Molecular Orientation and Energy Transfer Dynamics of a Metal Oxide Bound Self-Assembled Trilayer.

Self-assembly of molecular multilayers via metal ion linkages has become an important strategy for interfacial engineering of metalloid and metal oxide (MOx) substrates, with applications in numerous areas, including energy harvesting, catalysis, and chemical sensing. An important aspect for the rational design of these multilayers is knowledge of the molecular structure-function relationships. For example, in a multilayer composed of different chromophores in each layer, the molecular orientation of each layer, both relative to the adjacent layers and the substrate, influences the efficiency of vectorial energy and electron transfer. Here, we describe an approach using UV-vis attenuated total reflection (ATR) spectroscopy to determine the mean dipole tilt angle of chromophores in each layer in a metal ion-linked trilayer self-assembled on indium-tin oxide. To our knowledge, this is the first report demonstrating the measurement of the orientation of three different chromophores in a single assembly. The ATR approach allows the adsorption of each layer to be monitored in real-time, and any changes in the orientation of an underlying layer arising from the adsorption of an overlying layer can be detected. We also performed transient absorption spectroscopy to monitor interlayer energy transfer dynamics in order to relate structure to function. We found that near unity efficiency, sub-nanosecond energy transfer between the third and second layer was primarily dictated by the distance between the chromophores. Thus, in this case, the orientation had minimal impact at such proximity.

Nanomechanical Properties of Artificial Lipid Bilayers Composed of Fluid and Polymerizable Lipids.

Polymerization enhances the stability of a planar supported lipid bilayer (PSLB) but it also changes its chemical and mechanical properties, attenuates lipid diffusion, and may affect the activity of integral membrane proteins. Mixed bilayers composed of fluid lipids and poly(lipids) may provide an appropriate combination of polymeric stability coupled with the fluidity and elasticity needed to maintain the bioactivity of reconstituted receptors. Previously (Langmuir, 2019, 35, 12483-12491) we showed that binary mixtures of the polymerizable lipid bis-SorbPC and the fluid lipid DPhPC form phase-segregated PSLBs composed of nanoscale fluid and poly(lipid) domains. Here we used atomic force microscopy (AFM) to compare the nanoscale mechanical properties of these binary PSLBs with single-component PSLBs. The elastic (Young's) modulus, area compressibility modulus, and bending modulus of bis-SorbPC PSLBs increased upon polymerization. Before polymerization, breakthrough events at forces below 5 nN were observed, but after polymerization, the AFM tip could not penetrate the PSLB up to an applied force of 20 nN. These results are attributed to the polymeric network in poly(bis-SorbPC), which increases the bilayer stiffness and resists compression and bending. In binary DPhPC/poly(bis-SorbPC) PSLBs, the DPhPC domains are less stiff, more compressible, and are less resistant to rupture and bending compared to pure DPhPC bilayers. These differences are attributed to bis-SorbPC monomers and oligomers present in DPhPC domains that disrupt the packing of DPhPC molecules. In contrast, the poly(bis-SorbPC) domains are stiffer and less compressible relative to pure PSLBs; this difference is attributed to DPhPC filling the nm-scale pores in the polymerized domains that are created during bis-SorbPC polymerization. Thus, incomplete phase segregation increases the stability of poly(bis-SorbPC) but has the opposite, detrimental effect for DPhPC. Overall, these results provide guidance for the design of partially polymerized bilayers for technological uses.

Shear-Mediated Platelet Activation is Accompanied by Unique Alterations in Platelet Release of Lipids.

INTRODUCTION: Platelet activation by mechanical means such as shear stress exposure, is a vital driver of thrombotic risk in implantable blood-contacting devices used in the treatment of heart failure. Lipids are essential in platelets activation and have been studied following biochemical activation. However, little is known regarding lipid alterations occurring with mechanical shear-mediated platelet activation. METHODS: Here, we determined if shear-activation of platelets induced lipidome changes that differ from those associated with biochemically-mediated platelet activation. We performed high-resolution lipidomic analysis on purified platelets from four healthy human donors. For each donor, we compared the lipidome of platelets that were non-activated or activated by shear, ADP, or thrombin treatment. RESULTS: We found that shear activation altered cell-associated lipids and led to the release of lipids into the extracellular environment. Shear-activated platelets released 21 phospholipids and sphingomyelins at levels statistically higher than platelets activated by biochemical stimulation. CONCLUSIONS: We conclude that shear-mediated activation of platelets alters the basal platelet lipidome. Further, these alterations differ and are unique in comparison to the lipidome of biochemically activated platelets. Many of the released phospholipids contained an arachidonic acid tail or were phosphatidylserine lipids, which have known procoagulant properties. Our findings suggest that lipids released by shear-activated platelets may contribute to altered thrombosis in patients with implanted cardiovascular therapeutic devices. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00692-x.

Expression of truncated Kir6.2 promotes insertion of functionally inverted ATP-sensitive K+ channels.

ATP-sensitive K+ (KATP) channels couple cellular metabolism to electrical activity in many cell types. Wild-type KATP channels are comprised of four pore forming (Kir6.x) and four regulatory (sulfonylurea receptor, SURx) subunits that each contain RKR endoplasmic reticulum retention sequences that serve to properly translocate the channel to the plasma membrane. Truncated Kir6.x variants lacking RKR sequences facilitate plasma membrane expression of functional Kir6.x in the absence of SURx; however, the effects of channel truncation on plasma membrane orientation have not been explored. To investigate the role of truncation on plasma membrane orientation of ATP sensitive K+ channels, three truncated variants of Kir6.2 were used (Kir6.2ΔC26, 6xHis-Kir6.2ΔC26, and 6xHis-EGFP-Kir6.2ΔC26). Oocyte expression of Kir6.2ΔC26 shows the presence of a population of inverted inserted channels in the plasma membrane, which is not present when co-expressed with SUR1. Immunocytochemical staining of intact and permeabilized HEK293 cells revealed that the N-terminus of 6xHis-Kir6.2ΔC26 was accessible on both sides of the plasma membrane at roughly equivalent ratios, whereas the N-terminus of 6xHis-EGFP-Kir6.2Δ26 was only accessible on the intracellular face. In HEK293 cells, whole-cell electrophysiological recordings showed a ca. 50% reduction in K+ current upon addition of ATP to the extracellular solution for 6xHis-Kir6.2ΔC26, though sensitivity to extracellular ATP was not observed in 6xHis-EGFP-Kir6.2ΔC26. Importantly, the population of channels that is inverted exhibited similar function to properly inserted channels within the plasma membrane. Taken together, these data suggest that in the absence of SURx, inverted channels can be formed from truncated Kir6.x subunits that are functionally active which may provide a new model for testing pharmacological modulators of Kir6.x, but also indicates the need for added caution when using truncated Kir6.2 mutants.

Examining the influence of bilayer structure on energy transfer and molecular photon upconversion in metal ion linked multilayers.

Metal ion linked multilayers is a unique motif to spatially control and geometrically restrict molecules on a metal oxide surface and is of interest in a number of promising applications. Here we use a bilayer composed of a metal oxide surface, an anthracene annihilator molecule, Zn(II) linking ion, and porphyrin sensitizers to probe the influence of the position of the metal ion binding site on energy transfer, photon upconversion, and photocurrent generation. Despite being energetically similar, varying the position of the carboxy metal ion binding group (i.e. ortho, meta, para) of the Pt(II) tetraphenyl porphyrin sensitizer had a large impact on energy transfer rates and upconverted photocurrent that can be attributed to differences in their geometries. From polarized attenuated total reflectance measurements of the bilayers on ITO, we found that the orientation of the first layer (anthracene) was largely unperturbed by subsequent layers. However, the tilt angle of the porphyrin plane varies dramatically from 41° to 64° to 57° for the para-, meta-, and ortho-COOH substituted porphyrin molecules, which is likely responsible for the variation in energy transfer rates. We go on to show using molecular dynamics simulations that there is considerable flexibility in porphyrin orientation, indicating that an average structure is insufficient to predict the ensemble behavior. Instead, even a small subset of the population with highly favorable energy transfer rates can be the primary driver in increasing the likelihood of energy transfer. Gaining control of the orientation and its distribution will be a critical step in maximizing the potential of the metal ion linked structures.

Potential-Modulated Total Internal Reflection Fluorescence for Measurement of the Electron Transfer Kinetics of Submonolayers on Optically Transparent Electrodes.

An electroreflectance method to determine the electron transfer rate constant of a film of redox-active chromophores immobilized on an optically transparent electrode when the surface coverage of the film is very low (<0.1 monolayer) is described herein. The method, potential-modulated total internal reflection fluorescence (PM-TIRF) spectroscopy, is a fluorescence version of potential-modulated attenuated total reflection (PM-ATR) spectroscopy that is applicable when the immobilized chromophores are luminescent. The method was tested using perylene diimide (PDI) molecules functionalized with p-phenylene phosphonic acid (PA) moieties that bind strongly to indium-tin oxide (ITO). Conditions to prepare PDI-phenyl-PA films that exhibit absorbance and fluorescence spectra characteristic of monomeric (i.e., nonaggregated) molecules were identified; the electrochemical surface coverage was approximately 0.03 monolayer. The tilt angle of the long axis of the PDI molecular plane is 58° relative to the ITO surface normal, 25° greater than the tilt angle of aggregated PDI-phenyl-PA films, which have a surface coverage of approximately one monolayer. The more in-plane orientation of monomeric films is likely due to the absence of cofacial π-π interactions present in aggregated films and possibly a difference in PA-ITO binding modes. The electron transfer rate constant (ks,opt) of monomeric PDI-phenyl-PA films was determined using PM-TIRF and compared with PM-ATR results obtained for aggregated films. For PDI monomers, ks,opt = 3.8 × 103 s-1, which is about 3.7-fold less than ks,opt for aggregated films. The slower kinetics are attributed to the absence of electron self-exchange between monomeric PDI molecules. Differences in the electroactivity of the binding sites on the ITO electrode surface also may play a role. This is the first demonstration of PM-TIRF for determining electron transfer rate constants at an electrode/organic film interface.

Nanodomain Formation in Planar Supported Lipid Bilayers Composed of Fluid and Polymerized Dienoyl Lipids.

Polymerization of synthetic phospholipid monomers has been widely used to enhance the stability of lipid membranes in applications such as membrane-based biosensing, where the inherent instability of fluid-phase lipid bilayers can be problematic. However, lipid polymerization typically decreases membrane fluidity, which may be required to maintain the activity of reconstituted integral proteins and peptides. Prior work has shown that a bilayer composed of binary mixtures of poly(lipid) and fluid lipid exhibits enhanced stability and supports the function of incorporated biomolecules. This work examines the structural basis of these findings using planar supported lipid bilayers (PSLBs) composed of binary mixtures of a polymerizable lipid, 1,2-bis[10-(2',4'-hexadienoloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC), and a nonpolymerizable lipid, 1,2-diphytanoyl-sn-glycero-3-phosphocholine (DPhPC). Fluorescence recovery after photobleaching (FRAP) measurements showed that long-range lateral diffusion was minimally affected when the poly(lipid) mole ratio was ≤0.7. Atomic force microscopy, used to examine phase segregation in these PSLBs, showed that DPhPC forms a continuous lipid matrix that is 0.2-0.4 nm thicker than the island-like poly(bis-SorbPC) domains, with lateral dimensions of ≤200 nm. The nanoscale phase segregation allows for long-range lateral diffusion of lipid probes in the DPhPC matrix. The combination of fluidity and stability in these materials should make them useful in membrane-based biosensing applications.

Expression, purification, and electrophysiological characterization of a recombinant, fluorescent Kir6.2 in mammalian cells.

The inwardly rectifying K+ (Kir) channel, Kir6.2, plays critical roles in physiological processes in the brain, heart, and pancreas. Although Kir6.2 has been extensively studied in numerous expression systems, a comprehensive description of an expression and purification protocol has not been reported. We expressed and characterized a recombinant Kir6.2, with an N-terminal decahistidine tag, enhanced green fluorescent protein (eGFP) and deletion of C-terminal 26 amino acids, in succession, denoted eGFP-Kir6.2Δ26. eGFP-Kir6.2Δ26 was expressed in HEK293 cells and a purification protocol developed. Electrophysiological characterization showed that eGFP-Kir6.2Δ26 retains native single channel conductance (64 ±â€¯3.3 pS), mean open times (τ1 = 0.72 ms, τ2 = 15.3 ms) and ATP affinity (IC50 = 115 ±â€¯25 µM) when expressed in HEK293 cells. Detergent screening using size exclusion chromatography (SEC) identified Fos-choline-14 (FC-14) as the most suitable surfactant for protein solubilization, as evidenced by maintenance of the native tetrameric structure in SDS-PAGE and western blot analysis. A two-step scheme using Co2+-metal affinity chromatography and SEC was implemented for purification. Purified protein activity was assessed by reconstituting eGFP-Kir6.2Δ26 in black lipid membranes (BLMs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG), l-α-phosphatidylinositol-4,5-bisphosphate (PIP2) in a 89.5:10:0.5 mol ratio. Reconstituted eGFP-Kir6.2Δ26 displayed similar single channel conductance (61.8 ±â€¯0.54 pS) compared to eGFP-Kir6.2Δ26 expressed in HEK293 membranes; however, channel mean open times increased (τ1 = 7.9 ms, τ2 = 61.9 ms) and ATP inhibition was significantly reduced for eGFP-Kir6.2Δ26 reconstituted into BLMs (IC50 = 3.14 ±â€¯0.4 mM). Overall, this protocol should be foundational for the production of purified Kir6.2 for future structural and biochemical studies.

Axially Bound Ruthenium Phthalocyanine Monolayers on Indium Tin Oxide: Structure, Energetics, and Charge Transfer Properties.

The efficiency of charge collection at the organic/transparent conducting oxide (TCO) interface in organic photovoltaic (OPV) devices affects overall device efficiency. Modifying the TCO with an electrochemically active molecule may enhance OPV efficiency by providing a charge-transfer pathway between the electrode and the organic active layer, and may also mitigate surface recombination. The synthesis and characterization of phosphonic acid-ruthenium phthalocyanine (RuPcPA) monolayer films on indium tin oxide (ITO), designed to facilitate charge harvesting at ITO electrodes, is presented in this work. The PA group was installed axially relative to the Pc plane so that upon deposition, RuPcPA molecules were preferentially aligned with the ITO surface plane. The tilt angle of 22° between the normal axes to the Pc plane and the ITO surface plane, measured by attenuated total reflectance (ATR) spectroscopy, is consistent with a predominately in-plane orientation. The effect of surface roughness on RuPcPA orientation was modeled, and a correlation was obtained between experimental and theoretical mean tilt angles. Based on electrochemical and spectroelectrochemical studies, RuPcPA monolayers are composed predominately of monomers. Electrochemical impedance spectroscopy (EIS) and potential modulated-ATR (PM-ATR) spectroscopy were used to characterize the electron-transfer (ET) kinetics of these monolayers. A rate constant of 4.0 × 103 s-1 was measured using EIS, consistent with a short tunneling distance between the chromophore and the electrode surface. Using PM-ATR, ks,opt values of 2.2 × 103 and 2.4 × 103 s-1 were measured using TE and TM polarized light, respectively; the similarity of these values is consistent with a narrow molecular orientation distribution and narrow range of tunneling distances. The ionization potential of RuPcPA-modified ITO was measured using ultraviolet photoelectron spectroscopy and the results indicate favorable energetics for hole collection at the RuPcPA/ITO interface, indicating that this type of TCO modification may be useful for enhancing charge collection efficiency in OPV devices.

Characterization of Charge-Transfer Kinetics at Organic/Electrode Interfaces Using Potential-modulated Attenuated Total Reflectance (PM-ATR) Spectroscopy.

Potential-modulated attenuated total reflectance (PM-ATR) spectroscopy is a spectroelectrochemical method that utilizes the potential modulation approach and a waveguide ATR geometry. This unique combination enables measurements of electron-transfer (ET) kinetics of monolayer to submonolayer thin films on waveguide electrode surfaces. Selective probing of molecular subpopulations in a film can be achieved by choosing appropriate combinations of applied potential, wavelength, and polarization of light, which allows subpopulation structure to be correlated with ET kinetics. In this review, the basic theory of PM-ATR is introduced, and examples illustrating characterization of the structure and ET kinetics of organic semiconductor monolayers on electrode surfaces are presented, demonstrating the capabilities and applications of the PM-ATR technique.

Determining Band-Edge Energies and Morphology-Dependent Stability of Formamidinium Lead Perovskite Films Using Spectroelectrochemistry and Photoelectron Spectroscopy.

We show for the first time that the frontier orbital energetics (conduction band minimum (CBM) and valence band maximum (VBM)) of device-relevant, methylammonium bromide (MABr)-doped, formamidinium lead trihalide perovskite (FA-PVSK) thin films can be characterized using UV-vis spectroelectrochemistry, which provides an additional and straightforward experimental technique for determining energy band values relative to more traditional methods based on photoelectron spectroscopy. FA-PVSK films are processed via a two-step deposition process, known to provide high efficiency solar cells, on semitransparent indium tin oxide (ITO) and titanium dioxide (TiO2) electrodes. Spectroelectrochemical characterization is carried out in a nonsolvent electrolyte, and the onset potential for bleaching of the FA-PVSK absorbance is used to estimate the CBM, which provides values of ca. -4.0 eV versus vacuum on both ITO and TiO2 electrodes. Since electron injection occurs from the electrode to the perovskite, the CBM is uniquely probed at the buried metal oxide/FA-PVSK interface, which is otherwise difficult to characterize for thick films. UPS characterization of the same FA-PVSK thin films provide complementary near-surface measurements of the VBM and electrode-dependent energetics. In addition to energetics, controlled electrochemical charge injection experiments in the nonsolvent electrolyte reveal decomposition pathways that are related to morphology-dependent heterogeneity in the electrochemical and chemical stability of these films. X-ray photoelectron spectroscopy of these electrochemically treated FA-PVSK films shows changes in the average near-surface stoichiometry, which suggests that lead-rich crystal termination planes are the most likely sites for electron trapping and thus nanometer-scale perovskite decomposition.

Enhanced Temporal Resolution with Ion Channel-Functionalized Sensors Using a Conductance-Based Measurement Protocol.

The binding of a target analyte to an ion channel (IC), which is readily detected electrochemically in a label-free manner with single-molecule selectivity and specificity, has generated widespread interest in using natural and engineered ICs as transducers in biosensing platforms. To date, the majority of developments in IC-functionalized sensing have focused on IC selectivity or sensitivity or development of suitable membrane environments and aperture geometries. Comparatively little work has addressed analytical performance criteria, particularly criteria required for temporal measurements of dynamic processes. We report a measurement protocol suitable for rapid, time-resolved monitoring (≤30 ms) of IC-modulated membrane conductance. Key features of this protocol include the reduction of membrane area and the use of small voltage steps (10 mV) and short duration voltage pulses (10 ms), which have the net effect of reducing the capacitive charging and decreasing the time required to achieve steady state currents. Application of a conductance protocol employing three sequential, 10 ms voltage steps (-10 mV, -20 mV, -30 mV) in an alternating, pyramid-like arrangement enabled sampling of membrane conductance every 30 ms. Using this protocol, dynamic IC measurements on black lipid membranes (BLMs) functionalized with gramicidin A were conducted using a fast perfusion system. BLM conductance decreased by 76 ± 7.5% within 30 ms of switching from solutions containing 0 to 1 M Ca2+, which demonstrates the feasibility of using this approach to monitor rapid, dynamic chemical processes. Rapid conductance measurements will be broadly applicable to IC-based sensors that undergo analyte-specific gating.

Influence of Molecular Aggregation on Electron Transfer at the Perylene Diimide/Indium-Tin Oxide Interface.

Chemisorption of an organic monolayer to tune the surface properties of a transparent conductive oxide (TCO) electrode can improve the performance of organic electronic devices that rely on efficient charge transfer between an organic active layer and a TCO contact. Here, a series of perylene diimides (PDIs) was synthesized and used to study relationships between monolayer structure/properties and electron transfer kinetics at PDI-modified indium-tin oxide (ITO) electrodes. In these PDI molecules, one of the imide substituents is a benzene ring bearing a phosphonic acid (PA) and the other is a bulky aryl group that is twisted out of the plane of the PDI core. The size of the bulky aryl group and the substitution of the benzene ring bearing the PA were both varied, which altered the extent of aggregation when these molecules were absorbed as monolayer films (MLs) on ITO, as revealed by both attenuated total reflectance (ATR) and total internal reflection fluorescence spectra. Polarized ATR measurements indicate that, in these MLs, the long axis of the PDI core is tilted at an angle of 33-42° relative to the surface normal; the tilt angle increased as the degree of bulky substitution increased. Rate constants for electron transfer (ks,opt) between these redox-active modifiers and ITO were determined by potential-modulated ATR spectroscopy. As the degree of PDI aggregation was reduced, ks,opt declined, which is attributed to a reduction in the lateral electron self-exchange rate between adsorbed PDI molecules, as well as the heterogeneous conductivity of the ITO electrode surface. Photoelectrochemical measurements using a dissolved aluminum phthalocyanine as an electron donor showed that ITO modified with any of these PDIs is a more effective electron-collecting electrode than bare ITO.

Characterization of ZnO Interlayers for Organic Solar Cells: Correlation of Electrochemical Properties with Thin-Film Morphology and Device Performance.

This report focuses on the evaluation of the electrochemical properties of both solution-deposited sol-gel (sg-ZnO) and sputtered (sp-ZnO) zinc oxide thin films, intended for use as electron-collecting interlayers in organic solar cells (OPVs). In the electrochemical studies (voltammetric and impedance studies), we used indium-tin oxide (ITO) over coated with either sg-ZnO or sp-ZnO interlayers, in contact with either plain electrolyte solutions, or solutions with probe redox couples. The electroactive area of exposed ITO under the ZnO interlayer was estimated by characterizing the electrochemical response of just the oxide interlayer and the charge transfer resistance from solutions with the probe redox couples. Compared to bare ITO, the effective electroactive area of ITO under sg-ZnO films was ca. 70%, 10%, and 0.3% for 40, 80, and 120 nm sg-ZnO films. More compact sp-ZnO films required only 30 nm thicknesses to achieve an effective electroactive ITO area of ca. 0.02%. We also examined the electrochemical responses of these same ITO/ZnO heterojunctions overcoated with device thickness pure poly(3-hexylthiophehe) (P3HT), and donor/acceptor blended active layers (P3HT:PCBM). Voltammetric oxidation/reduction of pure P3HT thin films on ZnO/ITO contacts showed that pinhole pathways exist in ZnO films that permit dark oxidation (ITO hole injection into P3HT). In P3HT:PCBM active layers, however, the electrochemical activity for P3HT oxidation is greatly attenuated, suggesting PCBM enrichment near the ZnO interface, effectively blocking P3HT interaction with the ITO contact. The shunt resistance, obtained from dark current-voltage behavior in full P3HT/PCBM OPVs, was dependent on both (i) the porosity of the sg-ZnO or sp-ZnO films (as revealed by probe molecule electrochemistry) and (ii) the apparent enrichment of PCBM at ZnO/P3HT:PCBM interfaces, both effects conveniently revealed by electrochemical characterization. We anticipate that these approaches will be applicable to a wider array of solution-processed interlayers for "printable" solar cells.

Photopolymerization of Dienoyl Lipids Creates Planar Supported Poly(lipid) Membranes with Retained Fluidity.

Polymerization of substrate-supported bilayers composed of dienoylphosphatidylcholine (PC) lipids is known to greatly enhance their chemical and mechanical stability; however, the effects of polymerization on membrane fluidity have not been investigated. Here planar supported lipid bilayers (PSLBs) composed of dienoyl PCs on glass substrates were examined to assess the degree to which UV-initiated polymerization affects lateral lipid mobility. Fluorescence recovery after photobleaching (FRAP) was used to measure the diffusion coefficients (D) and mobile fractions of rhodamine-DOPE in unpolymerized and polymerized PSLBs composed of bis-sorbyl phosphatidylcholine (bis-SorbPC), mono-sorbyl-phosphatidylcholine (mono-SorbPC), bis-dienoyl-phosphatidylcholine (bis-DenPC), and mono-dienoyl phosphatidylcholine (mono-DenPC). Polymerization was performed in both the Lα and Lß phase for each lipid. In all cases, polymerization reduced membrane fluidity; however, measurable lateral diffusion was retained which is attributed to a low degree of polymerization. The D values for sorbyl lipids were less than those of the denoyl lipids; this may be a consequence of the distal location of polymerizable group in the sorbyl lipids which may facilitate interleaflet bonding. The D values measured after polymerization were 0.1-0.8 of those measured before polymerization, a range that corresponds to fluidity intermediate between that of a Lα phase and a Lß phase. This D range is comparable to ratios of D values reported for liquid-disordered (Ld) and liquid-ordered (Lo) lipid phases and indicates that the effect of UV polymerization on lateral diffusion in a dienoyl PSLB is similar to the transition from a Ld phase to a Lo phase. The partial retention of fluidity in UV-polymerized PSLBs, their enhanced stability, and the activity of incorporated transmembrane proteins and peptides is discussed.

Band Edge Energetics of Heterostructured Nanorods: Photoemission Spectroscopy and Waveguide Spectroelectrochemistry of Au-Tipped CdSe Nanorod Monolayers.

Conduction and valence band energies (ECB, EVB) for CdSe nanorods (NRs) functionalized with Au nanoparticle (NP) tips are reported here, referenced to the vacuum scale. We use (a) UV photoemission spectroscopy (UPS) to measure EVB for NR films, utilizing advanced approaches to secondary electron background correction, satellite removal to enhance spectral contrast, and correction for shifts in local vacuum levels; and (b) waveguide-based spectroelectrochemistry to measure ECB from onset potentials for electron injection into NR films tethered to ITO. For untipped CdSe NRs, both approaches show EVB = 5.9-6.1 eV and ECB = 4.1-4.3 eV. Addition of Au tips alters the NR band edge energies and introduces midgap states, in ways that are predicted to influence the efficiency of these nanomaterials as photoelectrocatalysts. UPS results show that Au tipping shifts EVB closer to vacuum by up to 0.4 eV, shifts the apparent Fermi energy toward the middle of the band gap, and introduces additional states above EVB. Spectroelectrochemical results confirm these trends: Au tipping shifts ECB closer to vacuum, by 0.4-0.6 eV, and introduces midgap states below ECB, which are assigned as metal-semiconductor interface (MSI) states. Characterization of these band edge energies and understanding the origins of MSI states is needed to design energy conversion systems with proper band alignment between the light absorbing NR, the NP catalyst, and solution electron donors and acceptors. The complementary characterization protocols presented here should be applicable to a wide variety of thin films of heterogeneous photoactive nanomaterials, aiding in the identification of the most promising material combinations for photoelectrochemical energy conversion.

Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors; however, the matrices and high-vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-sorbylphosphatidylcholine) (poly(bis-SorbPC)) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS on the basis of differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin subunit B as a model receptor-ligand pair, we estimated the minimal detectable concentration of toxin to be 4 nM. On-plate tryptic digestion of bound cholera toxin subunit B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner.

Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography.

The ability to rapidly screen complex libraries of pharmacological modulators is paramount to modern drug discovery efforts. This task is particularly challenging for agents that interact with lipid bilayers or membrane proteins due to the limited chemical, physical, and temporal stability of conventional lipid-based chromatographic stationary phases. Here, we describe the preparation and characterization of a novel stationary phase material composed of highly stable, polymeric-phospholipid bilayers self-assembled onto silica microparticles. Polymer-lipid membranes were prepared by photochemical or redox initiated polymerization of 1,2-bis[10-(2',4'-hexadieoyloxy)decanoyl]-sn-glycero-2-phosphocholine (bis-SorbPC), a synthetic, polymerizable lipid. The resulting polymerized bis-SorbPC (poly(bis-SorbPC)) stationary phases exhibited enhanced stability compared to particles coated with 1,2-dioleoyl-sn-glycero-phosphocholine (unpolymerized) phospholipid bilayers when exposed to chemical (50 mM triton X-100 or 50% acetonitrile) and physical (15 min sonication) insults after 30 days of storage. Further, poly(bis-SorbPC)-coated particles survived slurry packing into fused silica capillaries, compared to unpolymerized lipid membranes, where the lipid bilayer was destroyed during packing. Frontal chromatographic analyses of the lipophilic small molecules acetylsalicylic acid, benzoic acid, and salicylic acid showed >44% increase in retention times (P<0.0001) for all analytes on poly(bis-SorbPC)-functionalized stationary phase compared to bare silica microspheres, suggesting a lipophilic retention mechanism. Phospholipid membrane-functionalized stationary phases that withstand the chemical and physical rigors of capillary LC conditions can substantially increase the efficacy of lipid membrane affinity chromatography, and represents a key advance toward the development of robust membrane protein-functionalized chromatographic stationary phases.

Methacrylate Polymer Scaffolding Enhances the Stability of Suspended Lipid Bilayers for Ion Channel Recordings and Biosensor Development.

Black lipid membranes (BLMs) provide a synthetic environment that facilitates measurement of ion channel activity in diverse analytical platforms. The limited electrical, mechanical and temporal stabilities of BLMs pose a significant challenge to development of highly stable measurement platforms. Here, ethylene glycol dimethacrylate (EGDMA) and butyl methacrylate (BMA) were partitioned into BLMs and photopolymerized to create a cross-linked polymer scaffold in the bilayer lamella that dramatically improved BLM stability. The commercially available methacrylate monomers provide a simple, low cost, and broadly accessible approach for preparing highly stabilized BLMs useful for ion channel analytical platforms. When prepared on silane-modified glass microapertures, the resulting polymer scaffold-stabilized (PSS)-BLMs exhibited significantly improved lifetimes of 23 ± 9 to 40 ± 14 h and > 10-fold increase in mechanical stability, with breakdown potentials > 2000 mV attainable, depending on surface modification and polymer cross-link density. Additionally, the polymer scaffold exerted minimal perturbations to membrane electrical integrity as indicated by mean conductance measurements. When gramicidin A and α-hemolysin were reconstituted into PSS-BLMs, the ion channels retained function comparable to conventional BLMs. This approach is a key advance in the formation of stabilized BLMs and should be amenable to a wide range of receptor and ion channel functionalized platforms.

ITO/poly(aniline)/sol-gel glass: An optically transparent, pH-responsive substrate for supported lipid bilayers.

Described here is fabrication of a pH-sensitive, optically transparent transducer composed of a planar indium-tin oxide (ITO) electrode overcoated with a a poly(aniline) (PANI) thin film and a porous sol-gel layer. Adsorption of the PANI film renders the ITO electrode sensitive to pH, whereas the sol-gel spin-coated layer makes the upper surface compatible with fusion of phospholipid vesicles to form a planar supported lipid bilayer (PSLB). The response to changes in the pH of the buffer contacting the sol-gel/PANI/ITO electrode is pseudo-Nernstian with a slope of 52 mV/pH over a pH range of 4-9. Vesicle fusion forms a laterally continuous PSLB on the upper sol-gel surface that is fluid with a lateral lipid diffusion coefficient of 2.2 µm2/s measured by fluorescence recovery after photobleaching. Due to its lateral continuity and lack of defects, the PSLB blocks the pH response of the underlying electrode to changes in the pH of the overlying buffer. This architecture is simpler to fabricate than previously reported ITO electrodes derivatized for PSLB formation, and should be useful for optical monitoring of proton transport across supported membranes derivatized with ionophores and ion channels.