SAM. AFM Characterization As evident from Figure 2, there is a clear topological distinction between the CH19 SAM and the CH19/CS7 mixed SAM. The CH19 SAM is much rougher than the CH19/CS7 SAM. This can be seen in the difference in elevation points. The diameter of these elevation points is nearly 100 nm in diameter with a spherical shape. The CH19/CS7 mixed SAM in contrast is much smoother. The difference in elevation shape suggests that the orientation of the mimotopes in the SAM are different. After Trastuzumab is added to the monolayers, the same type of elevation shape can be observed. It is more pronounced in CH19 due to the roughness of the surface. The ellipsometer and AFM data complement each other in determining a structural conformational comparison between the CH19 SAM and the mixed monolayer CH19/CS7 SAM. The linker region CGSGSGS most likely exists as a helix due to its sequence homology with the GxxxG transmembrane motif which stabilizes protein secondary structures of helix to helix association in transmembrane proteins.24 The linker is in an aliphatic environment similar to that of the transmembrane domain which makes this speculation appropriate. Peptide SAMs containing an alpha helix conformation have been shown to have a vertical tilt like alkanethiol SAMs.25 If we considered the general rule of 3.4 amino acid residues per turn of an alpha helix and a 5.4 Å distance between each turn, then for a CH19 SAM, we would expect a thickness of approximately 30 Å. The deviations from this thickness are expected as the immobilized mimotopes are not simply an alpha helix throughout the entire peptide. The mimotope region is


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Leo et. al 13 likely in a more complex structure. The ellipsometer data shows the reduction in thickness when CH19 is prepared in a less packed environment as a mixed SAM. The decrease in thickness is evidence of a conformation change of the CH19 peptide mimotope as the packing density is changed. Using these observations, the conformation of the CH19 in the SAMs can be speculated. Figure 3 is the proposed structure of the CH19 SAM. The SAM is likely to consist of a helical linker region, and a functional loop binding region to Trastuzumab. The functional binding region is speculated to consist of a loop from the interactions of Glu13 with His19 of the peptide. This maximizes the interaction of neighboring peptides in a Glu13-His19-Glu13 sandwich. The proposed structure also indicates the possibility of pi stacking of the Tyr12 and His19 residues to further stabilized the overall SAM as this creates a sandwich of Tyr12 and His19 of one CH19 with an adjacent Tyr12 and His19 of another CH19 peptide.23 This model is from the perspective of maximizing the titratable site interactions which at this point is still speculated in its importance. This model however provides a foundation for how the conformation relates to packing density and to how the CH19 peptide mimotope interacts with Trastuzumab.

Characterization of the Effects of buffer and homogeneity on the affinity of Trastuzumab with CH19 SAM by QCM We have discussed a few fundamental principles behind peptide folding. We have determined that there are interactions between peptides in the SAM and that the packing density influences the mimotope conformation in the SAM. If the packing density can affect the


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Leo et. al 14 conformation of the peptide, it should also affect the binding behavior of the peptide with Trastuzumab. Figure 4 depicts the frequency change of the QCM frequency after several additions of Trastuzumab to the CH19 SAM and mixed CH19/CS7 SAM in undiluted PBS buffer and 1000x diluted buffer conditions. The data indicates two interesting relationships. First, CH19/CS7 mixed monolayer had a higher capacity to Trastuzumab binding than the CH19 SAM in undiluted PBS buffer conditions. This is evident by the higher overall change of the frequency (∆F) in QCM at about 11 ug/mL of added Trastuzumab for the respective monolayers. Secondly, both peptide SAMs in the 1000x diluted PBS buffer behave differently from the undiluted PBS buffer conditions. The curve for both peptide SAMs in diluted conditions appear sigmoidal. The capacity for Trastuzumab binding has further increased for both peptide SAMs. Dilution of the buffer greatly increased Trastuzumab binding capacity for the CH19 SAM (~500 Hz over undiluted CH19 SAM), while the CH19/CS7 gained a smaller Trastuzumab binding capacity (~80Hz over undiluted CH19/CS7 SAM.) This indicates that the PBS buffer may have stabilized CH19 to CH19 peptide interactions in the SAMs. The dilution of the buffer disrupted these interactions in CH19 and CH19/CS7 SAMS. However, the CH19 SAM had more CH19 to CH19 peptide interactions to disrupt, resulting in the creation of more potential CH19 binding sites for Trastuzumab, largely increasing avidity over the CH19/CS7 SAM. The data in Figure 4, summarizes the relationship between the packing density and ionic strength effects of the mimotope in the peptide SAM and Trastuzumab binding. The affinity constants, ka, are represented in Table 2 and determined by Figures S1 and S2. The 1000x diluted PBS buffer conditions are in units of M-2 compared to the undiluted conditions. The M-2 unit indicates cooperative binding of the peptide monolayer with Trastuzumab. The sigmoidal


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Leo et. al 15 curve in the 1000x diluted buffer conditions also supports cooperative binding. Trastuzumab as a monoclonal antibody has two arms available for binding (Figure S3). The concentration of buffer likely causes the shift of Langmuir adsorption to cooperative binding of Trastuzumab. The characterization by AFM and ellipsometer showed a distinct difference in the morphology of the SAM of CH19 and CH19/CS7, however the QCM data did not show a significant difference in affinity of Trastuzumab at a different CH19 packing density. This is important as the established thought process of the binding lies in the conformational and orientation of the peptide and protein. Instead, the results at two different buffer conditions show that it was ionic strength that changed the binding behavior of both conformational states of CH19. To understand how the ionic strength of the solution applies to the model proposed in Figure 3, the charge properties of the peptide/solution interface must be examined.

Electrochemical Impedance Spectroscopy Influence on the Properties of the Peptide SAMs Closely related to the ionic strength of the solution is the charge of the electrode interface. Charge refers to the electric double layer that forms around the peptide in the presence of an electric potential26 in buffered solutions. It has been documented that applied potential affects SAMs, facilitating their formation,27, 28 and modulating their structure.29 Under no potential, there exists a hydration sphere around the peptide which balances the charge of the peptide with the ions of the buffer and water molecules.30 By manipulating the applied potential across the peptide, we can change the electric charge surrounding the peptides and thus change the properties of the SAM. We used a small-scale electrode (

Ionic Strength, Surface Charge, and Packing Density Effects on the

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