Confocal-Raman Microscopy Characterization of Supported

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Confocal-Raman Microscopy Characterization of Supported Phospholipid Bilayers Deposited on the Interior Surfaces of Chromatographic Silica David A. Bryce, Jay P. Kitt, and Joel M. Harris J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b13777 • Publication Date (Web): 27 Feb 2018 Downloaded from http://pubs.acs.org on February 27, 2018

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Journal of the American Chemical Society

Confocal-Raman Microscopy Characterization of Supported Phospholipid Bilayers Deposited on the Interior Surfaces of Chromatographic Silica David A. Bryce, Jay P. Kitt, and Joel M. Harris* Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT 84112-0850 USA ABSTRACT A common approach to exploring the structure and dynamics of biological membranes is through the deposition of model lipid bilayers on planar supports by Langmuir-trough or vesicle-fusion methods. Planar-supported lipid bilayers have been shown to exhibit structure and properties similar to lipid-vesicle membranes and are suitable for biosensing applications. Investigations using these planar-membrane models are limited to high-sensitivity methods capable of detecting a small population of molecules at the interface between a planar support and aqueous solution. In this work, we present evidence that supported-lipid bilayers can be deposited by vesicle fusion onto the interior surfaces throughout the wide-pore network of chromatographic silica particles. The thickness of a 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) film and head-group spacing are consistent with a single bilayer of DMPC deposited onto the pore surfaces. The high specific surface area of these materials generates phospholipid concentrations easily detected by confocal-Raman microscopy within an individual particle, which allows the structure of these supported bilayers to be investigated. Raman spectra of porous-silica-supported DMPC bilayers are equivalent to spectra of DMPC vesicle membranes, both above and below their melting phase transitions, suggesting comparable phospholipid organization and bilayer structure. These porous-silica-supported model membranes could share benefits that planar-supported lipid bilayers bring to biosensing applications, but in a material that overcomes the limited surface area of a planar support. To test this concept, the potential of these porous-silica-supported lipid bilayers as high-surface-area platforms for label-free Raman-scattering-based protein-biosensing is demonstrated with detection of concanavalin A selectively binding to a lipid-immobilized mannose target.

*Corresponding author: [email protected]

ACS Paragon Plus Environment

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2 INTRODUCTION Interactions between molecules and phospholipid membranes play a major role in a variety of biologically relevant processes, ranging from cell signaling and enzyme regulation to the uptake of drugs and toxins.1 Given the importance of these interactions, developing a model system that allows investigation of molecular interactions at lipid bilayer interfaces can provide new insight into these processes.2,3 An effective model-bilayer system can be useful in a variety of applications including assessment of small-molecule partitioning and its relationship to phospholipid bilayer composition and structure, or as platforms for detection of membranelocalized protein association. Many efforts to understand such processes and to develop biosensing applications have relied on planar-supported-lipid bilayers,4-8 where a phospholipid bilayer is deposited on planar glass or silica substrates stabilized by interactions of the polar phospholipid head-groups with a thin water layer on the hydroxylated surface.4 A variety of measurement techniques have been employed to assess the behavior of phospholipids within these bilayers. Fluorescence microscopy techniques, including fluorescence-recovery-afterphotobleaching, fluorescence-correlation spectroscopy and single-molecule tracking have been used to study bilayer structure, interfacial populations and diffusion behavior of fluorescent or fluorescently-labeled molecules.9-14 Vibrational spectroscopy has been employed to assess planar-supported bilayer structure including vibrational sum-frequency generation,15-17 infrared reflection,13,18-20 and total-internal- reflection Raman spectroscopies.21 Despite these many investigations, applications of planar-supported-lipid bilayers face several challenges. Measurement sensitivity is a major issue because of the small number of molecules that can be observed within a limited surface area of a planar substrate. Planar lipid bilayers are also not very stable because the intermolecular and surface forces that organize the bilayer are weak, and the bilayer cannot survive if it is removed from water.22 One approach taken to improve the stability of planar-lipid bilayers is to replace the substrate-proximal leaflet of the supported bilayer with a self-assembled monolayer of acyl chains bound to the underlying support.23-25 Adsorption of a phospholipid monolayer on an underlying self-assembled n-alkane thiol monolayer on silver or gold produces a stable hybrid supported bilayer membrane that can be used to assess bilayer structure and as a model to detect peptide-membrane and ligand-protein interactions.23-31 While hybrid bilayers improve the stability of planar-supported-lipid bilayers and their ability to be handled in air, the sensitivity required for detecting the small number of molecules


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3 that can be probed within the area of a planar surface remains a challenge. Recently, hybrid bilayers have been formed within the pores of n-alkyl-chain (C18) modified chromatographic silica particles,32-35

producing a stable model membrane that can be used to measure the

lipophilicity of target compounds by means of chromatographic retention measurements. More recently, the structure of these hybrid-bilayers formed within C18 particles was investigated by confocal-Raman microscopy.36 Because of the high specific surface area of chromatographic silica used as the support (>100 m2/g), it was possible to overcome limited surface area of planar lipid bilayers, allowing Raman scattering characterization of the hybrid bilayer structure within individual porous particles.36 Based on comparisons with vesicle membranes of their phospholipid head-group spacing and Raman spectra,37-41 it was found that these within-particle hybrid bilayers exhibit interdigitation of the silica-bound n-alkyl chains of the lower leaflet with the acyl chains of the phospholipids of the upper leaflet.36 This interdigitated structure leads to differences in head-group spacing, chain packing, and phase-transition behavior compared to vesicle membranes, and limiting their utility as a model for studying the interactions of membranes with small-molecules or peptides. A more ideal lipid-bilayer model for small-volume spectroscopic or chromatographic applications would duplicate the structure of planar-supported-lipid bilayers4-6 while being deposited onto the interior surfaces of particle porous supports. The possibility of forming such supported bilayers in porous silica particles is suggested by the successful deposition of supported-lipid bilayers within the cylindrical pores of aluminum oxide filter disks formed by anodization.20,42-45 In these studies, lipid bilayers were deposited by vesicle fusion onto nanometer-scale cylindrical pore walls of porous aluminum oxide, and the structure of the lipid bilayer and proteins associated with these membranes were investigated by solid-state NMR.42-44 Differential scanning calorimetry showed that these lipid bilayers exhibited phase-transition behavior comparable to lipid-vesicle dispersions.20,45 Recent cryo-EM evidence shows that vesicle fusion can produce supported-lipid bilayers that conform to 100-nm diameter silica particles, even at the small-radius junctions between aggregated particles.46 In the present work, we investigate supported-lipid bilayers deposited in wide-pore chromatographic silica by vesicle fusion. We employ confocal-Raman microscopy to study the uniformity of lipid deposition, the presence of intact vesicles within the pores, the internal pore volume displaced by deposited lipid, the temperature-dependent structure of the supported bilayer, and potential applications in label-free protein biosensing.



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4 EXPERIMENTAL SECTION Reagents and materials Spherical, bare chromatographic silica particles used in this work were obtained from YMC America (YMC Sil, Allentown, PA). Silica particles used in structural studies of DMPC bilayers had a nominal diameter of 10-µm and pore diameter of 30nm, while silica particles used for the protein binding study had a nominal pore diameter of 100nm and particle diameter of 5-µm, as reported by the manufacturer. Water used in all experiments was filtered using a Barnstead GenPure UV water purification system (ThermoFisher Scientific, Waltham, MA) and had a minimum resistivity of 18.0MΩ·cm. 1,2dimyristoly-sn-glycero-3-phosphocholine (DMPC), and mannose head-group-functionalized lipid

(1,2-dipalmitoyl-sn-glycero-3-phospho((ethyl-1',2',3'-triazole)triethyleneglycolmannose),

ammonium salt) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL), diluted into chloroform, and stored at -15°C until use. Concanavalin A (ConA) from jack-bean was purchased from Vector Labs (Burlingame, CA) and stored as-received as a lypholized powder at -18 °C until just before use, at which point it was rehydrated in 2-mM Tris-buffered saline which contained 1 mM calcium chloride at the desired protein concentration by gentle stirring. Chloroform (Chromasolv Plus, >99.9%), octadecyl(dimethyl)chlorosilane (Aldrich, >95%), sodium chloride (NaCl), potassium chloride (KCl) and calcium chloride (CaCl2), 3nitrobenzenesulfonic acid (3-NBS), and 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) were purchased from Sigma-Aldrich and used without further purification. Within-particle bilayer preparation and characterization Supported DMPC bilayers were prepared within the pore network of silica particles by vesicle fusion. Particles were first washed in base-piranha solution (60/40 concentrated ammonium hydroxide/30% hydrogen peroxide), rinsed in deionized water, and dried at 160°C (warning: base-piranha solutions are highly corrosive and strong oxidizers and can react explosively with organics). The void volume of the prepared silica was determined by displacement of heavy water (D2O) that served as a Raman-active void-volume marker. Specific surface area and pore-diameter of the silica particles used in structural studies of DMPC bilayers were determined by Brunauer-Emmett-Teller (BET) nitrogen analysis and mercury porosimetry, respectively, by Porous Materials Inc. (Ithaca, NY), where the specific surface area was 220 m2/g and pore diameter (distribution mode) was 29-nm. Vesicles were prepared by re-hydration of DMPC films formed by drying a lipid-chloroform solution under nitrogen, and subsequently under vacuum for a minimum of 1 hour. Dried lipid films were rehydrated in Tris-buffered saline (TBS) buffer (10 mM Tris, 137 mM NaCl, 2.7 mM


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5 KCl) with 5 mM CaCl2 added to facilitate fusion; the final lipid concentration was 2 mg/mL. Vesicle fusion was carried out as follows: the above lipid suspension was mixed with silica particles to achieve a final concentration of 2mg lipid/1 mg silica. This slurry was then sonicated at 40°C (well above the DMPC melting transition) for 20 minutes and stirred at this temperature for ~12 hours. The particle-vesicle suspension was separated by centrifugation, leaving the particles in a pellet, while excess lipid could be removed in the supernatant. The particles were then re-suspended in water, and this rinsing process was repeated 3 times to ensure that excess lipid in solution was removed. The prepared particles were used immediately or refrigerated in water until use. To test for the presence of intact (unfused) vesicles within the pores of lipid-deposited particles, DMPC-supported-bilayers were formed within particles as above, but with 50-mM 3NBS tracer in the buffer solution which remains entrapped within intact vesicles.47 After the vesicle fusion step, the vesicle-particle suspension in the 3-NBS-containing buffer was cooled in an ice bath to below the bilayer melting transition and diluted 1000-fold with 3-NBS-free buffer, such that Raman scattering from free-solution 3-NBS was not detectable. Surface coverages of DMPC were determined by carbon analysis (MHW Laboratories, Phoenix, AZ). Particles were prepared as described above; after washing away excess lipid, the particle slurry was dried at ~120 °C overnight to remove water from the sample. Confocal-Raman microscopy. The confocal-Raman microscope used in this work has been described in detail elsewhere.48,49 Briefly, the beam of a Kr+ laser (Innova 90, Coherent Inc., Santa Clara, CA) operating at 647.1 nm was beam-expanded using a 4x beam expander (Special Optics Inc., Wharton, NJ) and directed into a Nikon TE-300 inverted microscope frame. The expanded beam was reflected by a dichroic beam splitter to slightly overfill a 1.4-NA, 100x oil-immersion objective (CFL PLAN APO, Nikon Inc., El Segundo, CA), which focused the beam to a ~600 nm diameter focus spot. Scattered light from the focus is collected by the objective, transmitted through the dichroic beam splitter, and passed through a holographic-notch filter (Kaiser Optical, Ann Arbor, MI). Scattered light was then collimated, focused through the entrance slit of a monochromator (500 IS, Bruker Corp., Billerica, MA) equipped with a diffraction grating having 300 lines/mm blazed at 750 nm, and dispersed onto a charge coupled device (CCD) detector (Andor iDus 420, Andor USA, South Windsor, CT). The entrance slit of the monochromator was set to 50 µm to define the horizontal dimension of the confocal aperture,



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6 while the vertical dimension was defined by limiting acquisition to 3 rows of pixels on the CCD chip (78 µm).50 Raman spectra were collected from the interior of individual chromatographic particles as follows: The focused laser beam was translated to the solution-coverslip interface where reflected light from the laser spot was visible. This reflection of the focused spot was then translated in x and y to beneath the center of a particle of interest. The microscope objective was then translated upward to bring the center of the particle into sharp focus, localizing the confocal probe volume at the particle center where spectra were collected. All spectra were baselinecorrected by subtraction of a 5th-order polynomial fit to peak-free regions of the spectrum. Spectral data analysis was carried out using custom Matlab routines (Mathworks, Natick, MA). Well cells for confocal-Raman microscopy were constructed by adhering a ~12-mm length of 10-mm i.d., 13-mm o.d. Pyrex glass tubing to a No. 1 glass coverslip using Devcon 5-min epoxy (ITW Devcon, Danvers, MA). The cell used for collection of temperature-dependent spectra has been described in detail previously.47,51,52 In short, samples were contained in a brass microscopy cell supported by a silver stage platform and jacketed by a copper block. Two Peltier heaters were fixed to the silver stage to heat the assembly. The temperature within the cell was maintained by a proportionalintegral-derivative (PID) controller, which switched a solid state relay to modulate current to the Peltier heaters, while an ethylene-glycol solution was flowed in a loop through a stainless steel chilling loop submerged in ice water to remove heat from the copper block. Optical-trapping Confocal-Raman microscopy. Optical-trapping confocal-Raman experiments were carried out on 200-nm diameter DMPC vesicles prepared by extrusion. Lipid suspensions were prepared as above, except that final lipid concentration after re-hydration was reduced to 1 mg/mL by dilution, and vesicle size was established by 11extrusions through 200nm track-etched polycarbonate membranes using an Avanti mini-extruder (Avanti Polar Lipids Inc., Alabaster, AL) heated to 40°C, well above the melting transition of DMPC. The vesicles were then transferred to temperature-controlled microscopy cell, and spectra were collected from individual vesicles that were trapped in the focus of the excitation beam;53-56 the spectra (normalized to the C-N head-group intensity) are compared to spectra from within-particle supported lipid bilayers to detect any structural differences. Differential Scanning Calorimetry. The temperature-dependent spectroscopic behavior of DMPC deposited in the pores of 30-nm pore-diameter silica was compared with the melting


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7 transition of these samples determined by differential scanning calorimetry (DSC). Samples for DSC analysis were prepared with final phospholipid concentration in excess of 2.5 mg/mL. Calorimetry experiments were carried out using a MicroCal VP-DSC (Malvern Instruments, Worcestershire, UK). Samples were degassed under mild vacuum and loaded into the calorimeter. The cell temperature was lowered to 10°C and allowed to equilibrate for 15 min. Calorimetry data were collected at a scan rate of 0.5°C/min and baseline corrected in OriginPro 8.5.1 (OriginLab, Northampton, MA). RESULTS AND DISCUSSION Structural characterization within-particle DMPC. Supported phospholipid bilayers are routinely prepared on planar silica, glass, and mica surfaces by vesicle fusion.5,57-59 In the present work, vesicle fusion is applied to the deposition of DMPC within wide-pore (29-nm pore-diameter) chromatographic silica particles by sonication of the particles for 20 minutes in a high-concentration lipid dispersion followed by annealing for 12 hours, all performed at 40°C, more than 15°C above the lipid melting transition. Confocal-Raman microscopy at the center of a 10-µm particle confirms deposition of DMPC within the silica pores, as shown in Figure 1; Raman bands are observed that correspond to phospholipid head-group C-N stretch (715 cm-1), and

carbon-carbon

and

carbon-

hydrogen modes from the lipid acyl chains, including the C-C stretching modes (~1050–1130 cm-1), CH2twisting mode (1295 cm-1), CH2bending mode (1440 cm-1) and C-H stretching modes (~2840–2980cm-1). While the above result shows that lipid can be found within a silica particle, questions arise about the uniformity of lipid deposition from particle-to-particle and

within

a

particle. These questions can be addressed through acquisition of spectra from particles dispersed in

Figure 1. Raman spectrum collected from within a silica particle with DMPC deposited onto the pore walls of the particle.



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8 an aqueous solution containing 20% D2O. Intra-particle spectra from three different particles were acquired and baseline corrected, but not normalized. The resulting intensities of phospholipid head-group C-N symmetric stretching and C-C stretching modes of the deposited lipid show less than 5% variation, while variations in the sampled pore volume can be assessed by the amplitudes of O-D stretching at 2,490 cm-1, which exhibits ~7% variation between particles (see Supporting Information, Figure S1). To determine whether DMPC deposition is uniform throughout the particle or restricted from some regions like the center of the particle, the lipid and mobile-phase within the pores were profiled in a ~14-µm particle immersed in a 20% D2O aqueous solution. Raman spectra were accumulated in 1-µm depth increments, and Raman scattering intensities from the bilayer C-N head-group stretch and from the O-D stretch of D2O in the pores are constant as a function of depth throughout the particle (see Supporting Information, Figure S2). When the confocal-probe volume emerges from the particle, the lipid signal falls off and the D2O signal increases as the probe volume samples the bulk solution. The uniform intensities of lipid both within an individual particle and between different particles do not establish that the DMPC signal derives from lipid molecules residing on the silica surface following vesicle-fusion. Given a typical ~20-nm diameter of sonicated vesicles,60 accumulated lipid might be present in intact vesicles present in the 30-nm diameter silica pores. To test for the presence of intact vesicles in the silica pores, an experiment was carried out using vesicles containing 3-nitrobenzenesulfonic acid (3-NBS) as a tracer. At neutral pH, the sulfonate group of 3-NBS is negatively charged preventing its escape from vesicles having gel-phase phospholipid membranes.47 For this experiment, DMPC vesicles were formed by sonication in a solution of 50-mM 3-NBS and allowed to interact with silica particles for a 12-hour period, as previously described. After 12 hours, the suspension was cooled to below the phase transition in an ice bath, and an aliquot of this particle/vesicle solution was withdrawn and diluted 1000-fold in an ionic-strength-matched buffer (no 3-NBS) and stored on ice. This dilution of the 3-NBS drops the solution-phase concentration to 50-µM, below the detection limit, so that any detectable 3-NBS must reside within intact vesicles. The dilute solution was transferred to a cold well cell, and Raman spectra were collected from both the lipid-containing particles and optically-trapped vesicles treated as above. Normalized spectra of optically-trapped vesicles and lipid-filled particles are compared in Figure 2, where the trigonal ring-breathing mode of 3-NBS (997 cm-1) and NO2 symmetric-stretch at 1359 cm-1 are clearly visible in optically-trapped vesicles, but absent in the spectrum from the lipid-containing particle. If the lipid within these


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9 particles

derived

from

a

significant population of intact DMPC vesicles, 3-NBS would be easily detected in the withinparticle Raman spectra. The absence of the 3-NBS scattering from within the silica particles indicates that the pore network is free of intact vesicles and that the lipid signal must derive from DMPC on the silica surface. Estimating

DMPC

surface coverage.

The above

results deposited

show in

that porous

DMPC silica

particles by vesicle fusion is uniform within a given particle

Figure 2. Evidence of vesicle-fusion during DMPC deposition. Spectra of a DMPC supported-bilayer in a silica particle following lipid-deposition with 3-NBS-filled vesicles (black), compared with an optically-trapped DMPC vesicles containing 50-mM 3-NBS (red); the 3-NBS ring-breathing and NO2-symmetric-stretching modes are -1 highlighted. Spectra are normalized to the C-N mode at 715 cm .

and from particle-to-particle, and that lipid is associated with the silica surface and not with residual vesicles trapped in the pores. The lipid present on the interior silica surface can be quantified by two independent approaches. First, from differences in the O-D stretching Raman scattering intensity in particles compared to the D2O solution in which they are immersed, one can determine the volume fraction of D2O displaced by the skeletal silica and by DMPC deposited within the particle. The D2O intensity of a bare silica particle is reduced by 19(±2)% due to displacement of D2O by the skeletal silica compared to the bulk solvent (Supporting Information, Figure S3). With accumulation of DMPC in the particle, the O-D signal decreases another 37(±3)% compared to D2O in the pores of bare particle. The reduction in pore volume by accumulated DMPC can be used to estimate the thickness of a DMPC layer under the assumption that the volume of the DMPC is uniformly distributed on the silica surface, an assumption supported by the uniform coverage by lipid within a given particle and between particles (see above). For a 1-mL total volume of material, the 0.19-mL volume occupied by the silica framework can be converted to surface area by multiplying by the density of amorphous silica (2.2 g/mL) and the specific surface area from the nitrogen-BET analysis (220m2/g). If the



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10 volume of lipid is distributed uniformly over this surface area, then the estimated thickness of the DMPC layer which that displaces D2O from the pore volume is 3.3(±0.5) nm. This result agrees within its uncertainty with the water-excluding acyl-chain thickness of DMPC bilayers, 3.03(±0.02) nm, measured by x-ray diffraction from a planar-supported-lipid bilayer.61 This displacement result supports the hypothesis that DMPC deposited by vesicle fusion forms a supported-bilayer on the interior silica surfaces. A second and independent method to quantify lipid deposited in the silica particles is through elemental (carbon) analysis carried out on lipid-containing particles, the results of which indicate a carbon mass fraction, 24.3±0.2%. This result when combined with the silica surface area from nitrogen-BET analysis, while accounting for the small difference in population of lipids on the proximal and distal leaflets of a curved bilayer (see Supporting Information, Page S5), yields a head-group area of 69±1 Å2. This result is ~18% larger than has been reported for a fluid-phase DMPC vesicle bilayer measured by small-angle neutron scattering experiments performed on vesicle dispersions.62 The small difference in head-group area may be due to the larger surface area sampled by nitrogen in a BET adsorption measurement than would be accessible to a much larger lipid head-group. Regardless, the close agreement of both the headgroup area with results from DMPC vesicles and the thickness of the deposited lipid from D2O displacement with a DMPC planar bilayer combine to provide strong evidence that the DMPC deposited by vesicle fusion into wide-pore silica is present as a lipid bilayer on the silica pore surfaces. Structure and organization of intraparticle DMPC bilayers. While the quantity and uniformity of lipid deposited into porous silica are consistent with a lipid bilayer deposited on the interior surfaces of the pores, the structure of this lipid film is critical to applications where membrane affinity is being assessed. For example, hybrid phospholipid bilayers, consisting of a proximal leaflet of covalently-bound octadecylsilane and a distal leaflet of phospholipids deposited in reversed-phase chromatographic silica32-35 have been found to differ considerably in structure from vesicle bilayers.36 These hybrid bilayers exhibit greater phospholipid head group spacing as compared to vesicle bilayers and show spectral features characteristic of interdigitation of the acyl-chains of the overlying lipid leaflet with the surface-bound C18 chains.36 To address the question of whether or not DMPC supported bilayers are structurally similar to bilayers of vesicle membranes, Raman spectra were collected from both DMPC deposited in wide-pore silica and optically-trapped DMPC vesicles below and above the melting


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11 transition (12°C and 35°C) for DMPC. Collected spectra were baseline corrected, normalized to the intensity of the C-N symmetric stretching mode, and presented in Figure 3, where important

spectral

regions

highlighted in the figure and will be addressed individually. Carbon-carbon stretching region (1030cm-11150cm-1)

The carbon-carbon

stretching region is dominated by three peaks indicative of the relative population of gaucheand trans- conformers in the

Figure 3. Spectral comparisons of DMPC supported bilayers (red) to DMPC vesicles (black) above and below the DMPC phase transition. The carbon-carbon stretching (blue), carbon-hydrogen bending (violet), and carbon-hydrogen stretching (green) regions are highlighted.

acyl chains of the phospholipid bilayer. Out-of-phase and in-phase carbon-carbon stretching modes of the acyl-chains at 1061 cm-1 and 1126 cm-1, respectively,63 are much higher in intensity from the trans-character of the acyl chains in the ordered, gel-phase.37,40,41,64-67 Changes in the broad peak at 1086 cm-1 correlate to changes in acyl-chain gauche conformers,37,40,67-69 where increases in intensity are typical of acyl chain disordering in the liquid-crystalline phase. Above the melting transition, in the liquid crystalline phase, we see nearly identical C-C spectral features for DMPC vesicle bilayers and those formed within particles. For both bilayers, the C-C region is dominated by gauche-conformers as is expected. Below the phase transition, we observe the expected higher relative intensity for trans-conformer peaks at 1061 cm-1 and 1126 cm-1 indicating that the silica-supported bilayer exhibits local extension of the chains that is equivalent to a gel-phase vesicle membrane. Carbon-hydrogen CH2 bending region (1260 cm-1 - 1500 cm-1) The carbon-hydrogen bending region is dominated by two peaks, centered at ~1300 cm-1 and 1440 cm-1. The lower frequency peak is from CH2-twisting, while the higher frequency peak corresponds to CH2bending.

66,67

The CH2-twisting mode is informative of both acyl-chain order as well as inter-

chain coupling,65,67 reflecting increases in acyl-chain conformational and rotational freedom with



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12 bilayer melting leading to peak asymmetry and broadening.40,65,66 These changes are observed in spectra of both vesicle and within-particle-supported bilayers with equivalent CH2-twist scattering from the two samples both above and below the melting transition. The CH2 bending mode (~1440 cm-1) is sensitive to lattice order in the acyl chains of the lipid bilayer.40,70 The band comprises scattering from several Raman active vibrational modes, including the antisymmetric methyl-bend at 1436 cm-1 and methylene-scissoring at 1455 cm-1.39,64,65,67 The comparison of CH2-bending between the supported and vesicle bilayers shows no differences above the melting transition. Below the phase transition, however, we observe slightly greater intensity and less splitting of the CH2-bending mode in the silica-supported bilayer compared to a vesicle bilayer. Splitting of the CH2-bending modes is attributed to lattice order and inter-chain coupling between acyl chains,67,71,72 which is greater for the gel-phase vesicle membrane compared to the supported bilayer. This result suggest that long-range ordering in the gel-phase of the phospholipid acyl chains in the supported bilayer is inhibited, either by interactions with the silica substrate or by the curved geometry of the 30-nm pores. Carbon-hydrogen stretching region (2800-3100 cm-1). The carbon-hydrogen stretching region appears as a broad band comprised of multiple Raman-active modes. Three bands in this region, the CH2-symmetric-stretching (2847 cm-1) and antisymmetric-stretching (2883 cm-1), and the terminal CH3-methyl stretching (2930 cm-1), provide information about acyl-chain coupling, disorder, and packing within the bilayer.37,38,64,65,67,73 The intensity of the CH2-antisymmetricstretching mode relative to the CH2-symmetric-stretching mode is indicative of acyl-chain lateral packing density. In the case of the silica-supported bilayers, we observe a small increase in ratio I2883/I2847 in both the fluid-phase and gel-phase compared to a vesicle bilayer. This result reflects a slightly higher packing density of acyl chains in the supported bilayer, which could be due to interactions of the proximal leaflet of the phospholipid head-group with the silica surface or compression of distal leaflet from the curvature of the pores. In summary, comparisons of Raman spectra of porous-silica supported DMPC with DMPC vesicles show that the structure and organization of these lipid bilayers are nearly identical, both above and below the main melting transition. A small decrease in the longer-range order in the gel-phase of the silica-supported bilayer is observed compared to a vesicle membrane, and the packing density of acyl chains in the supported bilayer appears to be slightly greater. Both of these effects could be due to interactions of the silica substrate with the


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13 proximal leaflet of the bilayer and/or disruption of order and distal leaflet compression from the geometry of the pores. Melting transition behavior of silica-supported lipid bilayers. Phospholipid bilayers in vesicle membranes74-76 exhibit a sharp gel-to-liquid-crystal melting transitions that correspond to disordering of the acyl-chains as revealed in their temperature-dependent Raman spectra;39,41 the melting of DMPC vesicle bilayers is well characterized, and appears as a sharp phase transition at 23.5°C.74 Planar-supported lipid bilayers on silica and mica supports have been investigated

by

atomic-force

microscopy

(AFM)

and

sum-

frequency generation and reveal a melting transition over a wider temperature range compared to lipid-vesicle membranes.77-79 This broad melting behavior has been attributed to interactions of the lipid-bilayer with the substrate, leading to sequential melting of the solution-exposed followed

distal

by

the

leaflet substrate-

associated proximal leaflet.77-81 To

assess

the

melting

transition behavior of lipid-bilayer films

deposited

within

porous

silica, Raman spectra of DMPC bilayers in porous silica were collected

as

a

function

of

temperature over a range of 12°C to 35°C,

baseline-corrected,

and

normalized to the area of the phospholipid

C-N

head-group

stretch at 715 cm-1; a sample of

Figure 4. A. Spectra of silica-supported DMPC at temperatures from 14°C to 30°C, acquired at 1°C increments; every other spectrum is plotted. B. Normalized trans-conformer spectral intensity (points, left-axis) and integrated change in heat capacity (red, right-axis) to determine the enthalpy of the supportedbilayer phase transition.



Page 14 of 27

14 these spectra is presented in Figure 4A. Features typical of lipid acyl-chain disordering are observed as the supported film passes through the melting transition. In the carbon-carbon stretching region, decreasing intensity of C-C trans-conformer peaks at 1061 and 1126 cm-1 and the growth of the broad symmetric C-C gauche peak at 1086 cm-1 are characteristic of bilayer melting.36,39-41

In the carbon-hydrogen twisting region at temperatures below the phase

transition, a sharp peak is observed at 1296 cm-1 that broadens and shifts to higher frequency upon melting. A correlation-field-induced splitting of the CH2 scissoring mode is observed in the gel phase, appearing as a sharp peak centered at 1437 cm-1 and a shoulder at 1454 cm-1, while the fluid-phase spectrum exhibits a single, broadened peak, centered at 1444 cm-1.41,72 The range of the melting transition of the silica-supported-lipid bilayers was investigated by quantifying the temperature-dependent Raman spectral changes and comparing the results with differential scanning calorimetry (DSC). A multidimensional least-squares analysis of the temperature-dependent Raman spectra (Supporting Information, Page S6) allows the relative concentration of the gel-phase component in the supported bilayer to be tracked across the melting transition. The results show the gel-phase component disappearing over a large temperature range, indicative of a ~8°C-wide melting transition from ~17°C to ~25°C centered ~21°C. A wide transition is also observed in the DSC results centered at 22°C with a slightly narrower range compared to the temperature-dependent spectral changes. A small shoulder at about ~24.5 °C is observed in the calorimetric endotherm. This feature matches the phasetransition of DMPC vesicle membranes and may be indicative of a small population of intact vesicles (below the detection limit of the 3-NBS tracer study above) or a small amount of multilamellar phospholipid film in the pores. In either case, the amplitude of the enthalpy change indicates that this population is very small, consistent with it not being detectable in the temperature-dependent composition results. The ~8°C width of the supported-bilayer phase transition of DMPC in porous silica is consistent with the main phase transition of DMPC bilayers on mica, determined by temperaturedependent AFM measurements, although the center temperature of the transition on mica was nearly 10°C higher.77 This higher melting temperature may reflect the greater ordering of the supported bilayer that is produced on the atomically-flat mica surface. The difference is also reflected in the enthalpy of the main phase transition, which is determined from integrated change in heat capacity.82 The enthalpy of the melting transition of the silica-pore supported bilayer is ∆H = 8.6±0.5 kJ/mol, which is smaller than enthalpy change reported for mica-


Page 15 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


15 supported DMPC, ∆H = 10.3 kJ/mol.83 The smaller enthalpy measured for melting a DMPC bilayer in 30-nm pores compared to DMPC on atomically-flat mica may reflect disruption of long-range order in the gel-phase bilayer due to the curved substrate geometry. The Raman spectra in the CH2-bending regions of the pore-supported bilayers reflected smaller lattice order and inter-chain coupling compared to 200-nm DMPC vesicles. Disruption of long-range order would lower the enthalpy barrier and melting temperature of the pore-supported bilayer compared to an atomically smooth planar substrate. Potential applications to label-free biosensing. Supported-lipid bilayers have been shown to be a nearly ideal platform for biophysical studies and for development of biosensors.4-7 These substrates are especially valuable for protein-based biosensing due to their modest nonspecific adsorption of proteins from solution.6-8 Using planar-supported lipid bilayers for these applications is challenged by the limited surface area and correspondingly small populations of molecules at the planar bilayer surface, requiring high sensitivity detection provided by nonlinear

optics,8,84

resonance,27,85

or

plasmonfluorescence-

labeling of the target molecules.6,7,14 Deposition of lipid bilayers within the pores of chromatographic silica might allow one to overcome the need for high-sensitivity detection by employing a very high-surfacearea support. To test the feasibility of using confocal Raman microscopy for detecting protein-ligand binding at

porous

phospholipid

silica-supported bilayers,

DMPC

bilayers were formed within silica particles containing a small fraction of ligand head-group-functionalized phospholipid that could capture an

Figure 5: Raman spectrum of silica-supported DMPC bilayers prepared in 5-µm diameter, 115-nm pore-diameter particles. DMPC bilayers before (black) and after (blue) exposure to 5-µM ConA show no detectable accumulation of protein. Bilayers prepared with 1-mol% PEG-mannose phospholipid and exposed to 5-µM ConA (red) show -1 the appearance of a phenylalanine ring-breathing mode at 1000 cm -1 as well as protein amide modes at 1240, 1550, and 1670 cm .



Page 16 of 27

16 unlabeled protein target from solution. The specific protein-ligand binding investigated was a lectin, concanavalin A (ConA), a 237-amino-acid protein that selectively binds to mannose ligands.27,86-89 Mannose sites were included in the deposited bilayer by adding a small (1-mol%) fraction of mannose-functionalized phospholipid to the DMPC-vesicle preparation prior to bilayer deposition. In this case, bilayers were formed in 5-µm diameter base-washed silica particles having a nearly 4-fold larger pore diameter (115-nm by BET measurements) to ensure sufficient pore space for the protein to diffuse into the particles and to avoid blockage of the pore network by ConA bound to the lipid-immobilized ligands. To test the capability of Raman microscopy to detect protein binding at a porous-silica supported bilayer, silica particles containing 1%-mannose in DMPC were exposed to 5-µM ConA in tris-buffered saline with 2-mM calcium ion for ~2 hours with mixing. A small aliquot of the suspension was transferred to a microscopy cell and Raman spectra were collected from the center of individual particles, and the results are plotted in Figure 5. A comparison with a Raman spectrum of DMPC blank particles reveals several Raman bands consistent with protein accumulation in the particle interior, including a prominent phenylalanine ring breathing mode (1000 cm-1), and several amide backbone modes (~1240 cm-1, 1554 cm-1, and 1664 cm-1).90,91 As a control, a DMPC supported bilayer (no mannose) was also exposed to 5-µM ConA, and the spectrum is included in Figure 5. There is no discernable difference in the Raman spectra of the DMPC blank particles before and after exposure to the ConA. This result indicates that the supported lipid bilayers within porous silica can be resistant to non-specific interactions with proteins. The selective capture by the mannose-functionalized lipid of ConA and its appearance in spectra from the center of particle shows that the protein can diffuse through the pore network and access mannose sites in the particle interior. Raman scattering provides both a label-free and structurally-informative method of detection; high-surface-area porous-silica-supported lipid bilayers are promising materials for use in a range of Raman microscopy-based experiments for detecting protein-ligand interactions that occur at phospholipid membranes. ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. Additional information is provided on testing particle-to-particle variability, depth profiling


Page 17 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


17 DMPC within a particle, determining bilayer thickness and head-group area, and multidimensional analysis of temperature-dependent Raman spectra.

ACKNOWLEDGMENTS This material is based upon work supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Energy, Chemical Sciences, Geosciences, & Biosciences under Award Number DE-FG03-93ER14333.



Page 18 of 27

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Raman scattering intensity


Page 22 of 27

C-H stretching modes

Out-of-phase C-C stretch (trans)

C-N symmetric stretch

800

CH2 twist In-phase C-C stretch (trans)

CH2 bending mode

C-C stretch (gauche)

1000

1200

1400

2800

3000

Raman shift (cm-1)

Figure 1. Raman spectrum collected from within a silica particle with DMPC deposited onto the pore walls of the particle.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Normalized Raman scattering intensity

Page 23 of 27

Silica-supported bilayer Optically-trapped vesicle 3-Nitrobenzenesulfonic acid

700

800

900 1000 1100 1200 1300 1400 1500

-1

Raman shift (cm )

Figure 2. Evidence of vesicle-fusion during DMPC deposition. Spectra of a DMPC supported-bilayer in a silica particle following lipid-deposition with 3-NBS-filled vesicles (black), compared with an optically-trapped DMPC vesicles containing 50-mM 3-NBS (red); the 3-NBS ring-breathing and NO2-symmetric-stretching modes are highlighted. Spectra are normalized to the C-N mode at 715 cm-1.


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Normalized Raman scattering intensity


Page 24 of 27

Within-particle bilayer Vesicle bilayer

Above Tm

Below Tm

800

1000

1200

1400

2800 2900 3000

Raman shift (cm-1)

Figure 3. Spectral comparisons of DMPC supported bilayers (red) to DMPC vesicles (black) above and below the DMPC phase transition. The carbon-carbon stretching (blue), carbon-hydrogen bending (violet), and carbon-hydrogen stretching (green) regions are highlighted. ACS Paragon Plus Environment

Page 25 of 27

Normalized Raman scattering

A

800

1000

1200

1400

2800

3000

Raman shift (cm-1)

B

Normalized gel-phase intensity

10 8 6 4 2

H (kJ/mol)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0 -2 12

14

16

18

20

22

24

26

28

Temperature (oC)

Figure 4. A. Spectra of silica-supported DMPC at temperatures from 14°C to 30°C, acquired at 1°C increments; every other spectrum is plotted. B. Normalized trans-conformer spectral intensity (points, left-axis) and integrated change in heat capacity (red, right-axis) to determine the enthalpy of the ACS Paragon Plus Environment supported-bilayer phase transition.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

normalized Raman scattering


Page 26 of 27

1 mol% mannose, 5 M ConA 0 mol% mannose, 5 M ConA 0 mol% mannose blank

Amide I

Phenylalanine ring breathing mode Amide III Amide II

700

1000

1200

1400

1600

1800

Raman shift (cm-1)

Figure 5: Raman spectrum of silica-supported DMPC bilayers prepared in 5-µm diameter, 115-nm pore-diameter particles. DMPC bilayers before (black) and after (blue) exposure to 5-µM ConA show no detectable accumulation of protein. Bilayers prepared with 1-mol% PEG-mannose phospholipid and exposed to 5-µM ConA (red) show the appearance of a phenylalanine ring-breathing mode at 1000 cm-1 as well as protein amide modes at 1240, 1550, and 1670 cm-1.


Page 27 of 27 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41


Tm

TOC Graphic


Confocal-Raman Microscopy Characterization of Supported

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