Biomolecular Structure at SolidâLiquid Interfaces ... - ACS Publications

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Biomolecular Structure at Solid−Liquid Interfaces As Revealed by Nonlinear Optical Spectroscopy Sandra Roy, Paul A. Covert, William R. FitzGerald, and Dennis K. Hore* Department of Chemistry, University of Victoria, Victoria, British Columbia, V8W 3V6 Canada Corresponding Author Notes Biography Acknowledgments References

1. INTRODUCTION The attachment of biomolecules onto solid surfaces is fundamental to a wide range of scientific, engineering, and industrial pursuits.1,2 For example, the adsorption of proteins onto polymer surfaces is an important aspect of the biocompatibility of materials designed for medical implants.3−5 Ideal materials from a mechanical standpoint may be hydrophobic polymers such as poly(methyl methacrylate); however, hydrophobic surfaces are undesirable in the body as contact with proteins often results in denaturation in order to maximize interactions with hydrophobic residues that may be buried in the proteins’ core in their solution conformation.6 To this end, the tailoring of medical implant surface properties via chemical (such as grafting hydrophilic components)7−9 or physical processes (such as plasma treatment)10,11 has been successful in rendering the surfaces more hydrophilic and hence discouraging protein adsorption in general.12 Another application for broadband inhibition is marine biofouling films.13,14 The additional drag arising from attachment of marine organisms to vessel hulls has been estimated to result in a 50% increase in fuel consumption after spending six months in the water.15 The development of antifouling formulations that are effective and, at the same time, minimize the risk of environmental pollution remains a considerable challenge.16,17 On the other hand, there are cases where surfaces are sought to encourage biomolecule adsorption, such as the attachment of antimicrobial peptides to vascular stents.18−20 An extreme example where favorable surface interactions are critical is in the design of biosensors, such as blood sugar monitors for the management of diabetes based on glucose oxidize.21 Enzyme immobilization not only requires a hospitable interfacial environment to maintain the active secondary and tertiary structure of the protein but it also demands that the orientation of the active site be controlled so as to maximize the catalytic activity.22 Water purification steps make extensive use of granular activated carbon filters where ozonation byproducts and other substances are removed by adsorption onto the carbon surfaces, often coupled with biological degradation in enzymatic processes at these surfaces.23−25

CONTENTS 1. Introduction 2. Overview of Techniques 2.1. Electronic Second-Harmonic Generation Spectroscopy 2.2. Electronic and Vibrational Sum-Frequency Generation Spectroscopy 2.3. Relationship between Structural Features and Optical Properties 3. Biomolecular Structure at Solid−Liquid Interfaces 3.1. Amino Acids 3.2. Peptides and Proteins 3.2.1. Side Chain Structure 3.2.2. Backbone Structure 3.3. Carbohydrates 3.4. DNA 3.4.1. ssDNA and dsDNA 3.4.2. Cation Effects 3.4.3. Complex Systems 3.5. Lipids 3.6. Molecules at Lipid Bilayers 3.6.1. Small Molecules 3.6.2. Peptides and Proteins 3.6.3. Complex Systems 3.7. Cells 4. Perspective 4.1. Experimental Advances 4.1.1. Enhanced Signal-to-Noise and Spectral Resolution 4.1.2. Phase Measurement 4.1.3. Microscopy 4.1.4. Combining Multiple Techniques 4.2. Electronic Structure Calculations and Molecular Simulations 5. Summary Author Information © 2014 American Chemical Society

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8388 8389 8390 8391 8391 8392 8392 8394 8394 8397 8399 8400 8400 8401 8402 8403 8405 8405 8405 8407 8408 8409 8409 8409 8409 8410 8410 8410 8411 8411

Special Issue: 2014 Aqueous Interfaces Received: August 2, 2013 Published: January 9, 2014 8388

dx.doi.org/10.1021/cr400418b | Chem. Rev. 2014, 114, 8388−8415

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our discussion to electronic second-harmonic generation (SHG) and vibrational sum-frequency generation (SFG) spectroscopy. While we will address a broad range of biomolecule−surface interactions, the common element is that these systems are all aqueous interfaces, and therefore the nature of the interfacial solvent structure is just as important as that of the solid substrate in determining the adsorbed biomolecule structure.64 Although this review will focus on the biomolecules, we acknowledge the importance of the solvent structure. Furthermore, while the interfacial structure of the liquid phase may not necessarily be addressed in many of the studies we highlight, it is nevertheless an important contributor. Although nonlinear optical methods are ideally suited to probing the buried solid−liquid interface, there are some additional experimental challenges when compared to studies of exposed solid−vapor interfaces. For example, at least one of the bulk phases must be transparent to each of the pump or probe beams. In some cases, this leads to more complex experimental geometries involving transmission and reflection. For example, when using infrared beams, it is generally desired not to go through the aqueous phase. Another challenge involves focusing the beams at the buried interface, through additional dispersive elements. Finally, the quantitative analysis of the signals obtained in such experiments requires the analysis of more complicated local field corrections, which take the adjacent solid and liquid optical properties into account. Owing to these additional experimental complexities, many of the systems that we describe here were first studied at vapor interfaces. For example, proteins may be adsorbed onto solid substrates from solution, and then dried on the substrates prior to measurement. Such studies are important, as many of them have been the pioneering efforts in this area. Although we draw attention to a small number of these studies, we have largely excluded them in the interest of focusing on the solid−liquid interface. In this review, we will provide examples that illustrate the state of the art in structural characterization for a wide range of biomolecules at solid−liquid interfaces. In section 2, we will first provide a brief description of the two experimental techniques that will be the focus of our review. Section 3 will highlight how advances in experimental techniques, aided by ever more sophisticated computation and analysis methods, gradually enable experiments that begin by providing unique qualitative information to become increasingly quantitative. We will begin with simple systems of small molecules such as amino acids and short peptides. We gradually make the transition to large proteins, and protein complexes embedded in membranes. In the end, we will address studies of live cells. Section 4 will provide some perspective on where this field is heading with continuing advances in experimental methods and supporting techniques.

Another area of considerable importance is the study of lipid monolayers, bilayers, and biological membranes. Characterization of the structure and activity of individual phospholipid molecules within membrane layers, including exchange between the top and bottom leaflets, is critical to an understanding of the chemical and mechanical properties of these complex assemblies.26,27 Furthermore, membranes are the host environment for many proteins and protein complexes. We will review nonlinear spectroscopic studies of lipids, covering simple constituents, mixed lipid monolayers and bilayers, and complex systems of peptides and proteins embedded in membranes. An emerging area at the interface between biology and nanotechnology is the control and manipulation of DNA and RNA at surfaces.28 Making use of the highly specific interactions resulting from the complementarity of nucleic acid strands, one is able to create an impressive array of biosensors.29,30 We will illustrate how nonlinear optical probes that are able to monitor these structures at the solid−liquid interface have played a major role in the advancement of this field. It should be emphasized that, given that the interfacial environment has historically been challenging to probe with sufficient selectivity, much of our knowledge to date comes from bulk gas phase, solution, or solid studies, either by magnetic resonance,31−34 or X-ray35−37 and neutron scattering.38−40 Because the structure of the same molecules may be significantly different at the solid−liquid interface, especially in the case of peptides and proteins with tertiary organization, this is akin to homology modeling,41 rather than solving the structures from scratch. Furthermore, for decades there has been an extensive compilation of indirect evidence of the surface structures. In the case of enzyme immobilization for biosensors, one can monitor the activity of the catalytic process while varying the surface conditions.42,43 Conditions that promote high activity may be associated with solution-like conformations and favorable orientations at the surface; conditions in which the sensor is essentially broken may be associated with denatured structures or blocked conformations. With the advent of surface-selective and structurally sensitive nonlinear spectroscopy, there is an opportunity to revisit these systems with a high degree of molecular detail. At the buried solid−liquid interface there are fewer methods available for structural characterization than for exposed surfaces. In particular, powerful techniques that require vacuum such as scanning tunnelling microscopy,44,45 and mass spectrometry2,46−48 cannot be used. Nevertheless, there are a number of methods for studying molecules adsorbed at solid− liquid interfaces, such as atomic force microscopy (including chemical force and Kelvin probe microscopy),49−52 fluorescence-based techniques,53,54 electrochemical methods,55,56 infrared absorption spectroscopy,57,58 and techniques based on Raman scattering.59,60 Spectroscopic methods are particularly attractive since they can access a wide variety of buried interfaces, so long as one of the materials (either the solid substrate or water layer) is transparent to the probe beam wavelength, or is sufficiently thin so as to allow the probe to enter and exit with appreciable intensity for detection. Among this subset of techniques, we will demonstrate that methods based on second-order nonlinearity have the advantage of offering an exquisite specificity for the interfacial environment.61−63 This is also advantageous in that such probes can report on the interfacial water structure in the presence of adsorbed biomolecules.64 For additional focus, we will restrict

2. OVERVIEW OF TECHNIQUES Before discussing the results of specific experiments, we will provide some brief background on the two primary techniques of interest here, electronic second harmonic generation (SHG), and vibrational sum-frequency generation (SFG) spectroscopy. As our focus will be on experiments that provide structural insight, we then give a short overview of the principles that relate molecular orientation and conformation to the measured signals and spectra. 8389


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2.1. Electronic Second-Harmonic Generation Spectroscopy

The molecular dipole induced by an external field may be quantified in terms of the dipole moment 1 (2) 1 (3) (1) μl = αlm Em + αlmn EmEn + αlmno EmEnEo + ··· 2 6 1 + α(n)En (1) n! where α(n) is the nth order polarizability and l, m, n, ... are any of the molecule-fixed Cartesian coordinates. At low field strengths, only linear optical properties are observed from α(1). This is the combined result of the reciprocal factorial in the weighting coefficients of the higher-order response tensors and the fact that the tensors themselves have diminishing values. Nonlinear effects are therefore observed only when large electric fields are applied, such as those from lasers. As we will not be addressing any single-molecule experiments in this review, we will need to concern ourselves with large ensembles of molecules in the focus of laser beams that are typically hundreds of micrometers in diameter. We are therefore primarily interested in the macroscopic analog of eq 1 that expresses the dipole moment per unit volume, or polarization, P. ⎛ 1 1 (3) Pi = ε0⎜χij(1) Ej + χijk(2) EjEk + χijkl EjEk El + ... ⎝ 2 6 ⎞ 1 + χ (n) En⎟ ⎠ n!

Figure 1. Sample experimental configurations illustrating (a) secondharmonic generation spectroscopy at the solid−liquid interface using a collinear geometry and (b) visible-infrared sum-frequency generation spectroscopy using a noncollinear geometry. In the collinear geometry, the two beams have been depicted as spatially offset for clarity. Also note that both experiments may be performed in either geometry; we are showing the most typical cases.

(2)

of the electron density reradiates a field at the second harmonic, whose intensity is proportional to the magnitude squared of the 2 effective susceptibility, |χ(2) eff | , and the intensity squared of the 2 pump beam, I .

Here χ is the nth order susceptibility, ε0 is the vacuum permittivity, and i, j, k, ... are placeholders for any of the laboratory-frame Cartesian coordinates. The relationship between the molecule-fixed properties, in which the response is generated, and the lab-frame, in which the excitation is applied and signal is measured, is described in section 2.3. Second harmonic generation (SHG) originates from secondorder contribution to the polarization 1 Pi(2ω) = ε0 χijj(2) Ej(ω)Ej(ω) (3) 2 This phenomenon is also known as frequency doubling, as two fields (most often from the same source) create a polarization at a frequency of twice the input field(s). In the most common implementation of the experiment, a single beam is directed to the solid−liquid interface of interest, the fundamental frequency is removed by means of band-pass and/ or notch filters, and the second-harmonic (SH) is detected. Such a configuration is illustrated in Figure 1a. Hence, both input beams are j-polarized as indicated in eq 3, and the i component of the SH intensity is sent to the detector. It is also possible to split the input beam into two separate paths, so the beams can approach the sample from different directions, or at different angles. This has the advantage that the SH, generated along the momentum-conserving direction, is not collinear with the transmitted or reflected pump beams, and so may spatially filtered from these more intense sources. Additionally, such a configuration allows the two pump beams to be polarized independently along j and k directions as indicated in eq 2. The strength of this interaction is governed by the second-order susceptibility, χ(2). This 27-element rank-3 tensor couples the input fields Ej and Ek to the ith component of the induced polarization. In the case of SHG, it is most common to provide both input fields from a single beam. These fields then have the same polarization, both indicated as Ej in eq 3. This oscillation (n)

Ii(2ω) ∝ |χe(2) |2 I 2(ω) ff, ijj j

(4)

The above expression explicitly states that the ijj-element of χ(2) eff couples two j-polarized input beams at frequency ω to the detected i-polarized beam at 2ω. Elements of the effective susceptibility are related to the actual χ(2) tensor elements through χe(2) = [Lii(2ω) ·eî (2ω)]χijk(2) [Ljj(ω) ·eĵ (ω)][Lkk (ω)·ek̂ (ω)] ff, ijk (5)

where L are the local field corrections and ê are the unit polarization vectors. The relationship between the χ(2) elements and molecular orientation and structure will be discussed in section 2.3. Up until now, the theory described has referred to uncharged interfaces. The addition of interfacial charge sets up a static, surface-bound electric field E0 whose strength is reflected in the magnitude of a third-order component of the induced polarization.65,66 Because the additional electric field is static, this third-order response is detected simultaneously with the second-order response 1 1 Pi(2ω) = ε0 χijj(2) Ej(ω)Ej(ω) + ε0 χijj(3) Ej(ω)Ej(ω)E0 2 6 0 (6)

This process, known as electric field induced second harmonic (EFISH) generation, has been demonstrated to be well suited to the determination of pKa values of protonation sites at oxide−water interfaces,67 the measurement of the isoelectric point,66,68 and, as shown later in this review, the measurement of cation−DNA binding energies.69−72 All of 8390


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these studies rely on measurment of increased signal that occurs with charge buildup at the interface. There are two approaches to nonlinear optical spectroscopy, and these are seen especially in the case of SHG studies. On the one hand, measured SH signals provide useful qualitative information, as they serve as markers for polar orientation, that is regions that have a break in centrosymmetry allowing χ(2) ≠ 0. On the other hand, it is possible to carry out the SH measurements in a quantitative way, calibrating the magnitude (and in some instances phase) of the response, and comparing the SH response obtained for different polarizations of the input beam(s) and output SH beam. This allows for a sensitive probe of molecular orientation. Our review will address both of these aspects.

(2) 2 Ii(ωvis + ωIR ) ∝ |χeff, | I (ω )I (ωIR ) ijk j vis k

In comparison to the SHG expression (eq 4) we note that in SFG, since the pump beams have different frequencies, it is often more convenient to prepare them in different polarization states (j-polarized visible, and k-polarized infrared), and therefore access additional elements of χ(2) eff . Since it is the vibrational signature of interfacial molecules that is desired, it is common that the sum-frequency intensity is plotted as a function of ωIR. 2.3. Relationship between Structural Features and Optical Properties

Once the χ(2) elements have been extracted from the measured elements of χ(2) eff with the help of eq 5, the relationship to the molecular structure can be seen in the relationship between the molecular optical properties and the macroscopic ensemble average in the laboratory frame. This entails representing the molecular hyperpolarizability tensors α(2) averaged over all molecular orientations to yield χ(2). In spherical polar coordinates, this amounts to

2.2. Electronic and Vibrational Sum-Frequency Generation Spectroscopy

Sum-frequency generation (SFG) is the nondegenerate analog of SHG. Its name reveals that the frequency doubling in SHG is actually better described as frequency adding, ω + ω = 2ω, in an energy-conserving process. Hence, if the experiment is performed with two lasers of different frequencies, ωSFG = ω1 + ω2. Figure 1b illustrates a typical noncollinear geometry which has an advantage in that the phase-matching direction of the reflected SFG beam may be spatially separated from the two pump beams. For studies of biomolecules at the solid−liquid interface, it is most popular to have one of the lasers in the visible region (532 or 800 nm, typically far from any electronic resonance), and the other laser either broadband or tunable throughout the mid-infrared, 1000−4000 cm−1. In this way, resonance enhancement is observed as the probe frequency ωIR approaches one of the q vibrational modes at ωq. This may be expressed as a frequency-dependent molecular hyperpolarizability (2) (2) αlmn (ωIR ) = αNR, lmn +

∑ q

χijk(2) = =

N ε0

N (2) ⟨αijk (θ , ϕ , ψ )⟩ ε0 2π

2π

∫0 ∫0 ∫0

π

fODF (θ , ϕ , ψ )

(2) (θ , ϕ , ψ ) sin θ dθ dϕ dψ × αijk

(9)

where θ is the tilt angle, ϕ is the azimuthal angle, and ψ is the twist. The angle-dependent lab frame α(2) ijk (θ, ϕ, ψ) is obtained from the molecular α(2) lmn via the coordinate transformation abc abc abc (2) αijk (θ , ϕ , ψ ) =

∑ ∑ ∑ Dil(θ , ϕ , ψ ) l

m

n

(2) × Djm(θ , ϕ , ψ )Dkn(θ , ϕ , ψ )αlmn

⟨0|αlm ̅ |1⟩⟨1|μn̅ |0⟩ ωq − ωIR − i Γq

(8)

(10)

where a, b, c are the molecular-frame unit vector and D is the direction cosine matrix.73 f ODF in eq 9 is an orientation distribution function that can take various forms. In the case of an isotropic distribution of molecules, as would be found in the bulk solution phase, f ODF = 1 resulting in ⟨α(2)⟩ = 0. This is the origin of the surface-selectivity of second-order (and all evenorder) nonlinear optical techniques. When f ODF ≠ 1, eq 9 indicates that the second-order susceptibility in the lab frame is a weighted average over molecular orientations. For example, if the azimuth angle ϕ is uniformly distributed in the plane of the interface, a Gaussian distribution of tilt and twist angles may be represented as

(7)

where α(2) NR is the nonresonant contribution, Γq is the Lorentzian line width, and i = √−1. Here we use l, m, n as placeholders for any of the molecule-fixed Cartesian coordinates. Taking |0⟩ as the vibrational ground state, |1⟩ as the vibrational first excited state, α̅ as the polarizability operator, and μ̅ as the dipole moment operator, one can see that the SFG response is a product of the Raman transition polarizability and IR transition dipole moment. In this respect, vibrational SFG spectra provide information akin to IR absorption and Raman scattering spectra, but while selectively probing molecules at the surface (or other environments that break the centrosymmetry of the bulk solid or solution phase). Strictly speaking, vibrational modes must be Raman- and IR-active in order to be observed in the SFG spectrum, but practically, exclusion based on molecular symmetry is generally observed only in the case of small molecules. In the case of large biomolecules, however, SFG spectra are often less congested than corresponding Raman or IR spectra. This is also due to the numerator in eq 7; even when Raman and IR selection rules are not mutually exclusive, if one of the transitions is weak, this greatly diminishes α(2), even before any orientational averaging is considered. In a traditional (homodyne) SFG experiment, the measured intensity is proportional to the magnitude squared of the effective susceptibility

⎡ (θ − θ )2 (ψ − ψ0)2 ⎤ 0 ⎥ fODF (θ , ψ ) = C N exp⎢ − − ⎢⎣ 2σθ 2 2σψ 2 ⎥⎦

(11)

where θ0 and ψ0 are the mean tilt and twist angles, σθ and σψ are the widths of the corresponding distributions, and CN is a normalization constant evaluated such that 2π

2π

∫0 ∫0 ∫0

π

fODF (θ , ϕ , ψ ) sin θ dθ dϕ dψ = 1

(12)

Using the above framework, the general procedure used in determining molecular structure from nonlinear optical spectra is to first extract elements of the second-order susceptibility tensor χ(2) from the experimental signals. In the case of SHG at 8391


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fixed frequencies, this will be directly proportional to the measured signal. In the case of vibrational SFG, spectra are fit to a line shape such as a Lorentzian to extract the peak amplitudes, frequencies, and widths. The amplitude of a particular vibrational band is then proportional to χ(2). The particular element(s) of χ(2) that is probed is determined by the polarization of the incoming pump beams and the selected polarization of the outgoing SHG or SFG beam. For example, in an experiment with a single s-polarized pump beam, detecting the p-component of the SHG reflected from the solid liquid interface (pss polarization combination), a linear combination of xyy and zyy elements of χ(2) are probed, where x and y directions are in the plane of the interface, and z is along the interface normal. In the often encountered case of azimuthal isotropy x and y directions are indistinguishable, χ(2) xyy = 0, and so pss experiments end up being a direct probe of χ(2) zyy . It is now desired to turn such χ(2) elements into descriptions of the transition dipole moment or vibrational normal mode orientation at the solid−liquid interface. One option for proceeding is to calibrate the experimental response so that the nonlinear susceptibility is measured in absolute units. This may be done by comparing the sample signal to that measured from a material with known response, such as a piece of quartz in known crystallographic orientation.74 Equation 9 reveals that there is still the molecular number density N that contributes to χ(2), in addition to the orientation dependence. It may be possible to estimate N from other experimental data.58 A more common route to the structural information is to ratio two independently measured elements of the susceptibility, such as (2) χ(2) zyy /χyzy . (Note that the yzy element could not be measured with only a single pump beam.) This removes the requirement of calibrating the experiment (including laser power, detector response) and also eliminates any dependence on N. However, it should be noted that one must still ensure that the response measured in different beam polarizations can be compared, as additional calibration may be required to account for polarization bias in the experimental configuration.75 Another consideration is that, in SFG experiments far from electronic resonance (and SHG with two pump beams), there are only three independent nonzero elements of χ(2). If ratios are formed, this results in a reduction to two independent pieces of experimental data that may be used to deduced the molecular orientation distribution. Despite this loss of information, the benefits from taking ratios generally outweigh this limitation. The final component that relates measured χ(2) elements to structural information of the biomolecules at solid−liquid interfaces is the molecular hyperpolarizability, α(2). Examining eqs 9 and 10 together shows that all (potentially 27) elements of α(2) in the molecular frame may contribute to a single element of χ(2). Furthermore, in the case of vibrational SFG experiments, the complete α(2) tensor must be known for each vibrational mode q of interest, as revealed in eq 7. These molecular properties can be measured in molecular crystals with known and narrow orientation distributions, or estimated using bond-additivity models.76−78 In certain cases where the local modes are of high symmetry and involve the same infrared transition, the ratio of α(2) elements may be inferred from Raman depolarization measurements in solution.79 If neither of these methods are suitable, one can estimate the hyperpolarizability using electronic structure calculations.80−85 This area will be addressed briefly in section 4.2.

3. BIOMOLECULAR STRUCTURE AT SOLID−LIQUID INTERFACES We will provide some examples of how the second-order nonlinear spectroscopic techniques SHG and SFG provide structural insight into molecules of biological relevance adsorbed at solid−liquid interfaces.86,87 We have organized our review in order of increasing complexity, starting with amino acids and carbohydrates and working our way to peptides and proteins. We discuss lipid bilayers and progress to studies where complex assembles of proteins are monitored within these layers. We will also cover the growing field of DNA at interfaces. Finally, we end up with a discussion of nonlinear optics of whole cells, illustrating the potential of these techniques in the characterization of complex, living systems. 3.1. Amino Acids

The adsorption of amino acids onto solid surfaces has significance on a variety of levels. From a practical standpoint, the separation of these small molecules using techniques such as ion exchange chromatography relies on subtle differences in their Coulombic interaction with a stationary phase. An enhanced molecular understanding of such interactions would lead to improved separation technologies.88−90 Also, clay surfaces are implicated in sequestering amino acids to form the first peptides in the primordial sea.91−93 Finally, the adsorbed structures of peptides and proteins are dictated by the interplay between the residue−residue and residue-solvent interactions that govern the structure in solution, and the residue-surface interactions (including solvent) introduced by the solid substrate. Studying individual amino acids allows these interactions to be probed more selectively and quantitatively without additional residues contributing to the response or congesting the vibrational spectra. It should be noted that amino acids are small and zwitterionic and often have roughly spherical solvation shells. As a result, adsorbed amino acid behavior may not be indicative of how a residue of the same type behaves in a peptide or protein. Nevertheless, they are often considered as a starting point in structural studies. Second-order nonlinear spectroscopic techniques have first been applied to amino acids at liquid−liquid94 and liquid− vapor interfaces,95,96 and subsequent studies have investigated them at solid−liquid interfaces.85,97−102 The first SFG report of amino acid adsorption at the solid−liquid interface was a study of phenylalanine at the glassy carbon electrode in 2002 by Kim et al.97 Figure 2 illustrates the effect of varying the applied potential on the C−H stretching region. The authors were able to conclude that, at the positively charged surface, phenylalanine was adsorbed by means of the carboxyl group. The methylene group was determined to adopt a conformation in which the dipole is oriented at an angle with respect to the surface normal. As the potential becomes highly positive, both the phenyl ring and the ammonium groups are oriented in such a way as to minimize the SFG signal. Pászti and Guczi have performed a detailed study of aspartic acid, glutamic acid, glutamine, and phenylalanine on hydrophilic TiO2 surfaces.103 The authors found that Asp and Glu formed an ordered layer, while Gln and Phe are not strongly adsorbed, leading them to conclude that acidic side chains play an important role in forming stable adsorbed layers on TiO2. In an SFG study, Hall et al. examined the orientation that Phe adopts when adsorbed at the water−polystyrene interface. Collecting spectra in three different polarization schemes revealed the amplitudes of the methylene symmetric and 8392


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Figure 2. SFG spectra of phenylalanine in the C−H stretching region of the glassy carbon electrode−water interface with various electrode potentials (a) 1100, (b) 900, (c) 600, (d) 300, (e) 0, and (f) −200 mV. Reprinted with permission from ref 97. Copyright 2002 American Chemical Society.

Figure 3. Molecular orientation analysis of phenylalanine adsorbed at the water-polystyrene interface. Comparing SFG spectra to parametrized orientation distribution functions resulted in families of solutions for (a) mean tilt and twist angles and (b) widths of the tilt and twist distributions considering two methylene stretching modes (shaded gray and black regions) or an additional five aromatic C−H modes (black regions only). (c) The tentative structures, of which only B and D remain as candidates. (d) Relationship between structure D and phenyl rings on the polystyrene surface. Adapted with permission from ref 85. Copyright 2010 American Chemical Society.

antisymmetric stretching tensor elements. Using these values together with hyperpolarizabilities obtained from electronic structure calculations, the authors considered orientation distribution functions that were Gaussian in the tilt and twist of the methylene group, as described in eq 11 The fourdimensional parameter space is therefore comprised of the mean tilt and twist angles (θ0 and ψ0), and the widths of these angular distributions (σθ and σψ). The gray and black regions together in Figure 3, panels a and b indicate values of these parameters that produce simulated SFG spectra consistent with the experimental observations. Corresponding orientations (sampled from the positions indicated by the red dots in Figure 3a) are shown in Figure 3c. As this was a homodyne SFG experiment, structures that differ by θ = 180° − θ and ψ = ψ + 180°, marked (A,C) and (B,D), could not be distinguished. However, although A and C would account for the observed methylene peak intensities in all three polarizations, such structures would orient the phenyl ring in a manner that would result in significant intensities of the five aromatic C−H stretching modes above 3000 cm−1. As negligible signal was observed in that spectral region, only B and D (shaded black in Figure 3, panels a and c) remain as candidates. It is interesting to note that structure D has its phenyl ring at an angle that allows for intercalation with the previously reported phenyl ring orientation from the polystyrene surface.104 This study demonstrated the utility of combining analysis of multiple resonant modes in SFG, electronic structure calculations, and a search over a wide orientation distribution parameter space to arrive at a detailed picture of adsorbed structure. In the previously mentioned study, the analysis was made possible by being able to identify specific functional groups (aliphatic CH2 and aromatic C−H) by their vibrational resonances. Those vibrational modes then serve as markers

for the orientation of parts of a molecule. Such an approach is generally limited to small molecules, as the spectrum of larger molecules becomes crowded, especially in the C−H stretching region. Even in cases where only a few bands are observed, as the frequency of methyl and methylene symmetric and antisymmetric modes are not that unique, there can be many modes contributing to each band of resonances. This problem was addressed in a subsequent study by Hall et al. examining leucine adsorption onto polystyrene.100 Although it would seem that unique vibrational modes could be assigned in this very small molecule, electronic structure calculations reveal 10 closely spaced aliphatic C−H stretching modes, in addition to any Fermi resonances. Although it is therefore not possible to fit the spectra to obtain the susceptibility tensor amplitudes on resonance for a particular mode, modeling approaches are still able to work backward to predict the SFG spectra. A previous molecular dynamics study of leucine adsorbed to model surfaces of varying hydrophobicity identified two favorable orientations on the surface, a dominant upright orientation with the isobutyl group directed toward the surface, and a secondary orientation in which Leu was laying on the surface.105 Hall et al. therefore mined the MD trajectories, seeking to map out all dominant Leu conformers in both orientations, and identifying five conformers as shown in Figure 4b. SFG responses were generated from electronic structure calculations of each of these conformers (filled circles in Figure 4b), projected into the surface frame using ensembles sampled from the trajectories. 8393


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spectroscopy. A combination of experimental and modeling techniques, involving both electronic structure calculations and molecular dynamics simulations has been able to resolve details of the orientation and conformation that these molecules adopt at various solid−liquid interfaces. 3.2. Peptides and Proteins

Compared with single amino acids, the secondary and tertiary structural features found in peptides and proteins add to the richness and complexity of their molecular spectra. In the past few decades, advances in protein NMR and X-ray crystallography have provided detailed structural information for these species residing in the bulk and at interfaces. However, these techniques are not interface specific, and considerable effort has been directed toward the use of nonlinear optical methods to study peptides, polypeptides, and proteins at solid−liquid interfaces.107−118 In this section, we trace the evolution of second harmonic and sum-frequency generation spectroscopies applied to larger, amino acid-based biomolecules. Despite the significant difference in complexity between peptides and proteins, nonlinear optical studies of these classes of molecules have proceeded in tandem; we likewise discuss them in tandem. 3.2.1. Side Chain Structure. Early nonlinear optical studies of interfacial protein structure probed the orientation of terminal methyl groups of several proteins (lysozyme, fibrinogen, and BSA) and the effect of surface coverage (submonolayer, monolayer, and multilayer) upon that orientation.107,119 Proteins in these studies were allowed to adsorb onto the surface from solution after which the substrate was removed from solution, rinsed, and kept at high humidity. While these studies were technically not of a solid−liquid interface, the experimental conditions were designed to approximate this type of interface. SFG spectra collected in ssp and sps polarizations (Figure 5) identify changes in surface structure upon increasing solution protein concentration. Clearly, the shapes of the BSA−silica and fibrinogen−silica spectra change as surface coverage is altered, while lysozyme− silica spectra remain unchanged. Shifts in the magnitudes of the CH3 symmetric stretch, ACH3(s) and the CH3 symmetric stretch to CH3 antisymmetric stretch ratio ACH3(s)/ACH3(a) (Figure 6a) were used to develop a quantitative picture of the surface. An increase in ACH3(s)/ACH3(a) indicates a decrease in the tilt of the side chain methyl groups with respect to the surface normal while an increase in ACH3(s) reflects an increase in the surface density of SFG-active methyl groups. The relatively constant nature of both ACH3(s) and ACH3(s)/ACH3(a) derived from the lysozyme spectra indicates a surface structure in which there is no change in methyl orientation or loading. In contrast, both fibrinogen and BSA witnessed an increase in ACH3(s) and ACH3(s)/ACH3(a) indicating both an increase in the number of SFG-active methyl groups and an overall decrease in their tilt angle. One further piece of information is available from the ratio of the antisymmetric response in the ssp and sps spectra. The theoretical relationship between CH3 tilt angle θ0, (2) response ratio χ(2) yyz,a/χyzy,a, and distribution width σ, is shown in Figure 6b. With the measured response ratios, the authors were able to determine exact tilt angles, given assumptions about the width of the tilt angle distribution. The cartoon shown at the bottom of Figure 6 presents a schematic of the evolution of the surface methyl density and orientation with increased surface protein loading.

Figure 4. (a) Molecules binned according to their dihedral angles (ξ1, ξ2) with region boundaries identified in yellow. The point corresponding to the largest population density in each region is marked with a white circle. The optimized geometries are indicated by the filled circles. (b) The five conformers in standing (top row) and laying (bottom row) orientations. The highlighted structures are those determined to contribute most significantly to the observed spectra. Adapted with permission from ref 100. Copyright 2011 American Chemical Society.

The ratios of the 10 species (five conformers in each of two orientations, as shown in Figure 4b) were varied in a search to obtain the closest agreement with the experimental spectra measured in three polarization schemes. It was determined that the best match to the experimental spectra was obtained by the two conformers highlighted in Figure 4b. The authors rationalized these results in terms of the relative (bulk) energies of the conformers, together with an idea of how these conformers would direct hydrophobic methyl groups into regions of low interfacial water density.100 This also highlights the importance of understanding surface water structure in accounting for the adsorption and adsorbed geometries of biomolecules.64,105 Amino acids at surfaces may also provide markers for specific ion interactions. An SHG study by Gibbs-Davis et al. has used amino acid-functionalized quartz surfaces to monitor the mobility of chromate ions.106 This study found that transport rates of Cr4+ at these surfaces was 50% slower than in bulk water, suggesting that peptidic substances in soil can significantly effect the residence time of this heavy metal pollutant. We have seen that amino acids, by virtue of their small size and isolated identifiable vibrational resonances, lend themselves to detailed quantitative structural investigations by SFG 8394


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Figure 5. SFG spectra collected in ssp (top) and sps (bottom) polarizations of lysozyme, fibrinogen, and BSA adsorbed on hydrophilic silica surfaces. Deposition of proteins is carried out in bulk protein solutions with concentrations of 1.0, 0.2, 0.04, 0.008, and 0.0 mg/mL. Filled circles denote collected data, and the solid lines represent fits to the data from which peak positions, widths, and oscillator strengths can be obtained. Note that the SFG signal intensity is independent of the lysozyme bulk concentration but changes with solution concentration for fibrinogen and BSA. Reprinted with permission from ref 107. Copyright 2003 American Chemical Society.

Figure 6. Analysis of magnitudes and ratios of the CH3 symmetric and antisymmetric peaks in the ssp spectra (a) alongside the ratios of the CH3 antisymmetric stretches in the ssp and sps spectra (b) led to a proposed model for the orientation of methyl groups in lysozyme, fibrinogen, and BSA adsorbed on hydrophilic silica surfaces (c). Reprinted with permission from ref 107. Copyright 2003 American Chemical Society.

Comparison of the above study with a study of BSA at solid− liquid interfaces illustrates the effect of solvent upon the structure of adsorbed proteins. BSA spectra were measured at several solid−liquid interfaces (varying hydrophobic character of both solid and liquid) and over a range of pH.117,118 Figure 7a shows the BSA spectra at the fused silica interface as the solid is repeatedly immersed into water and then removed from water. The initial spectrum (lower trace), collected immediately after adsorption and removal from solution, shows the same clear C−H modes seen in the solution−polymer experiment. Upon reintroduction to water, the C−H peaks disappear; they return upon removal once again from solution. If the assumption is made that BSA remains at the interface the entire time, the interpretation of these observations is that when BSA adsorbs in solution, the C−H groups have inversion symmetry, a result of BSA adopting a conformation that sequesters the hydrophobic groups in the interior of the structure. This symmetry is lost when the BSA is exposed to air, but is regained when exposed water again. Additionally, when the fused silica−BSA surfaces were exposed to hydrophobic liquids, similar response was seen as when exposed to air (Figure 7b). In a separate set of experiments, the authors exposed a polystyrene surface to an aqueous BSA solution at three different pH levels. SFG spectra (Figure 8a) show quite clearly peaks associated with C−H stretching, as well as the broad features associated with interfacial water. At first glance, it appears that the pH affects the relative intensities of the C−H modes, but upon fitting the data (Figure 8b) it is apparent that there is little change in the C−H modes, implying little change

in the ordering or conformation of BSA. Nearly all of the differences observed between the spectra can be accounted for by alteration of the interfacial water structure. This study, contrasted with the study by Kim et al.,107 highlights the effect of water upon surface structure. In that study, SFG spectra were observed when proteins were kept under a high humidity environment in order to try to closely approximate a solvated state, whereas when BSA was fully immersed in water, the C−H mode peaks disappear. In addition to studies of actual proteins, there is much to be gained from characterizing the adsorbed structures of model peptides with well-defined secondary structures. For example, LKα14 and LKβ15 are model amphiphilic peptides, consisting of a chain of 14 or 15 residues of leucine (Leu) and lysine (Lys), first synthesized and studied by DeGrado and Lear,120 with SFG studies originally by the Castner group.121 These peptides adopt either an α-helix (Ac-LKKLLKLLKKLLKL− OH) or β-sheet (Ac-LKLKLKLKLKLKLKL−OH) form, dependent upon the sequence of Leu and Lys. The vibrational signature of both peptides is simple as it contains only two species of amino acids; one, Leu, contributes mostly to the C−H stretching region, while the other, Lys, contributes to the NH3 stretching region. Studies of LKα14 adsorption have been done at polystyrene,31,122−124 silica,121,122,124 and different selfassembled monolayer (SAM) surfaces.125,126 Additionally, adsorption of LKβ15 on different SAM surfaces has been studied.125 In all of these studies, a strong orientation of the 8395


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Figure 8. (a) SFG spectra of d-PS/BSA solution interfaces with different solution pH values. (b) Fitting results for panel a. Reprinted with permission from ref 118. Copyright 2002 American Chemical Society.

Figure 7. (a) SFG spectra of fused silica (with adsorbed BSA)−air interface and fused silica (with adsorbed BSA)−water interface. (b) SFG spectra of fused silica (with adsorbed BSA)/benzene, fused silica (with adsorbed BSA)/CCl4, and fused silica (with adsorbed BSA)/FC75 interfaces. Reprinted with permission from ref 118. Copyright 2002 American Chemical Society.

challenge by measuring SFG spectra of LKα14 adsorbed to a self-assemble monolayer on gold.125 By using gold as the phase reference, they were able to obtain the relative phase of every peak in the C−H stretching region. One established method for obtaining solid liquid phase information is through the interference of the resonant signal of with a nonresonant SFG response from gold.128 When the resonant and nonresonant responses are in phase, a positive peak on resonance is observed. However, when the responses are 180° out of phase, a negative peak results. Following this method, Breen et al.129 and Weidner et al.,31 deposited a selfassembled monolayer on gold and observed LKα14 adsorption. With gold as the phase reference, the relative phase of every peak in the C−H stretching region was determined. In Weidner’s study they sequentially deuterated individual leucine CH3 groups of LKα14 adsorbed on polystyrene and measured the corresponding SFG for every leucine along the peptide (Figure 10c). From a quantitative analysis of the SFG signal,

hydrophobic side chains was observed when adsorbed on the hydrophobic surface. When adsorbed on hydrophilic surfaces, however, the consensus seemed to be that the spectra lose the hydrophobic features and exhibit a response around 3300 cm−1 from N−H stretching (Figure 9). Isotope labeling studies have shown that this peak originates from the side chain amine groups rather than from the backbone amide.127 These results infer that side chains with a higher surface interaction have a stronger orientation preference. On the other hand, the side chains oriented away from the surface appear to have a greater freedom of motion and thus yield little to no signal. Obtaining quantitative phase-resolved SFG spectra remains a challenge as capturing the phase of the SFG signal at solid− liquid interfaces is difficult. Weidner et al. approached this 8396


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structures from SFG data.116,134,136−144 The bulk of this work has been done by Chen and co-workers, who have developed methods and theory for obtaining and analyzing SFG spectra for overall peptide structure and orientation, as well as individual α-helix136 and β-sheet137 conformations. The first captured SFG spectra of protein amide I signals were reported in 2003.140 In this study, polystyrene−protein solutions and polystyrene−ubiquitin solutions of various concentrations and in D2O were investigated. It was observed that the peak shapes differed between BSA, FXIIa, Ubiquitin, and Mefp-2, all with very different backbone structural characteristics (Figure 11a). As these peaks appeared in the

Figure 9. (a) Structure of the α-helical LKα14 peptide in solution. (b) SFG spectrum of LKα14 adsorbed onto d8-PS (blue) and SiO2 (red). In the PS spectrum, the broad peak centered at 3092 cm−1 is assigned to structured water molecules. Peaks at 2869 (CH3 symmetric), 2895 (C−H or CH2 Fermi resonance), and 2935 cm−1 (CH3 Fermi resonance) are observed. The dominant peak at 3294 cm−1 in the SiO2 spectrum is assigned to an N−H stretching mode, and a weak OH peak is observed at ∼3190 cm−1. Reprinted with permission from ref 122. Copyright 2006 American Chemical Society.

the authors were able to calculate an average tilt and twist angle for each isopropyl side chain (Figure 10b). To infer the absolute orientation (toward or away from the surface) they combined their data with NMR studies (Figure 10a) and concluded that Leu1, Leu4, Leu8, and Leu11 are pointing toward the surface while Leu5, Leu7, Leu12, and Leu14 are mostly pointing away from the surface. A similar site-directed approach using fluorinated labels has been used to assess the orientation of phenyl rings in phenylalanine residues of a mineral protein.130 In addition, based on the same principle as LKα14, other SFG studies have been done on peptides with two amino acid of different hydrophilicity, varying the amino acids and peptide length.99,131−135 3.2.2. Backbone Structure. Much research has been done to develop methods to assess peptide and protein secondary

Figure 11. (a) SFG spectra collected from interfaces between PS and various protein solutions. (b) SFG spectra collected from interfaces between PS and ubiquitin H2O solution, ubiquitin D2O solution, and D2O solution with a much lower concentration. Reprinted with permission from ref 140. Copyright 2003 American Chemical Society.

water bending region of the IR spectrum, confirmation was needed that the observed shifts in peak structure were due to differences in protein structure and not water interfacial structure. The similarity between the PS−ubiquitin in H2O and PS−ubiquitin in D2O spectra (Figure 11b) provided this confirmation. This was the first report that demonstrated SFG

Figure 10. (a) Top plot is of the SFG twist and tilt angles for leucine at different locations within the peptide. The bottom plot shown NMR cone angle as a function of SFG tilt angle. (b) Tilt and twist angles obtained from ratios of the symmetric and antisymmetric peaks in the SFG spectra. (c) Schematic of leucine orientation, after adsorption, based on the results shown in panels a and b. Reprinted with permission from ref 31. Copyright 2010 National Academy of Sciences, USA. 8397


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perpendicular (E1) components of nonlinear susceptibility for a perfect α helix (Figure 14). These were derived from ab initio

can be used to study changes in secondary structure of peptides and proteins at interfaces. In subsequent publications, the position of the amide-I band peak center was shown to be unique for the α-helix, 310-helix, and β-sheet structures.136,139,141 A combined SFG and ATRFTIR study of tachyplesin I, a 17 amino acid residue that adopts a rigid antiparallel β-sheet structure, and MSI594, a 24 amino acid α-helix, at the polystyrene−solution interface, showed a distinct separation of the resonances associated with the two secondary structure types.141 Results from fitting the spectra seen in Figure 12, panels a and b, showed that the α-

Figure 14. Calculated susceptibility as a function of orientation angle θ (2) (2) for a δ distribution: (a) χ(2) yyz of an α helix; (b) χyzy of an α helix; (c) χzzz of an α helix. (d) Susceptibility tensor elements of a free CO group. Reprinted with permission from ref 138. Copyright 2008 American Chemical Society.

Figure 12. SFG spectra and fitting results for (a) 0.1 mg mL−1 MSI594, (b) tachyplesin I, and (c) tachyplesin I in the presence of 10 mM dithiothreitol at the polystyrene−solution interface. Squares representing the actual spectra, the dotted lines representing the fitted spectra, and the solid lines representing the component peaks used to fit the spectra. Reprinted with permission from ref 141. Copyright 2005 American Chemical Society.

calculations of the hyperpolarizability of each unit within the αhelix, which were then summed to arrive at the theoretical peptide susceptibility. For comparison, the resulting susceptibility tensor elements for a randomly coiled structure are also shown. The orientation angle θ describes the angle between the c axis of the α helix (Figure 13a) and the z axis of the surface. It is clear from this presentation of the theory that relative intensities of the A and E1 modes are both polarization and angle dependent. This behavior is advantageous in that it aids in the assignment of the amide I peaks when in the presence of overlapping response from, for example, “free CO” vibrations. In addition, polarization ratios allow for further constraint of θ. Chen and co-workers described the relationship between θ and a Gaussian distribution σ of the tilt for different polarization ratios (see Figure 15).

helix peak appears at 1650 cm−1 and the primary β-sheet band appears at 1688 cm−1. The disappearance of the peak at 1688 cm−1 upon addition of dithiothreitol (DTT; Figure 12c), used to reduce the disulfide bonds that maintain the β-sheet structure, provided further confirmation of this location of the β-sheet peak. In the perfect α-helix, first described by Pauling,145,146 all of the backbone carboxyl groups are oriented in the same direction. In contrast, backbone amide groups separated by four residues oppose each other; however, they are all oriented in the same fashion with respect to the α-helix main vector. The amide group orientation is a proven way to characterize the α helical conformation. The amide group contains two SFG active vibrational modes: the A mode is parallel to the helical axis (along c axis of Figure 13) and the E1 is perpendicular to the helical axis. In 2008, Chen and co-workers presented a detailed method to determine the in situ orientation and conformation of secondary structure of adsorbed peptides by SFG.138 Using fibrinogen on polystyrene as an example, they derived the theoretical angular dependencies of the parallel (A) and

(2) (2) Figure 15. Relationships between the (left) χ(2) zzz/χyyz or (right) χzzz/ (2) χyzy ratio and θ for an α-helix in terms of different Gaussian distribution widths σ. Black: σ = 0°; blue: σ = 5°; red: σ = 10°; green: σ = 20°; and pink: σ = 30°. When σ is 0, the distribution is a δ distribution. Reprinted with permission from ref 136. Copyright 2009 American Chemical Society.

Using the experimental data for θk, the angle of the dipole moment with respect to the helical axis, and taking into account the Higgs derivation of the selection rules for the α-helix amide I mode,147 Nguyen et al. determined the two amide mode dipole moments.136 For the A mode

Figure 13. Schematic of (a) α-helix and (b) β-sheet structures with molecular coordinate frame shown as a, b, and c axes. 8398


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absolute orientation at surfaces. Recently, a perspective by Weidner and Castner summarized the advancements made in adsorbed protein structure determination by SFG analysis.151 The authors illustrate that SFG can be a powerful tool to obtain protein structure via correlation with the results of other analysis tools such as NEXAFS, NMR, and molecular dynamics.

(13)

and for the E1 mode ⎛ 6.02 ⎞ ∂μ ⎜ ⎟ = ⎜ 6.02i ⎟ ⎜ ⎟ ∂Q ⎝ 0 ⎠

3.3. Carbohydrates

Carbohydrates are a much different class of compounds than peptides and proteins. Carbohydrate structural studies have remained in the domain of bulk nuclear and vibrational spectroscopic methods, along with X-ray diffraction. Recently, surface enhanced Raman spectroscopy has been used for detection of interfacial carbohydrates.152−154 However, these studies are limited to detection applications rather than structural studies. To date, the study of carbohydrate structure by SFG has been limited to a few papers. The first of these studies measured the CO, saturated C−H, and unsaturated C−H stretching regions of glucose bound to maleimide-terminated self-assembled monolayers (SAM) on gold.155 For the purposes of comparison between different length glucose chains, it was the saturated region that provided the most information (Figure 16). Three glucose adlayers were measured: C11-glucose, C16-glucose, and cysteineglucose. No difference between SFG spectra of the 11-carbon and 16-carbon glucose chains was observed. The implication of this result is that the orientation of the vibrational modes of the

(14)

Additionally, the Raman tensor elements of an 18-residue perfect α-helix were calculated. For the A mode ⎛ 6.1 0 0 ⎞ ∂α(1) ⎜ ⎟ = ⎜ 0 6.1 0 ⎟ ⎜ ⎟ ∂Q ⎝ 0 0 10.4 ⎠

(15)

and for the E1 mode ⎛0 0 3.6 ⎞ 1⎜ ∂α(1) ⎟ = ⎜0 0 3.6i ⎟ ⎟ 2⎜ ∂Q ⎝ 3.6 3.6i 0 ⎠

(16)

A similar study was completed for the antiparallel β-sheet structure.137 Using a bond additivity model, the dipole moment and polarizability tensor elements of the amide mode were calculated for a Pauling and Corey β-sheet148 structure. Focus was restricted to the B2 mode. Figure 13b identifies the orientation of the transition dipole moment (m) for this mode. Results of the calculatations are as follows: ⎛ 0 ⎞ ∂μ ⎜ ⎟ = ⎜ 3.80 ⎟ ∂Q ⎝ 0 ⎠

(17)

⎛ 0 0 −9.08 ⎞ ∂α(1) ⎜ ⎟ =⎜ 0 0 0 ⎟ ⎜ ⎟ ∂Q ⎝−9.08 0 0 ⎠

(18)

With this theory now in hand, detailed orientational and conformational analysis of protein structure was available from SFG data sets. Using the above methodology, Chen and co-workers probed chemically immobilized, modified cecropin (CP1c) on a polymer surface.149 They studied the solvent effects over time of this α-helix and found that the time it took the system to reach equilibrium was dependent on the peptide presence and concentration in the solvent. Two major results came of this work. The first was the demonstration that the adsorption of CP1c to polystyrene occurs in two steps, an adsorption step followed by a shift in orientation. This process was monitored (2) by time-dependent SFG in which χ(2) ppp/χssp was monitored over the course of 30 min. The second result is that the adsorption of CP1c dimers resulted in more than one orientation of the α helices. The source of this distribution is the formation of two layers of CP1c, one chemically immobilized and one physically adsorbed; it had been shown previously that chemically immobilized CP1c has a different orientation than its physically adsorbed counterpart.150 The real power in the backbone structure analysis described here come with the combination with other techniques. From these papers it is clear that combining SFG results with ATRFTIR is an essential tool for analyzing peptide structure and

Figure 16. (A) Precursor maleimide-terminated SAM. (B) Glucoseterminated SAM produced by covalent surface attachment. (C−E) Vibrational SFG spectra of the precursor SAMs (green, top traces) and product carbohydrate adlayers (blue, bottom traces) in three frequency regions: (C) CO stretch of the maleimide group, (D) saturated C−H stretch, and (E) unsaturated C−H stretch of the maleimide group. Green shades highlight the characteristic bands of the precursor maleimide SAM; blue shades highlight the bands of the product carbohydrate SAM. Spectra are vertically offset for clarity. Reprinted with permission from ref 155. Copyright 2009 American Chemical Society. 8399


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co-workers in 2005.159 The authors studied the phosphate charged group of ssDNA chemically immobilized on fused quartz by the SHG χ(3) technique. They were able to obtain interfacial charge density, potential and energy density. Sartenaer and co-workers shortly followed by publishing the first SFG study of a ssDNA monolayer on platinum.160 The authors compared the results in phosphate buffer solution (PBS) and a tris-EDTA (TE) solution. Results showed some intercalation of TE into the DNA monolayer, which was not the case for PBS. From the PBS solution, the authors were able to show the DNA SFG fingerprint. Single-stranded DNA films were further studied using SFG by Howell et al.161 Their study focused on 5-member oligonucleotides, thymine (T5) and adenine (A5), chemically immobilized on gold in air, D2O, and in PBS. Results from both the C−H stretching region as well as the amide region were presented. In the C−H region, the authors found two CH3 stretching modes: in plane at 2868 cm−1 and out of plane at 2954 cm−1. As the SFG signal was found to be strongest when the transition dipole moment is perpendicular to the surface, the authors were able to assess the CH3 group orientation from the ratio of those two modes. The results showed a significant difference between air and PBS. When placed in PBS, the methyl lost their orientation parallel to the surface in favor of a more random orientation. This was seen as a change from a predominance of out-of-plane stretching in air to an equal contribution of both modes upon adding PBS. Their results also showed that the thymine oligonucleotide behaves similarly in air and in D2O. They attributed the difference between the solvents to the ions present and the pH difference. In the amide I region, the peak around 1700 cm−1 is attributed to the amide in the presence of free CO. The authors related this to a horizontal brush-like structure, and it was present in the D2O and not at the air interface. For the adenine oligonucleotide, the results are all the more similar in the different environments caused by a preference of adenine to bind to the gold surface. This work illustrates the potential of SFG in assessing ssDNA orientation at surfaces by analyzing both amide I and C−H regions of the spectra. Boman et al. were the first to publish a study of both single strand and duplex oligonucleotides162 at a solid−liquid interface by SHG. Thymine oligonucleotides of different lengths were tethered to fused quartz, then the complementary adenine strand was added in solution. SHG analysis was performed before and after each step. +45° and −45° polarized light were used as the source for SHG analysis, and the ratio between the p-polarized components of the beams resulting from each incident beam was measured to obtain the SHG linear dichroism (SHG-LD). Figure 18 shows the time dependent SHG-LD response on (260 nm) and off (250 nm) resonance, respectively red and blue, of the hybridization. The hybridization of the thymine oligonucleotide is observed by a change in the LD ratio. In an SHG hybridization study of DNA at the silica−water interface, the Gibbs-Davis group demonstrated that they could distinguish between bound DNA and DNA in solution.163 The SHG response was monitored during the addition of complementary strands, and following several rinse cycles. An increase in signal during hybridization, and no significant drop in SHG after rinsing allowed the authors to conclude that the DNA complex is stable at room temperature, once formed. Varying the temperature enabled the points at which destabilization and dissociation occurred.

glucose group are the same regardless of the even or odd nature of the glucose. A separate study investigated the binding affinity of surfaceattached glucosamine to Zn2+ via SHG.156 In this study, SHG response at a glucosamide-functionalized surface was measured as a function of Zn2+ concentration in solution (Figure 17b). As described in section 2.1, this response is driven by the interfacial charge, which in this case is due to binding of the cation to the glucosamine groups.

Figure 17. (a) Schematic of the glucosamine-functionalize fused silica surface. (b) Adsorption isotherm for Zn2+ at glucosamide-functionalized fused silica−water interfaces. The SHG E-field is normalized to the water background signal. The solid line is a fit of the triple layer model. Reprinted with permission from ref 156. Copyright 2010 American Chemical Society.

A recent study has taken advantage of the noninversion symmetry (P21 space group) of the glucosyl group within cellulose to detect its crystalline form within plant cell walls.157 SFG has proven very effective at this measurement as it is sensitive to only the crystalline cellulose and not to the surrounding amorphous structures, which compromise the signal in both XRD and 13C NMR detection methods. The use of SHG and SFG in the study of carbohydrates has not matured to the extent that it has for peptides and proteins, yet the few studies in which they has been used have demonstrated their utility. We expect more studies of interfacial carbohydrates by nonlinear optical methods to be forthcoming. 3.4. DNA

DNA is the basis of evolution and has recently been an interesting target for new technologies, such as microarrays and biosensors. Hybridization, the process in which a DNA strand and its complementary strand combine, is key to most of these technologies, but little is known about the process. One challenge in studying DNA hybridization lies in the fact that labeling, commonly used to monitor the process, alters the DNA structure. Furthermore, for in situ hybridization studies, one DNA strand needs to be tethered to a surface, while the complementary strand remains in solution, thus calling for a surface specific technique. Nonlinear optical techniques, such as SHG and SFG, have been shown to be surface specific, labelfree techniques for the in situ study of single- and doublestranded DNA (ssDNA and dsDNA, respectively) at surfaces.158 In this section we discuss studies of ssDNA and their hybridization at solid−liquid interfaces as well as the possible effects of the environment on the hybridization process. Some more complex biological DNA systems studied by SFG and SHG will also be mentioned. 3.4.1. ssDNA and dsDNA. The first use of nonlinear optics for looking at DNA on a surface was published by Geiger and 8400


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often referred to as solid-phase hybridization. The challenge with solid-phase hybridization is the effect of cations, that can help or hinder the process by modifying the electrostatic field at the surface, and by stabilizing the charged DNA. The effect of metal cations on DNA hybridization was first studied by Asanuma et al. using SFG analysis.165 Monolayers of ssDNA were assembled on a silicon surface in the presence of ions, then complementary strands were added to initiate the hybridization process. Four different cations were considered: Mg2+, Ca2+, K+, and Na+. Figure 20 shows the resulting SFG

Figure 18. Time-dependent SHG-LD response on resonance (red) and off resonance (blue) from T25 ssDNA hybridized with A25, at 0.25 M NaCl and pH 7. Reprinted with permission from ref 162. Copyright 2009 American Chemical Society.

Howell et al. studied the hybridization of thymine and adenine homo-oligonucleotides and diblock-oligonucleotides by SFG164 combined with other techniques. The resonant contribution to the SFG spectrum was extracted (Figure 19)

Figure 20. SFG spectra of ssDNA before(red) and after(blue) hybridization in presence of different cations; (a) Na+, (b) K+, (c) Ca2+, and (d) Mg2+. Reprinted with permission from ref 165. Copyright 2008 American Chemical Society.

spectra before (red) and after (blue) addition of the complementary strand for each cation. From the ratio of CH3 to CH2 peak intensity in the ssDNA results, a trend is evident where Mg2+ seems to disrupt the orientation of the ssDNA the most. From correlation with XPS data, the authors conclude that the order is reversed for the double-stranded DNA, as Mg2+ seems to assist in the hybridization process. Monovalent cations, on the other hand, seemed to disrupt the hybridization. Most research on the interaction of ions with DNA at interfaces has been done by the SHG χ(3) method69−72 since its signal is related to the charge of the interface. In 2011 the Geiger group demonstrated that the Eisenthal method was well-suited to study metal ion interaction with ssDNA at the silica−water interface.70 They were able to quantify the number of Mg2+ ions adsorbed on the mono-oligonucleotide strand of adenine, cytosine, thymine and guanidine. From these results they were able to assess which nucleobase has the strongest affinity for this cation. Cytosine and guanine were shown to bind more than twice the amount of Mg2+ compared to adenine or thymine. The decay of the SHG signal with increasing ionic strength can be fitted to get the binding constant (Kobs) and the

Figure 19. Normalized SFG signal and its resonant contribution of different oligonucleotides before (black) and after (gray) hybridization with A15 or A6. The hybridization was performed at 1 M NaCl and pH ∼7. Reprinted from ref 164 with permission from PCCP Owner Societies.

along with the overall SFG spectrum. No signal change was observed for the short oligonucleotide when adding the A6 complementary strand. This result, in correlation with NEXFAS and XPS data, shows no hybridization occurring. The same can be said for the A10T15. The longer chain (T25) shows a lowering of the SFG signal, showing an overall loss of ordering when the A15 is added. The authors attribute this to a partial hybridization because of the length difference of the strands. On the other hand, the A5T15 shows an increase in ordering upon hybridization. 3.4.2. Cation Effects. The DNA hybridization process when one of the strands is chemically tethered to a surface is 8401


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maximum bound metal surface charge density (σm) which then allows a quantification of the number of ions bound to the interface as well as the binding free energy (ΔDbind). The Geiger group expanded their study of ssDNA at the silica−water interface to six other divalent cations: Sr, Ca, Ba, Mn, Zn, and Cd.69 This time, the oligonucleotide was A15T6. The authors also showed that the binding from all cations studied was a reversible process. A summary of the results of the SHG χ(3) analysis is shown in Figure 21. Stronger binding

Figure 22. Total free energy of binding of Mg cation on ssDNA composed of 10 adenine and 10 guanine nucleobases but of different sequencing. Reprinted with permission from ref 71. Copyright 2012 American Chemical Society.

sequencing. Combining the SHG results with AFM, the authors proposed that this drastic variation could be stemming from different spatial arrangements that can hinder the binding of Mg2+. A recent study looked at the binding of a trivalent metal cation, Y3+, on ssDNA tethered to silica. The strands, made of 20 guanine nucleotides, were analyzed by the SHG χ(3) method.72 Guanine was used because of it has the strongest binding with cations.70 The binding energy of Y3+ on G20 was found to be −39.5 kJ/mol, showing a much stronger binding than any of the cations studied previously. One to three ions were found to bind each strand. This value is much lower than for Mg2+, and the authors suggested that this significant difference may be due to the hydration sphere size difference between these ions. From these results, they also stipulate that the Y3+ is most likely in +3 or +2 (Y(OH)2+) form when bound to the 20-mer. 3.4.3. Complex Systems. DNA bound to microparticles are often preferred as they experience less steric interaction than on a flat surface and thus interact more freely with its environment. Adsorption of molecules on particles suspended in solution can still be assessed by surface-specific nonlinear optics in a scattering geometry.166−168 Here we show two examples of DNA studied using this technique. Using time-resolved SHG, Doughty et al. were able to observe the binding and cleavage of DNA by the EcoR1 enzyme.169 DNA were bound to polystyrene microparticle. Upon addition of EcoR1 (see Figure 23), a sudden increase of the SHG signal is caused by the bending of the DNA. Then, the signal slowly decreased with time upon cleavage of the DNA by the enzyme. They also noticed that a high salt concentration shifted the set equilibrium to rehybridize the cleaved DNA, shown by a regain of SHG signal intensity. Studies of DNA bound to silica bead in solution were performed by the Eisenthal group, assessing the relative orientation of the anticancer drug Daunomycin inserted in the DNA double strand.170,171 It was determined that daunomycin preferentially adhered to the TCG sequence of the DNA. From all these DNA studies, we can see that SFG and SHG allowed the study of ssDNA and DNA hybridization at surfaces.

Figure 21. Total free binding energy and bound ion density of different cations on ssDNA found by SHG. Reprinted with permission from ref 69. Copyright 2011 American Chemical Society.

can be seen for both Sr and Cd ions, whereas Zn and Ba has the lowest binding interaction with the ssDNA. The authors studied Mg2+ binding in more detail to deduce the binding process of the cations. This SHG speciation study consisted of measuring the binding energy at different ionic strengths; in this study, this was accomplished by varying the NaCl concentration. From the resulting data, combined with data from previous experiments, the authors concluded that Mg2+ binds as its fully hydrated form: Mg(H2O)62+. From the fitting of this speciation study, they were also able to extract a change in surface charge of +1.2. From this value, a predominant binding pathway was determined out of three proposed possibilities, where Na+ binds to the phosphate of the DNA before being dislodged by Mg2+, correlated to no observed change in surface charge. Holland et al. then assessed the effect of oligonucleotide length and sequence on the binding of Mg2+. They studied guanine and adenine nucleobases for their respective strong and weak binding with the metal cation, as found from a previous study.70 Again, the authors applied the Eisenthal χ(3) method to obtain the ion density and the binding energy. Monooligonucleotide length studies showed no significant changes in the binding energy. Corroborating earlier studies,70 the change in length of the strand only modified the ion density. For the guanine strand, a linear trend was observed between strand length and ion density, averaging one metal binding per phosphate negative charge. In the case of adenine, no change at all was observed, as only two cations bound on average, whether the strand had 5 or 21 nucleotides. From these data, the authors formulated a model to calculate the total free binding energy of any A-G strand by an additivity method. To assess the applicability of this method they studied, by SHG, oligonucleotides of 20 nucleobases with varying sequences. As seen in Figure 22, the total binding free energy varies greatly with the sequencing of the strand. This shows that an additivity method cannot be used directly to assess ssDNA of difference 8402


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Figure 23. (a) Cartoon of the experiment. (b) Time dependent SH intensity. EcoR1 is added at time zero; The red line represent the DNA bending then cleavage of the DNA by the enzyme. The green portion shown the results after addition of NaCl. Adapted with permission from ref 169. Copyright 2011 National Academy of Sciences, USA.

The development and application of techniques such as the Eisenthal χ(3) method and the use of scattering experiments, recently expanded DNA studies to more complex and realistic systems. 3.5. Lipids

Many of the important surface interactions of biomolecules occur naturally along lipid bilayers, which compose the membranes of cells, so it is important to develop surfacespecific techniques to study the structure and orientation of such interfaces. Historically, the best way to investigate the interactions of membranes with their environment, and with other biomolecules, was using exogenous labeling. However, concerns have been raised regarding the effects that such a process can have on the structure of the systems being analyzed. Recently, SFG techniques have been developed to provide information on lipids in monolayers and bilayers, as well as the interactions of other molecules with these interfaces. Though monolayers are typically studied at the air−liquid interface, where the hydrophobic tails of the lipids naturally orient themselves into the air, the use of SFG to study bilayers at liquid−substrate interfaces has garnered much interest in the scientific community.172,173 Due to the symmetry constraints of SFG analysis, it is expected that lipid monolayers produce a strong SFG signal whereas bilayers, where two monolayers oppose one another symmetrically, produce none. However, SFG has been found to be a useful technique for studying certain aspects of lipid bilayers when the symmetry of the bilayer is perturbed. The lipid molecules referenced in the highlighted examples are shown in Figure 24. Liu and Conboy174,175 have used SFG to study the phase change of a lipid bilayer from gel to liquid-crystalline phase around the phase transition temperature. In these studies, the important feature monitored in the SFG spectrum is the magnitude of the peaks as a whole, which reflects the level of asymmetry in the membrane. The premise of these studies is that the transition between phases occurs gradually over the

Figure 24. Structures of lipids mentioned in this section.

bilayer as a whole, and that effects of the transition, which involve changes to the orientation and energy of bonds, are distributed asymmetrically through the bilayer. This is shown in the upper portion of Figure 25, where transitioned red lipids arise in different places of the upper and lower bilayer, thus creating asymmetry. Liu and Conboy also propose that more may transition in one monolayer than another, thus creating another source of asymmetry. The resulting asymmetry is shown in the spike of SFG signal in the CH3 stretching region at the transition temperature, with results of analysis of three different lipids shown in the lower part of Figure 25. In another study, Liu and Conboy again used SFG and the symmetric tendency of a lipid bilayer in order to observe, for the first time without chemical labeling, the phenomenon of lipid flip-flop,26 the exchange of lipids from one monolayer to the other. Flip-flop is an important process which explains the mechanism by which lipids and proteins can cross the bilayer, but previously was unconfirmed due to the necessity of using invasive techniques to study it. For example, it was shown that fluorescent labeling techniques significantly slow down the flip flop process.176 In this novel approach, a lipid bilayer was created using two different lipid monolayers- DSPC and deuterated DSPC respectively- thus creating an SFG-active bilayer where in its initial, fully asymmetric state, the SFG signals for the C−H and C−D stretches were at a maximum. Exchange of lipids between layers- lipid flip-flop- was shown by 8403


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The Conboy group have investigated the exchange of lipids between monolayers in the presence of two transmembrane proteins, WALP23 and melittin,177 as well as cholesterol.178 The study of the effects of WALP23 and melittin177 was focused on determining whether the hydrophobicity of the transmembrane component of the embedded protein had an effect on the exchange rate of the lipids between layers. This might be expected because WALP23’s transmembrane component is more hydrophobic than melittin’s, which contains hydrophilic residues. The results showed that melittin facilitated flip-flop at a higher rate than WALP23, which is likely a result of the hydrophilic head groups of the lipids having an easier time crossing the membrane in the presence of the hydrophilic residues on melittin. The study concluded that the flip-flop was conducted via different mechanisms in each case based on thermodynamic analysis of flip-flop in the presence of each protein. Reduction of the energy needed for the transmembrane movement of lipids in the presence of WALP23 is thought to be a result of the destabilization of the initial lipid bilayer. With melittin, a toroidal pore formation of the proteins is suggested to occur, where head groups of lipids interact with the hydrophilic sites along the protein to form the sides of a channel. In this case, head groups would never need to be transported through the membrane at all, as the monolayers are essentially connected through the sides of the pore. This agrees with an earlier study conducted by the Chen group179 in which melittin’s interaction with an asymmetric DPPG bilayer was studied through SFG analysis of the C−H and C−D stretching modes. In the cholesterol study,178 investigations were made into the effect of cholesterol on lipid flip-flop, as well as the flip-flop of the embedded cholesterol itself. Similarly to WALP23 and melittin, cholesterol was shown to increase the rate of lipid flipflop. Thermodynamic data was used to suggest that cholesterol provides a lower-energy pathway for a lipid headgroup to traverse the membrane. To study the flip-flop of cholesterol itself, a symmetric bilayer was used, and cholesterol molecules were deposited in the membrane selectively oriented in one direction. The decay of the intensity of cholesterol’s characteristic SFG signal was used to show that cholesterol molecules became symmetrically oriented within about 10 min. The authors also concluded that asymmetry of the cholesterol molecules was not a factor in the study of the lipid flip-flop, which took significantly longer (hours) to equilibrate. Neivandt and colleagues investigated the perturbation of a DSPG hybrid bilayer membrane (HBM) by signal peptideless (SPL) proteins using SFG. In this experiment, a vesicle solution of DSPG was allowed to equilibrate with a surface coated with perdeuterated octodecanethiol (d-ODT), encouraging the spread of the vesicles to form a monolayer attached to the dODT through vesicle fusion. This approach negates the symmetry difficulties which arise when analyzing lipid bilayers themselves, given that all of the lipids are on one side of this hybrid bilayer. The experimental data revealed a drop in intensity of all SFG peaks in the C−H stretch region upon addition of the SPL protein FGF-1, and the subsequent reversal of this phenomenon upon rinsing with a PBS buffer solution. In all of the previously mentioned studies of lipid bilayers, and molecules interacting with lipid bilayers, the bilayers are supported by a solid surface, often silica. Chen and co-workers have examined the effect of placing such artificial membranes on a polymer cushion, so as better to better imitate the cell membrane environment.180 In this study, the SFG spectra of

Figure 25. Magnitudes of SFG peaks for lipid bilayers below, during and above phase transition temperature. Above picture shows justification for loss of symmetry at Tm. Reprinted with permission from ref 174. Copyright 2004 American Chemical Society.

the decay in the C−H stretching signal in the SFG spectrum. The signal approaches zero as the bilayer approaches an equal and symmetric distribution of lipids between the two monolayers. Figure 26 shows the experimental data collected

Figure 26. Intensity decay of CH3 signal over time. Blue line shows signal decay at 41.7 °C, green at 45.7 °C, and red at 50.3 °C. Dashed lines represent fits to the data. Reprinted with permission from ref 26. Copyright 2004 American Chemical Society.

from the experiment run three times at different temperatures. Naturally, the rate of decay of the SFG signal, corresponding to the rate of lipid flip-flop, increases with increasing temperature. Symmetry via lipid flip-flop is shown to be accomplished over a matter of hours. Analyzing the decay of the SFG signal has been used in investigations of different proteins on the rate of lipid flip-flop. 8404


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external surface of the liposomes’ bilayer membranes, the SHG signal increased. Importantly, the diameter of the liposomes is on the scale of one wavelength of the visible beam used. This means that opposing SHG signals emanating from MG+ molecules on opposite sides of the exterior of the liposome, in line with the incident laser, which mirror each other in terms of orientation, add constructively, rather than destructively, as shown in Figure 27a. Thus, despite the symmetry of the

asymmetric lipid bilayers (DPPG/d-DPPG) both on a PLLA cushion and on a calcium fluoride substrate were compared. DPPG is a fully saturated lipid, and thus centrosymmetry exists for all nonterminal C−H bonds, making only the terminal methyl group SFG-active on the hydrophobic tail. By examining the SFG spectrum, the orientation of this terminal group could be deduced and in both cases was determined to be around 17°off normal. Fitting the spectra in full suggested that the structures were equivalent in both cases, which means that the polymer cushion does not alter the structure of the lipid bilayer. In addition, the study was furthered by analyzing the orientation of cecropin P1 on both a cushioned and uncushioned bilayer, and no significant difference between the spectra was found. In studies where the investigation is focused on biomolecules interacting with the bilayer or monolayer, rather than the lipids themselves, the role of the lipids, often in the form of a planar supported lipid bilayer (PSLB), is to mimic a cell membrane with which the target molecule can interact. The major reason for using a polymer cushion instead of simply having a membrane on the substrate is that for studies involving transmembrane proteins, or other molecules which would in a real cell have room to move on both sides of the membrane, there is typically no room between the membrane and the surface on which it is supported. Polymer cushions create more room between the membrane and the surface. While they still support the lipid bilayer, they also allow the transgression of biomolecules through the membrane and provide some interstitial space between surface and membrane. This should allow for studies on such molecules in a more realistic environment. Alternatively, vesicles can be used as the surface, but given their overall centrosymmetry, their size must be precisely controlled in order to generate a χ(2) response, as will be seen in the following section.

Figure 27. (a) Constructive interference of SHG signals from externally oriented MG molecules and (b) destructive interference of SHG signals from internally oriented MG molecules. Reprinted with permission from ref 181. Copyright 2000 Biophysical Society.

liposome bilayer, the SHG signal is not canceled. While MG+ molecules oriented the same way on the external layer of the liposomes add their SHG signals constructively (Figure 27a), molecules at the same position of the liposome but on opposite sides of the bilayer add destructively (Figure 27b), and thus the movement of MG+ from the external layer to the internal layer causes a decay in the SHG signal. This work was more recently followed up by a study on the effects of different ionophores, which are lipid-soluble molecules responsible for transporting ions across a membrane, on the kinetics of MG+ diffusing across a model membrane.183 In this study, malachite green ions are initially added into a solution of the negatively charged DOPG liposomes, resulting in a steady decrease of SHG signal as they transport across the liposome membranes due to a concentration gradient. The transport of these organic cations into the liposomes creates a charge gradient which opposes further transport of MG+, and as such the decrease in signal is slowed down to just about steadystate. After 9−10 min, an ionophore was added, either valinomycin (VAL), gramicidin A (gA), or cyanide-mchlorophenylhydrazone (CCCP; Figure 28. These ionophores insert into the liposome membranes and facilitate the transport of inorganic cations from within the liposomes to the bulk solution, which balances the charge gradient and allows further diffusion of MG+ across the membrane to further satisfy the concentration gradient. This results in a second drop in SHG signal, which eventually again reaches steady-state as the concentration gradient becomes balanced. The same group also applied SHG to study the orientation of another dye molecule (Texas Red, DHPE) on a glass-supported DOPC/DOPG bilayer.184 A polarizer in the detector path was nulled in order to determine the orientation of the dye on the bilayer. Without a lipid bilayer, the dye molecules were found to orient themselves at about 33° relative to the surface normal, and on a bilayer, they were found to orient themselves at about 19°. 3.6.2. Peptides and Proteins. When applying nonlinear optics to peptides and proteins, the focus shifts from location to conformation and orientation. In many cases, the locations of specific peaks are analyzed with particular scrutiny as signatures

3.6. Molecules at Lipid Bilayers

Nonlinear optics are ideal for studying the interactions of biomolecules with lipid bilayers due to their surface-specificity and their ability to provide information on conformation and orientation. Given the symmetry restrictions of these techniques, only molecules interacting with an interface provide signal, and in the case of a lipid interface, this includes only molecules interacting with the lipids. With this in mind, nonlinear optics can determine the tendency of biomolecules to interact with and adsorb on lipid interfaces and in addition their conformation and orientation therein. The orientation of biomolecules can, in many cases, determine whether the molecule inserts into the lipid layer or stays separate. 3.6.1. Small Molecules. Some interesting techniques for studying molecules’ interactions with bilayers were developed by the analysis of dye molecules traversing bilayers. Just like the studies on lipid bilayers themselves, only the fluctuation in intensity of specific signals is used in order to compare levels of symmetry and thus deduce the presence of molecules on either side of the bilayer, which cancel each other out and deplete the signal due to the symmetric restrictions imposed by the technique. In one of the first applications of nonlinear optics to study molecules interacting with a bilayer, Eisenthal and co-workers used the symmetry-sensitivity of SHG in order to show the retarding effect of cholesterol on trans-membrane motion of an organic cation, malachite green (MG+).181,182 In a suspension of DOPG liposomes, MG+ was added, and as it adsorbed to the 8405


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orientation of MSI-78 was determined at different concentrations for each type of lipid bilayer composition, using a previously established method analyzing magainin 2.186 Figure (2) 29 shows the values for the strength ratio of χ(2) ssp /χppp for each

Figure 29. SFG intensity ratios for different concentrations of MSI-78 determine proposed average orientation of the AMP relative to the DPPG/d-DPPG surface normal. Reprinted with permission from ref 185. Copyright 2011 American Chemical Society.

concentration of MPI-78 in contact with a DPPG/d-DPPG asymmetric bilayer in the colored lines. The black line then shows this value as a function of the average tilt angle of a delta distribution of alpha-helices on the bilayer using their previously established method. The average tilt angle of the alpha-helices can be deduced for each concentration of the AMP by finding the angle where the colored line corresponding to that concentration intersects the black line. The results showed that as concentration increases, the angle between the principal axis of the α-helical AMP and the line normal to the bilayer decreases, which corresponds to the transition of the MSI-78 molecules from lying flat on the surface to inserting end-first into the membrane. In addition, the SFG peaks in the C−D and C−H stretching region were separately analyzed in order to look at the activity of the distal and proximal leaflets of the bilayer. As shown in Figure 30, the C−D stretching signal intensity from d-DPPG dropped off significantly compared to the C−H stretch from DPPG at just 400 nM of MSI-78. This indicates interaction of the AMP with just the distal leaflet, as opposed to insertion into the membrane, which would affect both leaflets. The signal is suggested to decrease as interactions disrupt the leaflet in such a way as to reduce the ordering of the terminal CD3. When the concentration of MSI-78 is increased to 600 nM, both the C−D and C−H stretch intensities drop, which indicates that both leaflets are slightly disrupted by the AMP. This corroborates the data collected from the amide I region, where the MSI-78 molecules appear to orient themselves perpendicular to the surface, and in a transmembrane position. At 2000 nM, both signals are weak, and the AMP is thought to adopt a toroidal pore conformation. MSI-78 was studied in contact with several different bilayer compositions in the study. DPPG is a model for a bacterial cell membrane, and given that bacterial cells are the target for AMPs, the results were as expected. When DPPC, a model for a mammalian cell membrane, was used instead, there was comparatively no SFG signal in the amide I region, indicating no interaction or ordering of the AMP along the bilayer interface, even at concentrations more than thirty times those

Figure 28. Drop in SHG signal due to diffusion of MG+ across lipid bilayer is reinitiated by addition of ionophores (a) VAL, (b) gA, and (c) CCCP. Adapted with permission from ref 183. Copyright 2008 American Chemical Society.

of specific secondary structures, and the phases of these peaks can then reveal orientation. Chen and co-workers have performed extensive studies of the orientation and structure of different antimicrobial peptides (AMPs) at lipid bilayers. One such study examined the interaction of the AMP MSI-78 on various lipid bilayer compositions using SFG.185 The lipid bilayers were asymmetrically prepared, as in the Conboy group when analyzing flip-flop, in order for them to generate SFG signal. The signal at around 1650 cm−1 of the amide group in MSI-78 was used to study the presence and orientation of the AMP, whereas the C−H stretching modes in the 2700−3100 cm−1 region and the C−D stretching modes in the 2000−2300 cm−1 region monitored the degree of order in the proximal and distal leaflets of the bilayer respectively. Using both ssp and ppp polarization schemes, the 8406


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orientations of both α-helices and β-sheets within the molecules at the bilayer. The results showed that increasing the concentration of the protein fragment increased the amount of β-sheet character in the secondary structure, where at low concentrations the fragment was arranged mostly in α-helices. The α-helices were oriented close to perpendicular to the surface normal, while the β-sheets were oriented parallel to the lipid bilayer. Koelsch and colleagues used SFG to study N-acyl-Lhomoserine lactones (AHLs) embedded within 1:1 POPC/ POPG bilayers during a process known as interkingdom signaling.198 In this experiment, deuterated AHLs of various lengths were placed both on bare silica and a supported lipid bilayer while the C-D stretching SFG region was monitored. No signal was observed for AHL-d9 without an SLB present, but three different signals were observed in the region for each of AHL-d9, AHL-d9, and AHL-d9 in the presence of the bilayer. Using a combination of IR and Raman spectroscopy, the conformation of the AHLs was determined to be all-trans in all cases, and by analyzing over a 5-h period, it was shown that the SFG signals in the region of interest did not drop off significantly, indicating a very low rate of flip-flop of the molecule in the bilayer. The group was interested in determining whether the molecule existed primarily in the proximal or distal leaflet, and suggested that this could be elucidated from the orientation “up” or “down” of the molecules within the SLB. By fitting the nonbackgroundsuppressed SFG spectra, they were able to determine that the molecule was likely oriented pointing toward the surface, indicating its presence is mainly in the distal leaflet. Yan and co-workers investigated the interaction of the polypeptide hIAPP with model membranes.199 The team relied on an ab initio model to predict the chiral SFG spectra resulting from the orientation of the polypeptide on a lipid bilayer, then used experimental SFG results to elucidate the actual orientation. Previously, Yan and co-workers had demonstrated the application of chiral SFG to determine protein secondary structure at membrane interfaces,200 and change over time of secondary structure of proteins,201 with both studies conducted at air−water interfaces in the presence of lipids. These earlier studies provided a series of SFG signatures which helped determine the secondary structures present in the molecule. 3.6.3. Complex Systems. Nonlinear optics has been applied to quite complex systems interacting with lipids. Such experiments enable investigation into the function and characteristics of complex molecules which are biologically relevant, such as DNA and G-protein coupled receptors. Once again, the surface-specific nature of SFG analysis is a significant aid in such studies. In gene therapy research, DNA is transported through lipid bilayers easily, but the release of the DNA (after crossing the boundary) is still an ongoing issue. To assess this, monolayers can be used as a model to study DNA interaction with lipids. Bonn and co-workers have been studying the adsoprtion of DNA to lipid monolayers in water, and the resulting orientations of the water and lipids, by SFG.202,203 Figure 31 illustrates the experiment where a lipid monolayer is formed at the water−air interface. DNA is then added in solution and adsorbs on the hydrophilic headgroups of the lipids. In their first paper on the subject,202 the authors show that both the lipid monolayer and the water show a strong SFG response and therefore a strong orientation preference. These features change upon adsorption of the DNA strands as the water

Figure 30. Different effects on C−H stretch (proximal leaflet, top) and C−D stretch (distal leaflet, bottom) of DPPG/d-DPPG bilayer with increased concentration of MSI-78. Reprinted with permission from ref 185. Copyright 2011 American Chemical Society.

of DPPG, which highlights the selectivity of the AMP. POPG (bacterial) and POPC (mammalian) were also studied. MSI-78 interacted very weakly with POPC, and quite strongly with POPG, with which it adopted a torroidal formation at lower concentration than with DOPG. Recently, Chen and co-workers have studied the orientation near membranes of the AMP LL-37, an AMP with a bent shape, given different membrane compositions.187 Chen and coworkers have studied alamethicin,188,189 melittin,190 tachyplesin I,137 cell-penetrating peptide Pep-1,191 G proteins,192,193 cytochrome b5,194 and arylamide oligomer 1.195 Both LL-37 and alamethicin are composed of two different α helices with a bend between them. Previous research was capable of determining the contributions of each helix to individual peaks in the amide I region. For example, in the case of alamethicin, one helix’ orientation can be elucidated from the (2) −1 χ(2) and the other from eff,ppp/χeff,ssp ratio of the peak at 1638 cm −1 the peak at 1671 cm , while the ordering of the peptide can be determined from the peak at 1705 cm−1. Luo and co-workers used SFG analysis to study the mechanism of insertion of the peptide mastoparan (MP), which has an α-helical secondary structure, into differently charged lipid bilayers with increasing phosphate buffer concentrations.196 The peptide was found to insert into negatively, neutrally and positively charged bilayers. The tilt angle of the helices in general became smaller in the case of neutrally and negatively charged bilayers, while adopting mutliple orientations in a positively charged bilayer. Luo and co-workers have also used SFG to analyze the protein fragment PrP 118−135 in the POPG bilayer,197 including using the position of the peak to determine the secondary structure of the molecule at the bilayer at different concentrations, and the 8407


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Figure 31. Cartoon of the SFG experiment showing the DNA in water adsorb on the hydrophilic headgroup of the lipids. Reprinted with permission from ref 202. Copyright 2007 American Chemical Society.

changes orientation. Furthermore, by varying DNA concentration and comparing to an ion screening study, the authors concluded that the addition of DNA squeezes out the water molecules, leaving less than a monolayer of water between the DNA and the lipid bilayer. They then studied the effects of varying the monolayer using different cationic and zwitterionic lipids203 in a D2O environment. They also looked at the response of those systems in the presence or absence of CaCl2. DNA was found to bind to the zwiterrionic lipid (DPPC) only via the preliminary binding of Ca2+. By looking at the CH stretching region of the SFG signal, the authors found that lipids have a greater orientation ordering in the presence of DNA. Another example of a complex biological system at a lipid interface studied by nonlinear optics is the study of proteinreceptor complex formation at a model membrane performed by Chen and co-workers.193 To study this large system, the authors calculated the theoretical χ(2) of all the α-helices of the protein for different tilt and twist orientations. Assuming no deformation of the protein upon complex formation, they then fitted their SFG spectrum with the calculated χ(2) to obtain specific population possibilities. They recently combined this analysis with ATR-FTIR dichroic data204 to narrow down orientation possibilities of Gαiβ1γ2 and Gβ1γ2 before and after complex formation. Figure 32 shows the possible orientations of Gαiβ1γ2 (Figure 32A) from the SFG χ(2) ratio of different polarization beams and (Figure 32C) from ATR-IR dichroism experiments. Even though the SFG experiment shows fewer orientation possibilities than IR, it is the combination of the two techniques (Figure 32D−E) that really narrows down the possibilities. In conclusion, use of nonlinear optical techniques has greatly enhanced the ability of researchers to analyze molecules specifically when interacting with lipid bilayers. The studies above show the application of these techniques to determine the presence, structural conformation, and orientation of molecules of various sizes, all using different aspects of SFG or SHG spectra.

Figure 32. Possible orientations of Gαiβ1γ2 determined by (A) the (2) SFG ratio of χ(2) zzz/χxxz (2.7 ± 0.3) and the possible orientations of myr(2) Gαiβ1γ2 determined by (B) the SFG ratio of χ(2) zzz/χxxz (2.5 ± 0.3). Orientations of Gαiβ1γ2 at which the calculated values best match experimentally measured values for (C) the ATR-FTIR dichroic ratio RATR (1.8 ± 0.2). The product of these two measurements of Gαiβ1γ2 (D) further narrows the range of possible orientations. (E) Same plot as panel D but only showing orientation areas with a score ≥70% (red). Reprinted with permission from ref 204. Copyright 2013 American Chemical Society.

between a whole cell and a gold surface (in this case being the fibronectin) can be assessed by SFG. Diesner et al. furthered this idea by probing a SAM surface through living cells.206 The same trend was observed (see Figure 33), as the SFG signal did not change with the presence of the cells, but the IRRAS signal did. The SFG signal was corresponding to the SAM. The authors conclude that SFG is a powerful technique to study interfacial region even in presence of living cells. Ordering of the extracellular matrix (ECM) of living cells was assessed by different techniques including SFG.207 They first studied the response of artificial FN fibrils, of collagen I fibers and of BSA adsorbed on substrates to understand any possible SFG signal originating from other sources than the ECM fibrils. Figure 34 shows the result of the time dependent SFG signal of the orientation of the ECM. The result show an evolution of the SFG signal with time with a peak at around 280 min, and after then the signal slowly goes back to noise level. The authors saw the same results with a different cell, but with a peak at different time. A parallel fluorescent microscopy study (see Figure 34) show the growth of the fibrils, which could be seen in the absence of the cell. The authors conclude that that ECM fibrils start developing and orienting, but after a certain time, the ECM fibrils formation

3.7. Cells

The development in nonlinear optic understanding and application allows for studies of systems of growing complexity. One recent breakthrough in SFG is the study of whole living cells. In 2008, Koelsch and co-workers studied the SFG response of a embryonic fibroblast adsorbed on fibronectin on a gold surface.205 Fibronectin (FN) is used to promote cell adhesion. Comparing to IRRAS results, the authors showed that adsorption of the cells does not significantly perturb the SFG signal but drastically changes the IRRAS signal. This proves that the cell did actually adsorb to the fibronectin but that its centrosymmetric properties makes it transparent to SFG. From these results, the authors conclude that the interface 8408


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addition to increased cost and operational complexity over their linear analogues, SHG and SFG traditionally suffer from reduced signal-to-noise (S/N) and reduced spectral resolution. One of the factors that is responsible for the low S/N is long acquisition times, especially in case of SFG experiments where vibrational resonance is often the only enhancement to the signal from weak NLO chromophores. In the case of scanning systems (nanosecond and picosecond pulse widths), recent advances in laser technology such as the use of solid-state saturable absorbers and air-sealed, diode pumped oscillators and amplifiers, have significantly reduced the shot-to-shot noise, reducing acquisition times or providing increased signal averaging for the same acquisition time. Broadband SFG setups (50−100 fs IR pulse widths) have been popular due to their ability to acquire a spectrum quickly, and without any moving components in the beam path. The latter point is especially important as it permits a close examination of the spectral line shape, including subtle shoulders on peaks, without the possibility of artifacts due to tuning the IR energy. However, broadband systems have typically obtained 10−15 cm−1 resolution, while scanning systems routinely achieve resolutions better than 5 cm−1. High resolution broadband SFG is an emerging technology that utilizes a narrow band visible laser (90 ps) synchronized to a 100 fs IR source.210,211 This results in ∼0.6 cm−1 resolution, with the spectra acquired at once on a CCD array. Such an experiment has recently been demonstrated to resolve spectral splitting as small as 2.8 cm−1 in the C−H stretching region.210 4.1.2. Phase Measurement. Although we have mentioned a couple of aspects in which SHG and SFG spectroscopy need to catch up in comparison with their linear analogous, the nonlinear techniques offer a major advantage that stems from their interfacial specificity. Since these techniques require that χ(2) ≠ 0, there must be a break in inversion symmetry. That is, only structures that exhibit polarity in their orientation give rise to SHG and SFG signals. In other words, if molecules are aligned at the solid−liquid interface, but there is no preference to their head-to-tail orientation with respect to the surface, χ(2) = 0. It is worth mentioning that a polarized IR absorption spectrum would probe a high order parameter for such an arrangement; there is simply no polar component of the orientation distribution. The corollary to this is that SHG and SFG are able to resolve the polarity of this orientation distribution, thereby providing further and unique information on the adsorbed structures. When the molecules are flipped in orientation, the sign of χ(2) changes. That is, the resulting SHG or SFG signal is shifted in phase by 180° compared to what would be generated in the opposite orientation. (This is the nature of the surface selectivity that results in χ(2) = 0 for equal populations of both orientations, or an isotropic distribution as in the bulk solution phase.) This also indicates that elucidating the absolute orientation of the molecules entails a measurement of the SHG or SFG phase, performed in an interferometric experiment. Several papers and review articles cover the basic concepts of phase resolved SHG and SFG spectroscopy.212−224 In brief, the two pump beams are used to create the nonlinear signal from the sample under investigation, and simultaneously from a reference material. The latter SFG field, termed the local oscillator (LO), may be generated in reflection or transmission geometry, before or after the sample. The two fields are then made to interfere at the detector, and analysis of the interference fringes reveals the phase difference, Δϕ, between sample and local oscillator SHG or SFG fields. The experiment

Figure 33. (A) IRRAS spectra and (B) SFG spectra of the fibronectin and SAM surface with and without the cells present. (Middle) Cartoon of the experiment. Reprinted with permission from ref 206. Copyright 2010 American Chemical Society.

Figure 34. Reprinted with permission from ref 207. Copyright 2011 Springer.

keep going, but with regions of different orientation, lowering the nonlinear signal.

4. PERSPECTIVE We have demonstrated that second-order nonlinear techniques such as SHG and SFG are uniquely suited to studies of biomolecules at the buried solid−liquid interface. For small molecules such as amino acids, fine structural details regarding their orientation and conformation at the surface may be resolved. In the case of larger molecules such as proteins, or more complex systems such as multiprotein assembles in membranes, the emphasis is currently shifted toward analysis of spectral changes. With ongoing developments in experimental techniques, aided by electronic structure calculations and molecular simulations, we are gradually observing an increased level of structural detail coming out of studies of larger biomolecules. We highlight a few of these advances below. 4.1. Experimental Advances

4.1.1. Enhanced Signal-to-Noise and Spectral Resolution. Compared to electronic and vibrational absorption spectroscopy, SHG and SFG can be label-free and are generally more sensitive to subtle structural variations.208,209 However, in 8409


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biaxial distributions where the tilt and twist angles of a particular chemical moiety are described, an extension of this formalism may be used.238 The Chen group has such such an approach to restrict the solutions of candidate orientation distributions of adsorbed proteins.204,239

is typically repeated with a material of known orientation or phase in place of the sample. The absolute phase of the sample ϕS is then given by the difference of these two measured phase shifts, offset by ϕR, the absolute phase of the standard ϕS = ΔϕS − LO − ΔϕR − LO + ϕR = (ΔΔϕ)S − R + ϕR

4.2. Electronic Structure Calculations and Molecular Simulations

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thereby removing the dependence of the LO phase shift. The majority of phase-resolved studies to date have been performed at exposed surfaces, owing to the additional complexity in the refractive index mismatch at a buried interface. Nevertheless, as techniques continue to progress, phase-resolution will play an important role in future studies of biomolecules at solid−liquid interfaces. 4.1.3. Microscopy. Nonlinear optical microscopy188,225,226 is an emerging and transformative field with the potential for great impact for biomolecule investigation. SH microscopy, in particular, has gained significant attention as it provides a contrast mechanism and therefore imaging modality that is complementary to what may be achieved using fluorescence and other techniques.227 However, it may also be performed quantitatively, thereby providing a spatial map of molecular orientations.228,229 When microscopy is extended to SFG, we add surface chemical mapping, as the response from specific vibrational modes may be plotted with respect to lateral dimensions of the sample.230−233 We have excluded these topics from our discussion, as they deserves special attention and are best reviewed together with other linear (fluorescence) and nonlinear (CARS) imaging techniques. In the future, we expect that microscopy based on SHG and SFG will not only contribute to our understanding of spatially resolved structure at surfaces, but will also enable multiplexed, high-throughput analysis based on arrays234−236 and microfluidic flow assemblies in the imaged regions. 4.1.4. Combining Multiple Techniques. Although we have illustrated that second-order nonlinear spectroscopy provides a unique structural probe for biomolecules at the solid−liquid interface, there are an increasing number of studies that combine the information content from SHG or SFG with that from other experiments. Among the studies that we have discussed, many have taken advantage of characterization tools such as NMR, near edge X-ray absorption fine structure (NEXAFS), time-of-flight secondary ion mass spectrometery (ToF-SIMS), quartz crystal microbalance (QCM), and atomic force microscopy (AFM). There are also complementary spectroscopic techniques. Of particular interest are those that probe the same vibrational modes as studied in SFG experiments. While techniques such as IR and Raman spectroscopy do not have the same interfacial selectivity as SFG, they provide orthogonal data that may be used to refine the experimentally determined orientation distribution. For example, if we consider a uniaxial distribution f(θ) expressed as a series of Legendre polynomials Pi(cos θ) ∞

f (θ ) =

∑ i=0

1 (i + 1)⟨Pi⟩Pi(cos θ ) 2

From the highlights we have provided, it is clear that SHG and SFG spectroscopy provide a sensitive probe of biomolecular structure at the solid−liquid interface, and that these experimental techniques alone cannot provide direct structural information. Throughout our examples, we have illustrated that computer simulations play a critical role in this effort. While a detailed discussion of these methods is beyond the scope of this review, we draw attention to the role of both static electronic structure and molecular dynamics simulations in complementing SHG and SFG studies. Many quantum chemistry packages provide facilities for computing α(2) in the case of electronic SHG. For vibrational SFG, this information is still accessible by invoking an harmonic approximation to the transition polarizability and transition dipole moment. The transitions appearing in the numerator of eq 7 may then be written as ⟨0|αlm ̅ , q|1⟩ ≈

⟨1|μn̅ , q |0⟩ ≈

∂αij(1) 1 2mqωq ∂Q ∂μk 1 2mqωq ∂Q

(21a)

(21b)

α(1) ij

where is an element of the linear polarizability tensor, μk is the k element of the dipole moment vector, m is the reduced mass, and Q is the coordinate of the qth vibrational mode. With this approach, all nine elements of the linear polarizability derivative, and 3 elements of the dipole moment derivative may readily be calculated for each of the 3N − 6 normal modes of interest, and any of the 27 elements of α(2) assembled via eq 7. In cases where the molecule is too large for such calculation, a local mode approximation may be used for a particular chemical group of interest, for example, methyl groups in a protein.107 Even in cases where individual normal modes cannot be experimentally isolated for analysis, it is possible to use calculations of α(2) for calculated normal modes to predict SFG spectra.81,240−249 Roy et al. have used this approach to simulate the aliphatic C−H and N−H stretching regions of LKα14, as illustrated in Figure 35a.247 The blue spectra are the (2) calculated Im[χxxz ] spectra (as would be obtained in a heterodyne ssp-polarized experiment) for LKα14 adsorbed onto a hydrophobic surface. Red spectra are the results for adsorption onto a neutral hydrophilic surface; charged hydrophilic surface results are shown in green. One of the advantages of such calculations is that one can readily isolate the contribution of a particular functional group or region of the molecule to the overall spectra. This has been done for the hydrophobic leucine side chains (Figure 35b) and hydrophilic lysine side chains (Figure 35c). At the charged surface, the authors noted that the signal originates primarily from a strong ordering of lysine side chains, with almost no contribution from the leucine side chains. Such analysis may be compared directly with results of isotopic labeling experiments to elucidate the change in structure accompanying adsorption.31 The continuing advancement of such modeling techniques will allow for

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experimental data may be used to provide the order parameters, ⟨Pi⟩. Second-order techniques such as SFG are sensitive to only polar orientation distributions, and provide a measure of ⟨P1⟩ and ⟨P3⟩. A polarized infrared absorption spectra can provide the missing ⟨P2⟩, while Raman spectra acquired under different beam polarizations will produce ⟨P2⟩ and ⟨P4⟩.237 In the case of 8410


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electronic structure calculations are tractable. Second, the orientation of the molecular hyperpolarizability within the nuclear framework is well-defined. When the molecular response (elements of α(2)) is related to the measured response (elements of χ(2)), essentially solving for parameters in the presumed orientation distribution function, the orientation of chemical bonds with respect to the surface is obtained. In the case of peptides and proteins, a local mode approximation enables sections of the molecule to be treated in much the same way as for small molecules, particularly for high frequency vibrational modes. When the systems become more complicated, as in the study of live cells, the modality becomes more focused on observing and accounting for changes in the response. The combination of experimental advances and complementary simulations is pushing studies of molecules of all sizes forward. In the next 5−10 years, we expect to see the equivalent level of structural detail as is currently possible for small molecules available for proteins at surfaces.

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. χ(2) xxz

Notes

Figure 35. Simulated signal contribution from the (a) lysine side chains, (b) leucine side chains, and (c) all side chains combined for LKα14 adsorbed on an hydrophobic (blue), neutral hydrophilic (red), and charged hydrophilic (green) surfaces. Reprinted with permission from ref 247. Copyright 2013 American Chemical Society.

The authors declare no competing financial interest. Biography

understanding of complicated experimental SFG spectra of larger systems.248 Molecular dynamics (MD) simulations may also be carried out using ab initio and density functional approaches. In such cases the same restrictions on number of atoms applies as in the case of static electronic structure calculations. However, in the case of MD, as such calculations are performed at every time interval in the simulation, additional constraints are imposed based on the practical length to which a trajectory may be calculated. As computers become faster and parallel algorithms improve, larger systems may be handed, and longer trajectories may be obtained. Another option is to perform classical MD simulations, in which parametrized force fields are employed. Here the challenge is in developing and subsequently choosing force fields that are appropriate for the system being studied. Although there are many coarse-grain and all-atom forcefields optimized for biomolecules, they are typically parametrized to reproduce bulk structural and thermodynamic properties. Using these to study molecules at the solid−liquid interface is a reasonable starting point, and this continues to be an active area of research.250−253

Sandra Roy (upper left) obtained a B.Sc. in Chemistry from Université Laval, Quebec City, in 2010 while working at Defense Research and Development Canada. In 2012 she obtained a M.Sc. in Chemistry in the Hore group. Her thesis entitled “Water and peptide structure at hydrophobic and hydrophilic surfaces” used molecular simulations to probe the interplay between surface charge, hydrophobicity, interfacial water structure, and adsorbed peptide structure. She is now pursuing a Ph.D. in the Hore group. Her current research is focused on biomolecule orientation at surfaces by vibrational spectroscopy and molecular modeling.

5. SUMMARY In this review, we have presented examples of biomolecules at the solid−liquid interface in order of increasing complexity, from amino acids to peptides and proteins to membrane embedded protein complexes. SHG and SFG offer insight into several aspects of the adsorption process and dynamics of the molecules. Structural insight is generally provided through studies that vary the polarization of the incident beam(s) and select an output polarization for detection. Partially due to the reliance on the electronic properties of the molecules, the most detailed quantitative studies are performed for small molecules. The advantage here is 2-fold. First, with less than ∼100 atoms

Paul Covert (upper right) received a B.A. in Chemistry from Reed College in 1995. In 2001, under the mentorship of Prof. Fredrick Prahl (Oregon State University), he completed a M.Sc. in Chemical Oceanography with a focus on chemical transformations of suspended particulate material in the Columbia river and estuary. Between 2001 and 2010, while working for OSU and the National Oceanic and Atmospheric Administration, he contributed to several research 8411


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programs investigating coastal nutrient dynamics, air−sea gas-exchange processes, and ocean acidification. Paul joined the Hore group in 2010 to pursue a Ph.D. in Chemistry. His current research interests include the development of sum-frequency generation methods for the study of aqueous−solid interfaces in the environment. William FitzGerald (bottom left) obtained a B.Sc. in Chemistry from the University of Victoria in 2013, and is presently working towards an M.Sc. in the Hore group. The focus of his research is bulk and surface characterization through ellipsometry and polarimetry. Dennis Hore (bottom right) received his Ph.D. from Queen’s University in 2003, under the mentorship of Profs. Almeria Natansohn (Queen’s, Chemistry) and Paul Rochon (Royal Military College, Physics). His dissertation examined the photoinduced orientation of light-responsive polymers. He was then a postdoc in Prof. Geri Richmond’s group at the University of Oregon, studying surfactant and water structure at the air−water interface. In 2006 he joined the Department of Chemistry at the University of Victoria. His research interests are centered around characterizing the structure of molecules adsorbed at solid surfaces. He is particularly interested in how spectroscopic experiments, electronic structure calculations, and molecular simulations may be combined to arrive at a feature-rich description of interfacial structure.

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