S Scott Saavedra
- Professor, Chemistry and Biochemistry-Sci
- Professor, BIO5 Institute
- Professor, Optical Sciences
- Professor, Biomedical Engineering
- Ph.D. Chemistry
- Duke University, Durham, North Carolina, United States
No activities entered.
Analytical SeparationsCHEM 527 (Fall 2020)
Exchange Chemical InfoCHEM 695B (Fall 2020)
ResearchCHEM 900 (Fall 2020)
ThesisCHEM 910 (Fall 2020)
Exchange Chemical InfoCHEM 695B (Spring 2020)
ResearchCHEM 900 (Spring 2020)
Analytical Mol SpectroscCHEM 526B (Fall 2019)
Exchange Chemical InfoCHEM 695B (Fall 2019)
ResearchCHEM 900 (Fall 2019)
Adv Analytical Chem LabCHEM 528B (Spring 2019)
DissertationCHEM 920 (Spring 2019)
Exchange Chemical InfoCHEM 695B (Spring 2019)
ResearchCHEM 900 (Spring 2019)
Analytical SeparationsCHEM 527 (Fall 2018)
DissertationCHEM 920 (Fall 2018)
Exchange Chemical InfoCHEM 695B (Fall 2018)
ResearchCHEM 900 (Fall 2018)
DissertationCHEM 920 (Spring 2018)
Exchange Chemical InfoCHEM 695B (Spring 2018)
ResearchCHEM 900 (Spring 2018)
Analytical Mol SpectroscCHEM 526B (Fall 2017)
DissertationCHEM 920 (Fall 2017)
Exchange Chemical InfoCHEM 695B (Fall 2017)
ResearchCHEM 900 (Fall 2017)
Adv Analytical Chem LabCHEM 528B (Spring 2017)
Analytical ChemistryCHEM 696A (Spring 2017)
Analytical SeparationsCHEM 527 (Spring 2017)
DissertationCHEM 920 (Spring 2017)
Exchange Chemical InfoCHEM 695B (Spring 2017)
ResearchCHEM 900 (Spring 2017)
Analytical ChemistryCHEM 696A (Fall 2016)
Directed ResearchCHEM 492 (Fall 2016)
DissertationCHEM 920 (Fall 2016)
Exchange Chemical InfoCHEM 695B (Fall 2016)
ResearchCHEM 900 (Fall 2016)
Adv Analytical Chem LabCHEM 528B (Spring 2016)
DissertationCHEM 920 (Spring 2016)
Exchange Chemical InfoCHEM 695B (Spring 2016)
Honors ThesisCHEM 498H (Spring 2016)
ResearchCHEM 900 (Spring 2016)
- Heitz, B., Aspinwall, C., & Saavedra, S. (2013). Highly Stable Polymerized Lipid Membranes for Ion Channel Recordings. In Molecular Self-Assembly: Advances in Chemistry, Biology and Nanotechnology(pp 159-182). Hauppauge, NY: Nova Science.More infoEditor: Comrie, JP
- Hall Jr, H., Jones, I. W., Orosz, K. S., Liang, B., Fonseka, M. N., Saavedra, S. S., Aspinwall, C. A., & Christie, H. S. (2019). Nanodomain Formation in Planar Supported Lipid Bilayers Composed of Fluid and Polymerized Dienoyl Lipids. Langmuir, 35, 12483-12491. doi:10.1021/acs.langmuir.9b02101
- Ehamparam, R., Oquendo, L. E., Liao, M. W., Brynnel, A. K., Ou, K., Armstrong, N. R., Mcgrath, D. V., & Saavedra, S. S. (2017). Axially Bound Ruthenium Phthalocyanine Monolayers on Indium Tin Oxide: Structure, Energetics, and Charge Transfer Properties. ACS Applied Materials & Interfaces, 9(34), 29213-29223. doi:http://doi.org/10.1021/acsami.7b07394
- Bright, L. K., Baker, C. A., Branstrom, R., Saavedra, S. S., & Aspinwall, C. A. (2015). Methacrylate Polymer Scaffolding Enhances the Stability of Suspended Lipid Bilayers for Ion Channel Recordings and Biosensor Development. ACS BIOMATERIALS-SCIENCE & ENGINEERING, 1(10), 955-963.
- Ehamparam, R., Pavlopoulos, N. G., Liao, M. W., Hill, L. J., Armstrong, N. R., Pyun, J., & Saavedra, S. S. (2015). Band Edge Energetics of Heterostructured Nanorods: Photoemission Spectroscopy and Waveguide Spectroelectrochemistry of Au-Tipped CdSe Nanorod Monolayers. ACS NANO, 9(9), 8786-8800.
- Gallagher, E. S., Adem, S. M., Baker, C. A., Ratnayaka, S. N., Jones, I. W., Hall, H. K., Saavedra, S. S., & Aspinwall, C. A. (2015). Highly stabilized, polymer-lipid membranes prepared on silica microparticles as stationary phases for capillary chromatography. JOURNAL OF CHROMATOGRAPHY A, 1385, 28-34.
- Liang, B., Ju, Y., Joubert, J. R., Kaleta, E. J., Lopez, R., Jones, I. W., Hall, H. K., Ratnayaka, S. N., Wysocki, V. H., & Saavedra, S. S. (2015). Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS. ANALYTICAL AND BIOANALYTICAL CHEMISTRY, 407(10), 2777-2789.
- Lin, H., Lin, H., MacDonald, G. A., MacDonald, G. A., Shi, Y., Shi, Y., Polaske, N. W., Polaske, N. W., McGrath, D. V., McGrath, D. V., Marder, S. R., Marder, S. R., Armstrong, N. R., Armstrong, N. R., Ratcliff, E. L., Ratcliff, E. L., Saavedra, S. S., & Saavedra, S. S. (2015). Influence of Molecular Orientation on Charge Transfer Processes at Phthalocyanine/Metal Oxide Interfaces and Relationship to Organic Photovoltaic Performance. Journal of Physical Chemistry C, 119, 10304-10313. doi:http://dx.doi.org/10.1021/acs.jpcc.5b02971
- Lin, H., MacDonald, G. A., Shi, Y., Polaske, N. W., McGrath, D. V., Marder, S. R., Armstrong, N. R., Ratcliff, E. L., & Saavedra, S. S. (2015). Influence of Molecular Orientation on Charge-Transfer Processes at Phthalocyanine/Metal Oxide Interfaces and Relationship to Organic Photovoltaic Performance. JOURNAL OF PHYSICAL CHEMISTRY C, 119(19), 10304-10313.
- Al-Obeidi, A., Ge, C., Orosz, K., & Saavedra, S. (2013). ITO/poly(aniline)/sol-gel glass: A pH-responsive substrate for supported lipid bilayers. J. Materials.More infodoi:10.1155/2013/676920
- Lin, H., Polaske, N. W., Oquendo, L. E., Gliboff, M., Knesting, K. M., Nordlund, D., Ginger, D. S., Ratcliff, E. L., Beam, B. M., Armstrong, N. R., McGrath, D. V., & Saavedra, S. S. (2012). Electron-Transfer Processes in Zinc Phthalocyanine Phosphonic Acid Monolayers on ITO: Characterization of Orientation and Charge-Transfer Kinetics by Waveguide Spectroelectrochemistry. JOURNAL OF PHYSICAL CHEMISTRY LETTERS, 3(9), 1154-1158.
- Ratnayaka, S. N., Ratnayaka, S. N., Elandaloussi, E. H., Elandaloussi, E. H., Korth, B. D., Korth, B. D., Bates, R. B., Bates, R. B., Pyun, J., Pyun, J., Hall Jr., H. K., Hall Jr., H. K., Saavedra, S. S., & Saavedra, S. S. (2012). Studies of mixed liposomes with novel sorbyl functionalized head group lipids. Chemistry and Physics of Lipids, 165(8), 809-817.More infoPMID: 23127336;PMCID: PMC3534896;Abstract: Three novel polymerizable amphiphiles with a sorbyl-substituted head group were synthesized and systematically characterized. These amphiphiles are neutral in charge. None of these molecules forms vesicles by itself, presumably due to lack of amphiphilicity and/or extensive head group interaction. Therefore, mixed vesicles were formed with other fluid lipids such as DPenPC, eggPC, or DOPC. We investigated the properties of these mixtures in both vesicles and Langmuir films. The Langmuir isotherms show formation of monolayers by all three molecules. However, the isotherms for mixed monolayers suggest that two components are largely immiscible to the mixing lipid. Under polymerization conditions, mixed vesicles of these amphiphiles form oligomers, suggesting that in spite of a larger head group, they form mesophases. © 2012 Elsevier Ireland Ltd.
- Araci, Z. O., Runge, A. F., Doherty, W. J., & Saavedra, S. S. (2011). Erratum: Correlating molecular orientation distributions and electrochemical kinetics in subpopulations of an immobilized protein film (Journal of the American Chemical Society (2008) 130 (1572-1573) DOI: 10.1021/ja710156d). Journal of the American Chemical Society, 133(33), 13205-.
- Heitz, B. A., Juhua, X. u., Jones, I. W., Keogh, J. P., Comi, T. J., Hall, H. K., Aspinwall, C. A., & Saavedra, S. S. (2011). Polymerized planar suspended lipid bilayers for single ion channel recordings: Comparison of several dienoyl lipids. Langmuir, 27(5), 1882-1890.More infoPMID: 21226498;PMCID: PMC3043114;Abstract: The stabilization of suspended planar lipid membranes, or black lipid membranes (BLMs), through polymerization of mono- and bis-functionalized dienoyl lipids was investigated. Electrical properties, including capacitance, conductance, and dielectric breakdown voltage, were determined for BLMs composed of mono-DenPC, bis-DenPC, mono-SorbPC, and bis-SorbPC both prior to and following photopolymerization, with diphytanoyl phosphocholine (DPhPC) serving as a control. Poly(lipid) BLMs exhibited significantly longer lifetimes and increased the stability of air-water transfers. BLM stability followed the order bis-DenPC > mono-DenPC > mono-SorbPC > bis-SorbPC. The conductance of bis-SorbPC BLMs was significantly higher than that of the other lipids, which is attributed to a high density of hydrophilic pores, resulting in relatively unstable membranes. The use of poly(lipid) BLMs as matrices for supporting the activity of an ion channel protein (IC) was explored using α-hemolysin (α-HL), a model IC. Characteristic i-V plots of α-HL were maintained following photopolymerization of bis-DenPC, mono-DenPC, and mono-SorbPC, demonstrating the utility of these materials for preparing more durable BLMs for single-channel recordings of reconstituted ICs. © 2011 American Chemical Society.
- Jones, I. W., Simon, A. M., Saavedra, S. S., & Hall Jr., H. K. (2011). Synthesis of a diverse library of N,N-dimethylamino containing monomers appropriate as lipid head groups. Tetrahedron Letters, 52(43), 5547-5549.More infoAbstract: We report on the synthesis of a diverse library of N,N-dimethylamino containing monomers. Subjecting these monomers to Chabrier reaction conditions would yield lipids with polymerizable head groups. This library of lipid head groups is equipped with arms of various lengths containing reduction-oxidation polymerizable groups at the terminus. © 2011 Elsevier Ltd. All rights reserved.
- Lin, H., Polaske, N. W., McGrath, D. V., & Saavedra, S. S. (2011). Electron transfer kinetics of phosphonic acid functionalized zinc phthalocyanine monolayers on ITO by potential modulated attenuated total reflectance. ACS National Meeting Book of Abstracts.More infoAbstract: Phthlocyanines are candidate building blocks for organic photovoltaic (OPV) devices. Crucial to the performance of OPV devices is the charge-injection barrier between the indium tin oxide (ITO) electrode and the organic semiconductor that arises from poor wettability of ITO surface towards the organic layer. Phosphonic acid (PA)-linked Pcs are an excellent candidate in this context because of the strong affinity of PAs towards ITO. Here monolayer films of zinc phthalocyanine (ZnPc-PA), tethered to ITO via the PA moiety, were characterized by polarized attenuated total reflectance (ATR) spectroscopy. The monomeric form of ZnPc-PA exhibit a more flat orientation, whereas the aggregated form of ZnPc-PA is oriented in a more upright arrangement. The electron transfer rate constants across the monolayer-ITO interface are measured using potential modulated attenuated total reflectance (PM-ATR) in TE and TM polarizations. This allows rate constants to be determined for sub-populations of molecules that are oriented predominately in-plane (monomers) and out-of-plane (aggregates), respectively.
- Page, J., Heitz, B. A., Joubert, J. R., Keogh, J. P., Sparer, T., Saavedra, S. S., & Wei, H. e. (2011). In vitro assessment of macrophage attachment and phenotype on polymerized phospholipid bilayers. Journal of Biomedical Materials Research - Part A, 97 A(2), 212-217.More infoPMID: 21391298;PMCID: PMC3076933;Abstract: Phosphatidyl choline (PC)-based materials have been found to be resistant to nonspecific protein adhesion in vitro. In this study, a PC-based planar supported phospholipid bilayer composed of 1,2-bis[10-(2′,4′- hexadienoyloxy)decanoyl]-sn-glycero-3-phosphocholine (bis-SorbPC or BSPC) was generated on piranha-treated silicon wafers by vesicle deposition. The bilayer was polymerized with redox initiation forming a stable 4-nm thick coating. Polymerized lipid bilayers (PLBs) were characterized and tested for uniformity, with ellipsometry and contact angle. Cellular adhesion and morphological changes in RAW 264.7 macrophages were investigated in vitro on PLBs and compared to bare silicon controls. Fluorescent and scanning electron microscopy were used to observe changes in cellular morphology. The PLBs showed much lower cellular adhesion than bare silicon controls. Of the cells that attached to the PLBs, a very low percentage showed the same morphological expressions seen on the controls. It is hypothesized that proteins adsorb to the defects in the PLBs, caused by incomplete polymerization, and this mediates the observed minimal cellular attachment and morphological changes. © 2011 Wiley Periodicals, Inc.
- Simon, A. M., Marucci, N. E., & Saavedra, S. S. (2011). Measuring photochemical kinetics in submonolayer films by transient ATR spectroscopy on a multimode planar waveguide. Analytical Chemistry, 83(14), 5762-5766.More infoPMID: 21651043;Abstract: Understanding the kinetics of reactions in molecular thin films can aid in the molecular engineering of organic photovoltaics and biosensors. We have coupled two analytical methods, transient absorbance spectroscopy (TAS) and attenuated total reflectance (ATR), in a relatively simple arrangement when compared with previous TAS/ATR instruments to interrogate molecular structure and photochemistry at interfaces. The multimode planar waveguide geometry provides a significant path length enhancement relative to a conventional transmission geometry, making it feasible to perform measurements on low-surface-coverage films. The performance of the instrument was assessed using a thin film composed of purple membrane (PM) fragments containing bacteriorhodopsin deposited onto PDAC, a positively charged polymer. The surface coverage of retinal chromophore in this film is ∼0.1 monolayer and its orientation distribution is anisotropic, with a mean tilt angle of 68° from surface normal. After photoinduced formation of the transient M state, the chromophore decays to the ground state in 4.4 ± 0.6 ms, equivalent to the decay of suspended PM fragments, which shows that deposition on PDAC does not alter M-state photokinetics. The surface coverage of the M state is calculated to be 2 pmol/cm 2, which is ∼1% of a close-packed monolayer. This work demonstrates that TAS/ATR can be used to probe structure and photochemical kinetics in molecular films at extremely low surface coverages. © 2011 American Chemical Society.
- Heitz, B. A., Jones, I. W., Hall Jr., H. K., Aspinwall, C. A., & Saavedra, S. S. (2010). Fractional polymerization of a suspended planar bilayer creates a fluid, highly stable membrane for ion channel recordings. Journal of the American Chemical Society, 132(20), 7086-7093.More infoPMID: 20441163;PMCID: PMC2882248;Abstract: Suspended planar lipid membranes (or black lipid membranes (BLMs)) are widely used for studying reconstituted ion channels, although they lack the chemical and mechanical stability needed for incorporation into high-throughput biosensors and biochips. Lipid polymerization enhances BLM stability but is incompatible with ion channel function when membrane fluidity is required. Here, we demonstrate the preparation of a highly stable BLM that retains significant fluidity by using a mixture of polymerizable and nonpolymerizable phospholipids. Alamethicin, a voltage-gated peptide channel for which membrane fluidity is required for activity, was reconstituted into mixed BLMs prepared using bis-dienoyl phosphatidylcholine (bis-DenPC) and diphytanoyl phosphatidylcholine (DPhPC). Polymerization yielded BLMs that retain the fluidity required for alamethicin activity yet are stable for several days as compared to a few hours prior to polymerization. Thus, these polymerized, binary composition BLMs feature both fluidity and long-term stability. © 2010 American Chemical Society.
- Zhang, H., Joubert, J. R., & Saavedra, S. S. (2010). Membranes from polymerizable lipids. Advances in Polymer Science, 224(1), 1-42.More infoAbstract: Potential technological applications for artificial lipid and proteo-lipid membranes, such as capsules for controlled delivery of drugs and coatings for biosensors and biomaterials, are in many cases limited by the inherent instability of lipid lamellar phases. Development of methods to stabilize artificial lipid membranes has therefore been a focus of research efforts since the 1970s. Linear and cross-linking polymerization of synthetic lipid monomers is a well-studied strategy. Several comprehensive reviews on polymerizable lipids and supramolecular structures derived from them appeared between 1985 and 2002. Consequently, this review focuses on significant developments in this field during 2000-2008. These include synthesis of new types of polymerizable lipids, creation and characterization of novel poly(lipid) membrane systems, and applications of polymerized vesicles and membranes in chemical sensing, separations science, drug delivery, materials biocompatibility, and energy storage. Polymerization of membranes to achieve stability and their functionalization for technological applications are emphasized. © 2009 Springer-Verlag Berlin Heidelberg.
- Heitz, B. A., Juhua, X. u., Hall Jr., H. K., Aspinwall, C. A., & Saavedra, S. S. (2009). Enhanced long-term stability for single ion channel recordings using suspended poly(lipid) bilayers. Journal of the American Chemical Society, 131(19), 6662-6663.More infoPMID: 19397328;PMCID: PMC2682430;Abstract: (Figure Presented) Black lipid membranes (BLMs) are widely used for recording the activity of incorporated ion channel proteins. However, BLMs are inherently unstable structures that typically rupture within a few hours after formation. Here, stabilized BLMs were formed using the polymerizable lipid bis-dienoyl phosphatidylcholine (bis-DenPC) on glass pipettes of ∼10 μm (I.D.). After polymerization, these BLMs maintained steady conductance values for several weeks, as compared to a few hours for unpolymerized membranes. The activity of an ion channel, A-hemolysin, incorporated into bis-DenPC BLMs prior to polymerization, was maintained for 1 week after BLM formation and polymerization. These lifetimes are a substantial improvement over those achievable with conventional BLM technologies. Polymerized BLMs containing functional ion channels may represent an enabling technology for development of robust biosensors and drug screening devices. Copyright © 2009 American Chemical Society.
- Joubert, J. R., Smith, K. A., Johnson, E., Keogh, J. P., Wysocki, V. H., Gale, B. K., Conboy, J. C., & Saavedra, S. S. (2009). Stable, ligand-doped, poly(bis-SorbPC) lipid bilayer arrays for protein binding and detection. ACS Applied Materials and Interfaces, 1(6), 1310-1315.More infoPMID: 20355927;PMCID: PMC2857382;Abstract: A continuous-flow microspotter was used to generate planar arrays of stabilized bilayers composed of the polymerizable lipid bis-SorbPC and dopant lipids bearing ligands for proteins. Fluorescence microscopy was used to determine the uniformity of the bilayers and to detect protein binding. After UV-initiated polymerization, poly(lipid) bilayer microarrays were air-stable. Cholera toxin subunit b (CTb) bound to an array of poly(lipid) bilayers doped with GM1, and the extent of binding was correlated to the mole percentage of GM1 in each spot. A poly(lipid) bilayer array composed of spots doped with GM1 and spots doped with biotin-DOPE specifically bound CTb and streptavidin to the respective spots from a dissolved mixture of the two proteins. Poly(bis-SorbPC)/GM1 arrays retained specific CTb binding capacity after multiple regenerations with a protein denaturing solution and also after exposure to air. In addition, these arrays are stable in vacuum, which allows the use of MALDI-TOF mass spectrometry to detect specifically bound CTb. This work demonstrates the considerable potential of poly(lipid) bilayer arrays for high-throughput binding assays and lipidomics studies. © 2009 American Chemical Society.
- Roberts, D. L., Yaning, M. a., Bowles, S. E., Janczak, C. M., Pyun, J., Saavedra, S. S., & Aspinwall, C. A. (2009). Polymer-stabilized phospholipid vesicles with a controllable, pH-dependent disassembly mechanism. Langmuir, 25(4), 1908-1910.More infoPMID: 19154125;PMCID: PMC2654230;Abstract: In this letter, we report a facile method to prepare robust phospholipid vesicles using commonly available phospholipids that are stabilized via the formation of an interpenetrating, acid-labile, cross-linked polymer network that imparts a site for controlled polymer destabilization and subsequent vesicle degradation. The polymer network was formed in the inner lamella of the phospholipid bilayer using 2,2-di(methacryloyloxy-1-ethoxy)propane (DMOEP) and butyl methacrylate (BMA). Upon exposure to acidic conditions, the highly cross-linked polymer network was partially converted to smaller linear polymers, resulting in substantially reduced vesicle stability upon exposure to chemical and physical insults. Isolated polymers had pH-dependent-solubility in THF. Transmission electron microscopy and dynamic light scattering revealed time-dependent enhanced vesicle stability in high concentrations of surfactant and vacuum conditions at elevated pH, whereas exposure to acidic pH rapidly decreased the vesicle stability, with complete destabilization observed in less than 24 h. The resultant transiently stabilized vesicles may prove useful for enhanced drug delivery and chemical sensing applications and allow for improved physiological clearance. © Copyright 2009 American Chemical Society.
- Sui, Z., Hanan, N. J., Phimphivong, S., Wysocki Jr., R. J., & Saavedra, S. S. (2009). Synthesis, characterization and sol-gel entrapment of a crown ether-styryl fluoroionophore. Luminescence, 24(4), 236-242.More infoPMID: 19253273;PMCID: PMC3244207;Abstract: The synthesis and initial evaluation of a new dye-functionalized crown-ether, 2-[2-(2,3,5,6,8,9,11,12,14,15-decahydro-1,4,7,10,13,16-benzohexaoxacyclooctadecin)ethenyl]-3-methyl benzothiazolium iodide (denoted BSD), are reported. This molecule contains a benzyl 18-crown-6 moiety as the ionophore and a benzothiazolium to spectrally transduce ion binding. Binding of K+ to BSD in methanol causes shifts in the both absorbance and fluorescence emission maxima, as well as changes in the molar absorptivity and the emission intensity. Apparent dissociation constants (Kd) in the range 30-65 mM were measured. In water and neutral buffer, Kd values were approximately 1 mM. BSD was entrapped in sol-gel films composed of methyltri-ethoxysilane (MTES) and tetraethylorthosilicate (TEOS) with retention of its spectral properties and minimal leaching. K+ binding to BSD in sol-gel films immersed in pH 7.4 buffer causes significant fluorescence quenching, with an apparent response time of approximately 2 min and an apparent Kd of 1.5 mM. © 2009 John Wiley & Sons, Ltd.
- Zhang, H., Orosz, K. S., Takahashi, H., & Saavedra, S. S. (2009). Broadband plasmon waveguide resonance spectroscopy for probing biological thin films. Applied Spectroscopy, 63(9), 1062-1067.More infoPMID: 19796490;PMCID: PMC2912159;Abstract: A commercially available spectrometer has been modified to perform plasmon waveguide resonance (PWR) spectroscopy over a broad spectral bandwidth. When compared to surface plasmon resonance (SPR), PWR has the advantage of allowing measurements in both s-and p-polarizations on a waveguide surface that is silica or glass rather than a noble metal. Here the waveguide is a BK7 glass slide coated with silver and silica layers. The resonance wavelength is sensitive to the optical thickness of the medium adjacent to the silica layer. The sensitivity of this technique is characterized and compared with broadband SPR both experimentally and theoretically. The sensitivity of spectral PWR is comparable to that of spectral SPR for samples with refractive indices close to that of water. The hydrophilic surface of the waveguide allows supported lipid bilayers to be formed spontaneously by vesicle fusion; in contrast, the surface of an SPR chip requires chemical modification to create a supported lipid membrane. Broadband PWR spectroscopy should be a useful technique to study biointerfaces, including ligand binding to transmembrane receptors and adsorption of peripheral proteins on ligand-bearing membranes. © 2009 Society for Applied Spectroscopy.
- Araci, Z. O., Runge, A. F., J., W., & Saavedra, S. S. (2008). Correlating molecular orientation distributions and electrochemical kinetics in subpopulations of an immobilized protein film. Journal of the American Chemical Society, 130(5), 1572-1573.More infoPMID: 18193877;Abstract: Understanding the relationship between the structure and electrochemical activity of a protein film immobilized on an electrode surface is a prerequisite to the rational design of protein-based bioelectronic devices. In a monolayer of horse heart cytochrome c (cyt c) adsorbed to an indium-tin oxide (ITO) electrode, only about half of the film is electroactive, which makes it difficult to correlate the broad orientation distribution (measured spectroscopically on the entire film) with the electron transfer rate constant (measured electrochemically on the electroactive portion of the film) [Runge, A. F.; Mendes, S. B.; Saavedra, S. S. J. Phys. Chem. B 2006, 110, 6732-6739]. To address this problem, a novel form of electroreflectance spectroscopy, potential-modulated, attenuated total reflectance (PM-ATR), is used to monitor changes in the absorbance of a cyt c while a modulated potential is simultaneously applied to the ITO-coated, planar waveguide electrode. From measurements as a function of the light polarization and modulation frequency, electron transfer rate constants for differently oriented subpopulations of cyt c molecules are obtained. The apparent rate constant measured using transverse magnetic (TM) polarized light was 3.3-fold greater than that measured using transverse electric (TE) polarized light, while the rate constant measured electrochemically was intermediate between the TM and TE constants. These data are consistent with a shorter heme-electrode tunneling distance for molecules adsorbed in a vertical orientation (probed with TM) versus molecules adsorbed in a horizontal orientation (probed with TE). This is the first study to correlate a distribution of molecular orientations with a distribution of electron transfer rate constants in a redox-active molecular film. Copyright © 2008 American Chemical Society.
- Ratnayaka, S. N., Wysocki Jr., R. J., & Saavedra, S. S. (2008). Preparation and characterization of asymmetric planar supported bilayers composed of poly(bis-sorbylphosphatidylcholine) on n-octadecyltrichlorosilane SAMs. Journal of Colloid and Interface Science, 327(1), 63-74.More infoPMID: 18755471;PMCID: PMC2622739;Abstract: Planar supported lipid bilayers (PSLBs) have been widely studied as biomembrane models and biosensor scaffolds. For technological applications, a major limitation of PSLBs composed of fluid lipids is that the bilayer structure is readily disrupted when exposed to chemical, mechanical, and thermal stresses. A number of asymmetric supported bilayer structures, such as the hybrid bilayer membrane (HBM) and the tethered bilayer lipid membrane (tBLM), have been created as an alternative to symmetric PSLBs. In both HBMs and tBLMs, the inner monolayer is covalently attached to the substrate while the outer monolayer is typically composed of a fluid lipid. Here we address if cross-linking polymerization of the lipids in the outer monolayer of an asymmetric supported bilayer can achieve the high degree of stability observed previously for symmetric PSLBs in which both monolayers are cross-linked [E.E. Ross, L.J. Rozanski, T. Spratt, S.C. Liu, D.F. O'Brien, S.S. Saavedra, Langmuir 19 (2003) 1752]. To explore this issue, HBMs composed of an outer monolayer of a cross-linkable lipid, bis-sorbylphosphatidylcholine (bis-SorbPC), and an inner SAM were prepared and characterized. Several experimental conditions were varied: vesicle fusion time, polymerization method, and polymerization time and temperature. Under most conditions, bis-SorbPC cross-linking stabilized the HBM such that its bilayer structure was largely preserved after drying; however these films invariably contained sub-micron scale defects that exposed the hydrophobic core of the HBM. The defects appear to be caused by desorption of low molecular weight oligomers when the film is removed from water, rinsed, and dried. In contrast, poly(bis-SorbPC) PSLBs prepared under similar conditions by Ross et al. were nearly defect free. This comparison shows that formation of a cross-linked network in the outer leaflet of an asymmetric supported bilayer is insufficient to prevent lipid desorption; inter-leaflet covalent linking appears to be necessary to create supported poly(lipid) assemblies that are impervious to repeated drying and rehydration. The difference in stability is attributed to inter-leaflet cross-linking between monolayers which can form in symmetric bis-SorbPC PSLBs. © 2008 Elsevier Inc. All rights reserved.
- Senarath-Yapa, M. D., Phimphivong, S., Coym, J. W., Wirth, M. J., Aspinwall, C. A., & Saavedra, S. S. (2007). Preparation and characterization of poly(lipid)-coated, fluorophore-doped silica nanoparticles for biolabeling and cellular imaging. Langmuir, 23(25), 12624-12633.More infoPMID: 17975939;Abstract: The fabrication, characterization, and implementation of poly(lipid)-coated, highly luminescent silica nanoparticles as fluorescent probes for labeling of cultured cells are described. The core of the probe is a sol-gel-derived silica nanoparticle, 65-100 nm in diameter, in which up to several thousand dye molecules are encapsulated (Lian, W.; et al. Anal. Biochem. 2004, 334, 135-144). The core is coated with a membrane composed of bis-sorbylphosphatidylcholine, a synthetic polymerizable lipid that is chemically cross-linked to enhance the environmental and chemical stability of the membrane relative to a fluid lipid membrane. The poly(lipid) coating has two major functions: (i) to reduce nonspecific interactions, based on the inherently biocompatible properties of the phosphorylcholine headgroup, and (ii) to permit functionalization of the particle, by doping the coating with lipids bearing chemically reactive or bioactive headgroups. Both functions are demonstrated: (i) Nonspecific adsorption of dissolved proteins to bare silica nanoparticles and of bare nanoparticles to cultured cells is significantly reduced by application of the poly(lipid) coating. (ii) Functionalization of poly(lipid)-coated nanoparticles with a biotin-conjugated lipid creates a probe that can be used to target both dissolved protein receptors as well as receptors on the membranes of cultured cells. Measurements performed on single nanoparticles bound to planar supported lipid bilayers verify that the emission intensity of these probes is significantly greater than that of single protein molecules labeled with several fluorophores. © 2007 American Chemical Society.
- Araci, Z. O., Runge, A. F., J., W., & Saavedra, S. S. (2006). Potential modulated attenuated total reflectance spectroscopy of prussian blue films on ITO. Israel Journal of Chemistry, 46(3), 249-255.More infoAbstract: Potential modulated attenuated total reflectance (PM-ATR) spectroscopy has been employed to study charge transfer processes in Prussian blue (PB) films deposited on indium tin oxide (ITO) electrodes. PM-ATR is a planar waveguide-based spectroelectrochemical technique in which the optical response of an electroactive film is measured as a function of applied potential and modulation frequency. The multiple internal reflection geometry of PM-ATR provides a significant sensitivity advantage over the single external reflectance geometry that has been employed in most prior electroreflectance studies. The apparent electron transfer rate of PB on ITO obtained using PM-ATR was compared to that obtained with conventional cyclic voltammetry; the respective rates, 0.33 ± 0.15 s-1 (n = 3) and 0.71 ± 0.37 s-1 (n = 10), are in good agreement.
- Ross, E. E., Joubert, J. R., Wysocki Jr., R. J., Nebesny, K., Spatt, T., O'Brien, D. F., & Saavedra, S. S. (2006). Patterned protein films on poly(lipid) bilayers by microcontact printing. Biomacromolecules, 7(5), 1393-1398.More infoPMID: 16677019;Abstract: The use of polymerized lipid bilayers as substrates for microcontact printing (μCP) of protein films was investigated. We have previously shown that vesicle fusion of bis-SorbPC, a dienoate lipid, on glass and silica substrates, followed by redox-initiated radical polymerization, produces a planar supported lipid bilayer (PSLB) that is ultrastable [Ross, E. E.; Rozanski, L. J.; Spratt, T.; Liu, S.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 1752] and highly resistant to nonspecific adsorption of dissolved proteins [Ross, E. E.; Spratt, T.; Liu, S.; Rozanski, L. J.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 17661.] Here we demonstrate that μCP of bovine serum albumin (BSA) onto a dried poly(bis-SorbPC) PSLB from a poly(dimethylsiloxane) (PDMS) stamp produces a layer of strongly adsorbed protein, comparable in surface coverage to films printed on glass surfaces. Immobilization of proteins on poly(PSLB)s has potential applications in biosensing, and this work shows that direct μCP of proteins is a technically simple approach to create immobilized monolayers, as well as multilayers of different proteins. © 2006 American Chemical Society.
- Runge, A. F., Mendes, S. B., & Saavedra, S. S. (2006). Order parameters and orientation distributions of solution adsorbed and microcontact printed cytochrome c protein films on glass and ITO. Journal of Physical Chemistry B, 110(13), 6732-6739.More infoPMID: 16570979;Abstract: The structure of solution adsorbed and microcontact printed (μCP) cytochrome c (cyt c) films on glass and indium tin oxide (ITO) was investigated using attenuated total reflectance (ATR) and total internal reflectance fluorescence (TIRF) spectroscopies to determine the orientation of the heme groups in the films. The second and fourth order parameters of the heme as well as information on the angle between the absorption and emission dipoles of the heme, γ, were experimentally determined. The order parameters of the heme are related to the order parameters of the protein molecule using the known angle between the heme plane and the electrostatic dipole moment of the cyt c protein. The effect of the surface roughness of the substrates (glass and ITO) was also taken into account quantitatively using AFM data. Physically possible order parameters were obtained for the heme group in both solution adsorbed and μCP films, but not for the electrostatic dipole moment of the protein. In addition, the experimental values of 〈cos 2 γ〉 for immobilized zinc-substituted cyt c are greater than the values of 〈cos 2 γ〉 determined in viscous solutions, which could be an indication that the environment of the heme groups changes upon adsorption. The electron transfer behavior of solution adsorbed and μCP films on ITO, determined using electrochemical methods, is compared to their orientation distribution and surface coverage as determined by spectroscopic methods. © 2006 American Chemical Society.
- Runge, A. F., Saavedra, S. S., & Mendes, S. B. (2006). Combination of polarized TIRF and ATR spectroscopies for determination of the second and fourth order parameters of molecular orientation in thin films and construction of an orientation distribution based on the maximum entropy method. Journal of Physical Chemistry B, 110(13), 6721-6731.More infoPMID: 16570978;Abstract: This article describes two mathematical formalisms for the determination of the second and fourth order parameters of molecular films using optical spectroscopy. Method A uses polarized total internal reflection fluorescence (TIRF) to calculate the second and fourth order parameters, 〈P 2(cos θ)〉 and 〈P 4(cos θ)〉, using an independently determined value for the angle between the absorption and emission dipoles, γ. Method B uses 〈P 2(cos θ)〉 obtained from attenuated total reflectance (ATR) data, along with polarized TIRF measurements to calculate 〈P 4(cos θ)〉 and (cos 2 γ). The choice of a specific method should rely on experimental considerations. We also present a method to separate the contributions of substrate surface roughness and dipole orientation with respect to the molecular axis from the spectroscopically determined second and fourth order parameters. Finally, a maximum entropy approach for construction of an orientation distribution from order parameters is compared with the commonly used delta and Gaussian distributions. © 2006 American Chemical Society.
- Bradshaw, J. T., Mendes, S. B., & Saavedra, S. S. (2005). Planar integrated optical waveguide spectroscopy. Analytical Chemistry, 77(1), 28 A-36 A.More infoAbstract: Integrated optical waveguides (IOWs) exhibit a proportionally longer path length relative to thick waveguides. John Thomas Bradshaw, Sergio B. Mendes, and S. Scott Saavedra at the University of Arizona focus on IOWs with a planar geometry to study thin films and interfaces.
- Lawton, R. A., Price, C. R., Runge, A. F., J., W., & Saavedra, S. S. (2005). Air plasma treatment of submicron thick PDMS polymer films: Effect of oxidation time and storage conditions. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 253(1-3), 213-215.More infoAbstract: The effect of oxidation time and storage conditions on the surface wettability of thin films of polydimethylsiloxane (PDMS) spun cast onto glass slides was studied. Films were oxidized in a low temperature air plasma for either 1 min or 6 s and stored in either air, water, or hexadecane. Analysis of the sessile water contact angle with time shows that partial to complete hydrophobic recovery occurs within days under all conditions. © 2004 Elsevier B.V. All rights reserved.
- McBee, T. W., & Saavedra, S. S. (2005). Stability of lipid films formed on λ-aminopropyl monolayers. Langmuir, 21(8), 3396-3399.More infoPMID: 15807579;Abstract: The stability of supported lipid membranes (SLMs) deposited on planar substrates derivatized with (λ-aminopropyl)silane (GAPS) was examined. Ellipsometry, fluorescence microscopy, and atomic force microscopy were used to characterize SLMs exposed to repeated drying and rehydration. Vesicle fusion on GAPS-coated substrates produced SLMs with a thickness significantly greater than that of a single lipid bilayer. Exposure to even one cycle of drying/rehydration significantly decreased the thickness of a SLM on GAPS, and repeated drying/rehydration resulted in near quantitative lipid desorption. Thus SLMs on GAPS do not appear to be significantly more stable than the single bilayer SLM that is formed on bare glass or SiO 2 under equivalent conditions. © 2005 American Chemical Society.
- Runge, A. F., Rasmussen, N. C., Saavedra, S. S., & Mendes, S. B. (2005). Determination of anisotropic optical constants and surface coverage of molecular films using polarized visible ATR spectroscopy. Application to adsorbed cytochrome c films. Journal of Physical Chemistry B, 109(1), 424-431.More infoPMID: 16851032;Abstract: This article describes a method to determine the anisotropic optical constants and surface coverage of molecular films using polarized attenuated total reflectance (ATR) absorbance measurements. We have extended the transfer-matrix formalism to describe birefringent and dichroic films in ATR geometries and have combined it with an iterative numerical procedure to determine the anisotropic values of both the real (n) and imaginary (k) parts of the complex refractive index of the film under investigation. Anisotropic values of the imaginary part of the refractive index (k) allow for the determination of the surface coverage and one order parameter of the film. To illustrate this approach, we have used cytochrome c (cyt c) protein films adsorbed to glass and indium tin oxide (ITO) surfaces. Experimental results show that cyt c films on these surfaces, which were formed under identical conditions, have significant differences in their surface coverages (11.2 ± 0.4 pmol/cm 2 on glass and 21.7 ± 0.9 pmol/cm 2 on ITO); however, their order parameters 〈(cos 2θ)〉 are similar (0.30 ± 0.02 on glass and 0.36 ± 0.04 on ITO).
- Runge, A. F., Rasmussen, N. C., Saavedra, S. S., & Mendes, S. B. (2005). Erratum: Determination of anisotropic optical constants and surface coverage of molecular films using polarized visible ATR spectroscopy. Application to adsorbed cytochrome c films (Journal of Physical Chemistry B (2005) 109B (427)). Journal of Physical Chemistry B, 109(18), 9194-.
- Mendes, S. B., Bradshaw, J. T., & Saavedra, S. S. (2004). Technique for determining the angular orientation of molecules bound to the surface of an arbitrary planar optical waveguide. Applied Optics, 43(1), 70-78.More infoPMID: 14714646;Abstract: A technique to determine the angular orientation of a molecular assembly bound to the surface of a planar optical waveguide of arbitrary structure is described. The approach is based on measuring the absorption dichroic ratio by using the waveguide evanescent fields with orthogonal polarizations (TE, TM) and the same mode order to probe two molecular assemblies, (i) a reference sample composed of an isotropic orientation distribution of dipoles and (ii) a sample of interest. The isotropic sample is used to characterize the waveguide structure, which then allows the orientation parameters of a molecular assembly under investigation to be determined from a measured dichroic ratio. The method developed here is particularly important for applications in gradient-index and multilayer planar waveguide platforms because in those cases the extension of previously reported approaches would require a full experimental characterization of the guiding structure, which would be problematic and may yield inaccurate results.
- Conboy, J. C., Liu, S., O'Brien, D. F., & Saavedra, S. S. (2003). Planar supported bilayer polymers formed from bis-diene lipids by Langmuir-Blodgett deposition and UV irradiation. Biomacromolecules, 4(3), 841-849.More infoPMID: 12741807;Abstract: Substrate-supported lipid bilayers have been prepared from bis-diene functionalized phosphorylcholine (PC) lipids and polymerized by UV irradiation. The overall bilayer structure is largely preserved upon removal from water, although significant loss of material occurs from the upper leaflet of the bilayer, likely due to desorption at the air/water interface. The morphology and surface structure of the bilayer, as observed by AFM, indicate a substantially different arrangement of the lipids in the hydrated and dehydrated states, presumably due to the loss of water from the near surface region. These changes have been correlated with infrared spectral shifts sensitive to the conformation of the hydrocarbon chains. Protein adsorption studies show that rehydrated, polymerized bilayers retain a degree of resistance to BSA adsorption intermediate between model hydrophobic and fluid PC lipid bilayer surfaces. The degree of protein adsorption is correlated with desorption of material from the upper leaflet of the bilayer upon drying, which produces voids at which hydrophobically driven protein adsorption occurs.
- Du, Y., & Saavedra, S. S. (2003). Molecular orientation distributions in protein films. V. Cytochrome c adsorbed to a sulfonate-terminated, self-assembled monolayer. Langmuir, 19(16), 6443-6448.More infoAbstract: Controlling the molecular orientation of protein molecules immobilized on insoluble substrates is now recognized as an important issue in numerous fields such as biosensing. In this study, we have investigated adsorption of horse heart cytochrome c (hcyt c) to a silane-based, self-assembled monolayer (SAM) as a strategy for preparing an oriented protein film. Previous attempts in our laboratory to create oriented protein films using this strategy were unsuccessful because multiple types of adsorptive interactions between the protein and the SAM produced a broad range of adsorbed orientations. In contrast, here we show that when a single type of site-directed interaction between hcyt c molecules and the SAM predominates, an oriented protein film results. Sulfonate-terminated SAMs were prepared by in situ oxidation of thioacetate SAMs. Electrostatically mediated adsorption of positively charged hcyt c produced a film with a porphyrin tilt angle distribution of 41° ± 13°, which is significantly narrower than distributions measured previously for cytochrome c films formed on other types of silane-based SAMs.
- Ross, E. E., Rozanski, L. J., Spratt, T., Liu, S., O'Brien, D. F., & Saavedra, S. S. (2003). Planar supported lipid bilayer polymers formed by vesicle fusion. 1. Influence of diene monomer structure and polymerization method on film properties. Langmuir, 19(5), 1752-1765.More infoAbstract: Although fluid lipid films have been used widely in biosensing devices, they lack the high stability desired for technological implementation because the noncovalent forces between the constituent lipids are relatively weak. In this work, polymerized, planar supported lipid bilayers ((poly)PSLBs) composed of diene-functionalized lipids have been prepared and characterized. Several parameters relating (poly)PSLB structure and stability to observations made in studies of polymerized bilayer vesicles were examined, including a comparison of UV photopolymerization and redox-initiated radical polymerization, the number and location of the polymerizable moieties in the lipid monomer, and a comparison to PSLBs produced with diacetylene lipids. Redox-initiated polymerization of films composed of bis-substituted diene lipids with at least one polymerizable moiety located near the acyl terminus produced dried PSLBs that were highly uniform and stable. All other conditions yielded PSLBs that contained a high density of defects after drying, including those formed from diacetylene lipids. In most cases, defect formation is attributed to desorption of unreacted monomers or low molecular weight polymers when the film was passed through the air/water interface. Studies on highly stable (poly)PSLBs doped with nonpolymerizable lipids showed that 40-80% of the dopants are retained when the film is dried. Thus to ensure quantitative lipid retention upon PSLB removal from water, all of the lipid monomers must be covalently anchored to the polymer network.
- Ross, E. E., Spratt, T., Liu, S., Rozanski, L. J., O'Brien, D. F., & Saavedra, S. S. (2003). Planar supported lipid bilayer polymers formed by vesicle fusion. 2. Adsorption of bovine serum albumin. Langmuir, 19(5), 1766-1774.More infoAbstract: Planar supported lipid bilayers (PSLBs) composed of phosphorylcholine (PC) lipids are known to be highly resistant to nonspecific adsorption of soluble proteins. However, these structures lack the stability desired for implementation in molecular devices (e.g., biosensors). In part 1 of this report [Ross, E. E.; Spratt, T.; Liu, S.; Rozanski, L. J.; O'Brien, D. F.; Saavedra, S. S. Langmuir 2003, 19, 1752-1765], we described preparation and characterization of (poly)PSLBs composed of diene-functionalized lipids. Here we have examined nonspecific adsorption of bovine serum albumin (BSA) to these (poly)PSLBs and several reference surfaces, using total internal reflection fluorescence and atomic force microscopy. The results show that the protein resistance of a cross-linked (poly)PSLB composed of bis-sorbyl phosphatidylcholine (bis-SorbPC) is equivalent to that of a fluid PSLB composed of 1-palmitoyl-2-oleoylphosphatidylcholine, even after the former has been dried and rehydrated. Since the lipids in a (poly)bis-SorbPC PSLB are laterally immobile, these results also demonstrate that fluidity is not required for a PC lipid bilayer to resist nonspecific protein adsorption. Relative to (poly)bis-SorbPC, BSA adsorption to other types of (poly)-PSLBs was significantly greater, including PSLBs formed from a diacetylene-functionalized lipid. The increased protein adsorption is attributed to a higher density of structural defects.
- Runge, A. F., & Saavedra, S. S. (2003). Comparison of Microcontact-Printed and Solution-Adsorbed Cytochrome c Films on Indium Tin Oxide Electrodes. Langmuir, 19(22), 9418-9424.More infoAbstract: Three different methods for preparing cytochrome c (cyt c) films on planar indium tin oxide (ITO) electrodes have been characterized and compared using electrochemical and surface spectroscopy methods. Protein films formed by adsorption of dissolved cyt c and by microcontact printing with both hydrophobic and hydrophilic poly(dimethylsiloxane) (PDMS) stamps are compared in terms of the total and electrochemical surface coverage, standard reduction potential, and rate of direct electron transfer with the ITO surface. Plasma treatment of the PDMS stamps, which renders them hydrophilic, dramatically increases the surface coverage of printed films to a level comparable to that of films formed by adsorption of dissolved cyt c. Their electrochemical properties are also very similar. This work is the first report of the direct electrochemical activity in a metalloprotein film formed by microcontact printing on an electrode surface.
- Bradshaw, J. T., Mendes, S. B., & Saavedra, S. S. (2002). A simplified broadband coupling approach applied to chemically robust sol-gel, planar integrated optical waveguides. Analytical Chemistry, 74(8), 1751-1759.More infoPMID: 11985305;Abstract: A new generation waveguide spectrometer with broadband coupling capabilities has been developed. As opposed to previous devices, this attenuated total reflection (ATR) spectrometer is much simpler in design, is more chemically robust, and transmits light down to at least 400 nm. The attenuated total reflection element consists of a single-mode, planar integrated optical waveguide fabricated by dip-coating a ∼ 300 nm thick, sol-gel composite layer on a glass substrate. A commercially available prism is used as the incoupler with an integral holographic diffraction grating acting as the dispersive outcoupling element. The transmission of narrow band-pass filters was used to compare the response of the waveguide spectrometer to that of a conventional transmission instrument. Spectral resolution was assessed by measuring the fwhm of various laser lines, which were found to range from 0.5 to 1.3 nm. The measured limits of detection for the waveguide spectrometer from 400 to 600 nm are 8.0 and 10.1 milliabsorbance units for TE and TM polarizations, respectively. Finally, to demonstrate the application of this technology to a molecular film confined to a solid-liquid interface, visible ATR spectra of an adsorbed submonolayer of horse heart cytochrome c were acquired. A procedure to correct the waveguide spectra for the wavelength dependence in ATR path length is described.
- Conboy, J. C., McReynolds, K. D., Gervay-Hague, J., & Saavedra, S. S. (2002). Quantitative measurements of recombinant HIV surface glycoprotein 120 binding to several glycosphingolipids expressed in planar supported lipid bilayers. Journal of the American Chemical Society, 124(6), 968-977.More infoPMID: 11829604;Abstract: The interaction of recombinant HIV-1 surface glycoprotein gp120 (rgp120) with natural isolates of lactosylceramide (LacCer), glucosylceramide (GcCer), and galactosylceramide (GaCer) has been quantitatively measured under equilibrium conditions using total internal reflection fluorescence (TRF) spectroscopy. The binding affinity (Ka) of rgp120 to these glycosphingolipids (GSLs), reconstituted at 5 mol % in supported planar lipid bilayers composed of 95 mol % POPC, is ca. 106 M-1 for dissolved rgp120 concentrations greater than 25 nM. In contrast, at concentrations of rgp120 between 0.2 and 15 nM, rgp120 does not bind significantly to LacCer and GlcCer, but has a high affinity for GalCer with a measured Ka value of 1.6 × 109 M-1. However, protein surface coverage measurements show that this strong binding process accounts for very little of the total protein adsorbed over the entire concentration range studied. At a protein concentration of ca. 20 nM, the surface coverage is only 3% of that achieved at apparent saturation (i.e., when the protein concentration is ca. 220 nM). Thus the "high affinity" binding sites comprise only a small fraction of the total number of binding sites. Several other variables were investigated. Rgp120 binding behavior at membranes doped with α-hydroxygalactosylceramide (α-GalCer) was very similar to that observed with GalCer, showing that the presence/absence of an α-hydroxy moiety does not significantly affect galactosylceramide recognition. Phase segregation of GalCer, which occurs when the mole fraction of this GSL in a POPC bilayer exceeds ca. 0.1, was also investigated and showed no effect on binding affinity at low rgp120 concentrations. To investigate the influence of fatty acid chain length, GSLs with monodisperse C18 and C24 chain lengths, both with and without an α-hydroxy moiety, were synthesized, and their binding affinity to rgp120 was examined. Relative to the natural isolates (which contain a mixture of chain lengths), minimal differences were observed; thus among the compounds tested, fatty acid chain length does not affect GSL recognition. The results of this work should aid efforts to design anti-HIV-1 agents based on membrane-tethered, carbohydrate-based receptors for rgp120.
- Kasmi, A. E., Leopold, M. C., Galligan, R., Robertson, R. T., Saavedra, S., Kacemi, K. E., & Bowden, E. F. (2002). Adsorptive immobilization of cytochrome c on indium/tin oxide (ITO): Electrochemical evidence for electron transfer-induced conformational changes. Electrochemistry Communications, 4(2), 177-181.More infoAbstract: The adsorptive immobilization and electrochemistry of horse and yeast cytochrome c on indium/tin oxide (ITO) electrodes is reported. Near-monolayer coverage was achieved in pH 7 phosphate buffers of ionic strength equal to 10 and 50 mM, respectively, for the horse and yeast species. The layers exhibit very well-behaved voltammetry and are stable on the timescale of hours to days. Cyclic voltammetry revealed quasireversible behavior that is a product of both electron transfer (ET) kinetics and ET-induced conformational changes. A square scheme mechanism linking the redox states and the conformational states is proposed. Using a simple ET kinetic model that adequately describes the voltammetry at higher scan rates, a standard ET rate constant of 18 s-1 was determined for adsorbed horse cytochrome c. With decreasing scan rate, we observed a limiting peak separation of approximately 10 mV, an example of unusual quasireversibility (UQR) that we attribute to the effect of conformational changes. Finally we note that the intrinsic cytochrome c ET rate on ITO is some 6 orders of magnitude less than for gold. © 2002 Published by Elsevier Science B.V.
- McReynolds, K. D., Bhat, A., Conboy, J. C., Saavedra, S., & Gervay-Hague, J. (2002). Non-natural glycosphingolipids and structurally simpler analogues bind HIV-1 recombinant gp120. Bioorganic and Medicinal Chemistry, 10(3), 625-637.More infoPMID: 11814851;Abstract: Interactions of recombinant gp120 (rgp120) with non-natural glycosphingolipids (GSLs) and structurally simpler analogues have been studied using a competitive adhesion assay. Conjugates of cellobiosyl ceramide and melibiosyl ceramide were synthetically prepared as water-soluble GSL analogues. These ligands were screened against a panel of biologically relevant analogues, and the results show that their interactions with rgp120 are comparable to natural cellular receptors. Glycolipid interactions with rgp120 were probed further by the synthesis and testing of structurally simpler analogues that were obtained by reductive amination of lactose, cellobiose, and melibiose with a biotinylated amino ethylene glycol moiety. RGp120 did not recognize conjugates lacking a lipid component. However, palmitoylation of the secondary amino alditols yielded compounds with comparable rgp120 affinity to the natural cellular receptor, galactosyl ceramide (GalCer). Taken together, the SAR showed that both a hydrophobic and a hydrophilic component are required for rgp120 recognition. Moreover, structural variability in the carbohydrate headgroup did not significantly alter rgp120 recognition indicating that this interaction is not highly specific. © 2002 Elsevier Science Ltd. All rights reserved.
- Ross, E. E., Bondurant, B., Spratt, T., Conboy, J. C., O'Brien, D. F., & Saavedra, S. S. (2001). Formation of self-assembled, air-stable lipid bilayer membranes on solid supports. Langmuir, 17(8), 2305-2307.More infoAbstract: A successful strategy for the self-assembly and stabilization of a substrate-supported, phospholipid bilayer is described. The bilayer is self-organized by fusion of fluid vesicles, composed of bissorbylphosphatidylcholine, on an oxide surface. The supported bilayer is then polymerized in situ to produce a cross-linked structure that is stable to surfactant solutions, organic solvents, and to transfer across the air/water interface, yet retains the resistance to nonspecific protein adsorption characteristic of a fluid phosphatidylcholine bilayer.
- Senarath-Yapa, M. D., & Saavedra, S. S. (2001). Dye leaching from a doped sol-gel is eliminated by conjugation to a dendrimer. Analytica Chimica Acta, 432(1), 89-94.More infoAbstract: The extent of leaching of dye molecules following encapsulation into sol-gel glasses was investigated. Covalent attachment of erythrosin isothiocyanate (EITC) to a polyamidoamine generation 4 (PAMAM) dendrimer, having a molecular weight of 14,215 Da, eliminated all detectable leaching from hydrated monoliths prepared from tetramethylorthosilicate (TMOS). In contrast, a prior study showed that significant leaching takes place when the dye is conjugated to 70 kDa dextran. To eliminate dye leaching via attachment to a macromolecular carrier, an inherently globular macromolecule having an organized secondary structure is therefore required. © 2001 Elsevier Science B.V.
- Yingmei, G. u., LaBell, R., O'Brien, D. F., & Saavedra, S. S. (2001). Quantitative studies of binding between synthetic galactosyl ceramide analogues and HIV-1 gp120 at planar membrane surfaces. Angewandte Chemie - International Edition, 40(12), 2320-2322.More infoAbstract: A critical spacer arm length necessary to promote efficient binding of the HIV-1 surface glycoprotein rgpl20 to several synthetic galactosyl-conjugated lipids, reconstituted into planar lipid bilayers, was identified (see figure). This should aid the design of anti-HIV-1 agents based on membrane-tethered, carbohydrate-based receptors for gpl20.
- Conboy, J. C., McReynolds, K. D., Gervay-Hague, J., & Saavedra, S. S. (2000). Gp120 binds cooperatively to several biologically relevant glycosphingolipids: Quantitative measurements at equilibrium by total internal reflection fluorescence microscopy. Angewandte Chemie - International Edition, 39(16), 2882-2884.More infoAbstract: The binding of The HIV-1 surface glycoprotein gp120 to several glycosphingolipides, reconstituted into planar lipid bilayers (see scheme; small circle= 2-oleoyl-1-palmitoylphosphatidylcholine (POPC), arrow = glycosylceramide recognition element; left: low and right: high protein concentrations), has been quantitatively measured under quilibrium conditions. Complex binding behavior was observed, which suggests that the induced aggregation of gp120 molecules at a ligand-bearing membrane surface may be an important step in the mechanism of HIV-1 infection of a host cell.
- Du, Y., Wood, L. L., & Saavedra, S. S. (2000). Growth behavior and structure of alkyltrichlorosilane monolayers bearing thioacetate and acetate tailgroups. Materials Science and Engineering C, 7(2), 161-169.More infoAbstract: The growth behavior of self-assembled monolayer (SAM) films formed from Cl3SiC11H22OCOCH3 (C11Ac) and Cl3SiC16H32SCOCH3 (C16Sc) on oxidized Si wafers at room temperature was investigated using atomic force microscopy (AFM), water contact angle measurements, and ellipsometry. Both silanes form thin films via nucleation of islands, similar to the growth behavior of n-octadecyltrichlorosilane (OTS) SAMs, although the island morphology is distinct for each molecule. Near-confluent monolayer films are obtained for both C11Ac and C16Sc after substrate immersion periods of 10-60 min. The ellipsometric thickness of both types of SAMs is less than that expected for a close-packed, all-trans ensemble of methylene chains aligned parallel to the substrate surface normal, suggesting a liquid crystalline-like structure in which methylene groups are present at the outer film surface. This model is supported by surface wettability data obtained on C11Ac SAMs that were reduced in situ to generate hydroxy-terminated films. When the deposition time is greater than 1 h, C11Ac forms highly disordered films of multilayer thickness that are much rougher than the films obtained at shorter deposition times. Multilayer formation is not observed with C16Sc, although increased deposition times also result in adsorption of additional, submonolayer amounts of material. © 2000 Elsevier Science S.A.
- Mendes, S. B., & Saavedra, S. S. (2000). Comparative analysis of absorbance calculations for integrated optical waveguide configurations by use of the ray optics model and the electromagnetic wave theory. Applied Optics, 39(4), 612-621.More infoPMID: 18337933;Abstract: Focusing on the use of planar waveguides as platforms for highly sensitive attenuated total reflection spectroscopy of organic thin films, we extend the ray optics model to provide absorbance expressions for the case of dichroic layers immobilized on the waveguide surface. Straightforward expressions are derived for the limiting case of weakly absorbing, anisotropically oriented molecules in the waveguide - cladding region. The second major focus is on the accuracy of the ray optics model. This model assumes that the introduction of absorbing species, either in the bulk cladding or as an adlayer on the waveguide surface, only causes a small perturbation to the original waveguide-mode profile. We investigate the accuracy of this assumption and the conditions under which it is valid. A comparison to an exact calculation by use of the electromagnetic wave theory is implemented, and the discrepancy of the ray optics model is determined for various waveguide configurations. We find that in typical situations in which waveguide-absorbance measurements are used to study organic thin films (kl/nl ≤ 10-1, h/λ ≈ 10-2) the discrepancy between the ray optics and the exact calculations is only a few percent (2-3%). © 2000 Optical Society of America.
- Mendes, S. B., & Saavedra, S. S. (1999). On probing molecular monolayers: A spectroscopic optical waveguide approach of ultra-sensitivity. Optics Express, 4(11), 449-456.More infoPMID: 19396302;Abstract: A broadband, multichannel, single-mode, planar waveguide based spectrometer was developed for probing molecular monolayers. A protein sub-monolayer (thickness h ≅ 3 nm, imaginary part of refractive index kl ≤ 0.01) immobilized on the waveguide surface was characterized by the waveguide attenuated total reflection (ATR) spectrometer. A sensitivity enhancement of 4 orders of magnitude, compared to conventional transmission measurements, has been experimentally achieved in the characterization of ultra-thin films. In addition, polarized spectroscopic measurements at the TE and TM waveguide modes were implemented to determine the average orientation angle of the adsorbed molecules. The work developed here is a new research tool for the investigation of some fundamental aspects of molecular films and a novel platform to develop new technological devices of high sensitivity and selectivity such as biosensors. © 1999 Optical Society of America.
- Saavedra, S., Edmiston, P. L., Lee, J. E., Wood, L. L., Dunphy, D. R., Robertson, R. T., Gabbard, E. A., & Mendes, S. B. (1999). Probing structure and function in planar supported protein films. Proceedings of SPIE - The International Society for Optical Engineering, 3858, 146-150.More infoAbstract: The development of molecular devices based on immobilized protein films is currently a very active area of research worldwide. Detailed structural and functional characterization of these films is a prerequisite to the rational development of deposition methods that produce bioactive structures, but is a technically difficult challenge. Two recent thrusts in our work have been investigation of methods designed to create macroscopically ordered arrays of protein molecules, and development of new optical techniques to characterize the ensemble properties of these arrays.
- Edmiston, P. L., & Saavedra, S. S. (1998). Molecular orientation distributions in protein films: III. Yeast cytochrome c immobilized on pyridyl disulfide-capped phospholipid bilayers. Biophysical Journal, 74(2 I), 999-1006.More infoPMID: 9533712;PMCID: PMC1302580;Abstract: Molecular orientation in a hydrated monolayer film of yeast cytochrome c, immobilized via disulfide bonding between Cys-102 and a pyridyl disulfide- capped phospholipid bilayer deposited from an air-water interface onto glass substrates, was investigated. The orientation distribution of the heme groups in the protein film was determined using a combination of absorption linear dichroism, measured in a planarintegrated optical waveguide-attenuated total reflection geometry- and fluorescence anisotropy, measured in a total internal reflection geometry. A gaussian model for the orientation distribution was used to recover the mean heme tilt angle and angular distribution about the mean which were 40 and 11°, respectively. Additional experiments showed that a large fraction of the cytochrome c was disulfide bonded to the bilayer, which correlates with the high degree of macroscopic order in the protein film. However, a subpopulation of yeast cytochrome c molecules in the film (~30% of the total) appeared to be nonspecifically adsorbed. The orientation distribution of this subpopulation was found to be much broader than the specifically bound fraction.
- Phimphivong, S., & Saavedra, S. S. (1998). Terbium chelate membrane label for time-resolved, total internal reflection fluorescence microscopy of substrate-adherent cells. Bioconjugate Chemistry, 9(3), 350-357.More infoPMID: 9576809;Abstract: A chelating agent conjugated to a lipid was synthesized by reacting the dianhydride form of diethylenetriaminepentaacetic acid sequentially with 4-aminosalicylate and dioleoylphosphatidyl-ethanolamine. The product, DOPE-YAS-Tb, exhibits photophysical properties characteristic of a chelated Tb3+ ion bound to an organic triplet donor: an excitation maximum at 310 nm, narrow emission bands at 490, 545, 590, and 625 nm, and a lifetime of 1.57 ms. The suitability of DOPE-YAS-Tb as a membrane-staining agent for morphological studies of cultured cells using total internal reflection fluorescence microscopy (TIRFM) was investigated. Swiss albino mouse 3T3 cells were cultured on silica and polystyrene substrates. Time-resolved detection was employed to reject short-lived background emission (autoemission from the cells and/or the polymer substrate), which allowed the long-lived Tb3+ emission to be selectively imaged. The results show that time-resolved TIRFM of cells stained with DOPE-YAS-Tb is an effective method of quantitatively examining the cell morphology in situations where background due to autoemission from cells and/or the substrate material is problematic.
- Plowman, T. E., Saavedra, S. S., & Reichert, W. M. (1998). Planar integrated optical methods for examining thin films and their surface adlayers. Biomaterials, 19(4-5), 341-355.More infoPMID: 9677149;Abstract: Thin film integrated optical waveguides (IOWs) have gained acceptance as a method for characterizing ultrathin dielectrical films and adlayers bound to the film surface. Here, we present the expressions that govern IOW methods as well as describe the common experimental configurations used in attenuated total reflection, fluorescence and Raman applications. The applications of these techniques to the study of adsorbed or surface-bound proteins to polymer and glass waveguides are reviewed.
- Edmiston, P. L., & Saavedra, S. S. (1997). Fabrication and Characterization of Uranium Oxide Doped Sol-Gel Planar Waveguides for Attenuated Total Reflectance Spectrometry. Chemistry of Materials, 9(11), 2599-2603.More infoAbstract: Planar integrated optical waveguides doped with fluorescent hexavalent UO22+ ions were fabricated by a sol-gel dip-coating process. Excitation of a mode in these waveguides produced fluorescence emission that decayed in a highly uniform manner as a function of propagation distance. A comparison of methods for determining waveguide attenuation coefficients by digital photography of the mode demonstrated that imaging the UO22+ fluorescence emission is significantly more precise than imaging the Rayleigh and Mie scattered light. The utility of these waveguides for performing attenuated total reflectance measurements on substrate-supported molecular films was also assessed. Both the fluorescence and scattering methods were used to determine the attenuation coefficient of an adsorbed heme protein monolayer. The fluorescence imaging method was clearly superior; its use enables attenuation measurements of very weakly absorbing, waveguide-supported films to be performed with significantly improved precision.
- Edmiston, P. L., Lee, J. E., Cheng, S., & Saavedra, S. S. (1997). Molecular orientation distributions in protein films. 2. Site-directed immobilization of yeast cytochrome c on thiol-capped, self-assembled monolayers. Journal of the American Chemical Society, 119(3), 571-576.More infoAbstract: Molecular orientation in films of yeast cytochrome c immobilized via disulfide bonding between cysteine 102 and the thiol tail groups of self-assembled monolayers (SAMs) coated on planar glass substrates was investigated. The orientation distribution of the heme groups in the protein film was determined using a combination of absorption linear dichroism, measured in a planar integrated optical waveguide-attenuated total reflection geometry, and emission anisotropy, measured in a total internal reflection fluorescence geometry. The mean heme tilt angle and angular distribution about the mean were recovered using a Gaussian model for the orientation tribution. These data are the first orientation distribution measurements reported for a protein film immobilized using a site-directed bonding strategy. The results show that the molecular architecture examined in this study does not produce a highly oriented protein film. A significant fraction of the immobilized cytochrome c is nonspecifically adsorbed to the SAM surface, which produces a relatively broad distribution of heme orientations.
- Mendes, S. B., Lifeng, L. i., Burke, J., & Saavedra, S. S. (1997). Achromatic prism-coupler for planar waveguide. Optics Communications, 136(3-4), 320-326.More infoAbstract: We derive the required optical properties of a prism for coupling light achromatically into a planar waveguide. Efficient coupling over a 110 nm spectral bandwidth was experimentally demonstrated with a prism-coupler designed to match the dispersion of a particular waveguide. The design tools described here should be useful in the implementation of broadband attenuated total reflection spectroscopy in the waveguide regime to probe ultrathin and very weakly absorbing molecular submonolayers.
- Wenzler, L. A., Moyes, G. L., Raikar, G. N., Hansen, R. L., Harris, J. M., Beebe Jr., T. P., Wood, L. L., & Saavedra, S. S. (1997). Measurements of single-molecule bond-rupture forces between self-assembled monolayers of organosilanes with the atomic force microscope. Langmuir, 13(14), 3761-3768.More infoAbstract: Atomic force microscopy (AFM) tips and glass surfaces were modified with various organosilanes to determine magnitude and dispersion information about single-molecule bond-rupture forces. X-ray photoelectron spectroscopy (XPS) and contact-angle measurements were used to study and quantify organosilane adsorption on the glass surface and on SiO2-coated AFM tips. Hydrogen bond interactions between hydroxyl- and thiol-terminated groups on the tip and surface were detected and measured. Differentiation between the functionalities of the acetate- and thioacetate-terminated silanes and their reduced forms produced by on-surface reduction (the alcohol and thiol, respectively) was also accomplished. The experiments demonstrate the complementary information that can be obtained from AFM and XPS and illustrate how they can be used to determine the nature of the surface after an organic transformation has occurred to the functional groups present. They also represent a first step in detecting chemical reactions on a localized scale and in measuring the dispersion in the single-molecule bond-rupture force when it exists.
- Wood, L. L., Cheng, S., Edmiston, P. L., & Saavedra, S. S. (1997). Molecular orientation distributions in protein films. 1. Cytochrome c adsorbed to substrates of variable surface chemistry. Journal of the American Chemical Society, 119(3), 560-570.More infoAbstract: Molecular orientation in hydrated cytochrome c (cyt c) films formed by adsorption to substrates of differing surface chemistry was investigated. The orientation distribution of the heme groups in the protein films was determined using a combination of two techniques: absorption linear dichroism, measured in a planar integrated optical waveguide attenuated total reflection geometry, and emission anisotropy, measured in a total internal reflection fluorescence geometry. The mean heme tilt angle and angular distribution about the mean were recovered using a Gaussian model for the orientation distribution. These data are the first orientation distribution measurements reported for protein film assemblies. The results show that a macroscopically ordered film of adsorbed cyt c molecules is produced when a single, high-affinity type of noncovalent binding occurs between the surface of the protein and the substrate surface. For example, electrostatic adsorption of the positively charged protein to the negatively charged head groups of a Langmuir-Blodgett film of arachidic acid produces a narrow orientation distribution. When multiple, competing adsorptive interactions are operative, which is the case when cyt c adsorbs to a clean glass surface, a relatively disordered film is produced.
- Edmiston, P. L., Lee, J. E., Wood, L. L., & Saavedra, S. S. (1996). Dipole orientation distributions in Langmuir-Blodgett films by planar waveguide linear dichroism and fluorescence anisotropy. Journal of Physical Chemistry, 100(2), 775-784.More infoAbstract: A method to determine the orientation distribution of fluorescent molecules in a thin, substrate-supported film is described. Attenuated total reflection spectrometry on the surface of a planar waveguide is used to measure absorption LD, from which the mean dipole tilt angle in the film is obtained. Steady-state fluorescence anisotropy is measured in a total internal reflection geometry on a film supported on fused silica but prepared under otherwise identical conditions. The angular distribution about the mean can be recovered from the anisotropy measurement by modeling the distribution as a probability density that is specified by two adjustable parameters. The method was tested on Langmuir-Blodgett (LB) films of arachidic acid doped with the fluorescent amphiphiles DiI and BODIPY. In the Dil-doped films, the mean tilt angle was 75° from the surface normal. Assuming a Gaussian distribution, the standard deviation was 12°, indicating a high degree of macroscopic order. In the BODIPY-doped films the distribution was 59 ± 17°, which indicates a less ordered assembly. Larger angular distributions were calculated using a step function model. The results show that dipoles in the headgroup region of arachidic acid LB films are more ordered than dipoles in the alkyl chain region. The method should prove useful in studying relationships between assembly technique, structure, and function in two-dimensional molecular arrays. © 1996 American Chemical Society.
- Lee, J. E., & Saavedra, S. S. (1996). Molecular orientation in heme protein films adsorbed to hydrophilic and hydrophobic glass surfaces. Langmuir, 12(16), 4025-4032.More infoAbstract: Due to the heterogeneous distribution of chemical functionalities present on the surface of most proteins, adsorption to solid materials of differing surface chemistry may produce different bound molecular orientations. Differences in molecular orientation may in turn produce differences in adsorbed biofunction, which has important implications for fabrication of protein-based molecular devices. Aspects of this topic were addressed here by investigating molecular orientation in submonolayer to monolayer thick films of myoglobin (Mb) and cytochrome c (cyt c) adsorbed to hydrophilic and hydrophobic glass substrates. Orientation was determined by measuring the mean tilt angle of the heme moiety in protein films supported on a planar integrated optical waveguide. The results show (i) mean molecular orientation in monolayer films of both Mb and cyt c on both substrates is anisotropic rather than random (ii) molecular orientation in monolayer cyt c films is dependent on the wettability of the substrate and (iii) on both substrates, molecular orientation in submonolayer Mb films is substantially different than that in monolayer films.
- Mendes, S. B., Lifeng, L. i., Burke, J. J., Lee, J. E., Dunphy, D. R., & Saavedra, S. S. (1996). Broad-band attenuated total reflection spectroscopy of a hydrated protein film on a single mode planar waveguide. Langmuir, 12(14), 3374-3376.More infoAbstract: The development of a multichannel spectrometer based on single mode planar integrated optical waveguide and capable of measuring broad-band visible absorbance spectra of a weakly absorbing molecular film at a solid-liquid interface is reported. The system was successfully tested by measuring spectra, over a 100 nm bandwidth, of a hydrated film of cytochrome c adsorbed to a glass waveguide surface. The approach should prove useful in spectral characterization of structure and function in thin film molecular assemblies at condensed phase interfaces.
- Wambolt, C. L., & Saavedra, S. S. (1996). Iodide fluorescence quenching of sol-gel immobilized BSA. Journal of Sol-Gel Science and Technology, 7(1-2), 53-57.More infoAbstract: Several potential applications of sol-gel immobilized proteins may require drying the gel before use. The attendant shrinkage may sterically restrict the penetration of solutes to the entrapped protein. In this study, collisional quenching of intrinsic bovine serum albumin (BSA) fluorescence by dissolved iodide was used to quantitatively compare the accessibility of protein molecules entrapped in hydrated and dried sol-gel monoliths. The results show that iodide penetration to immobilized BSA is sterically restricted by approximately 25% in hydrated gels and 50% in dried gels, relative to the quenching behavior of dissolved BSA. The further decrease upon drying is consistent with the overall degree of monolith shrinkage. © 1996 Kluwer Academic Publishers.
- Phimphivong, S., Kölchens, S., Edmiston, P. L., & Saavedra, S. S. (1995). Time-resolved, total internal reflection fluorescence microscopy of cultured cells using a Tb chelate label. Analytica Chimica Acta, 307(2-3), 403-417.More infoAbstract: A total internal reflection fluorescence microscope with time-resolved spectroscopy and CCD imaging capabilities is described. The capability of the microscope to selectively detect, via temporal resolution, the presence of a long-lived luminescence label in biological cells in vitro was evaluated. Tb chelates having radiative lifetimes of ca. 1.5 ms were employed as long-lived, extrinsic labels. Microspectroscopy and imaging were performed on mouse 3T3 cells stained with Tb chelates, stained with DiI (an extrinsic membrane label of ns lifetime), and in the absence of an extrinsic label. The results demonstrate the efficiency of using time-resolved detection to discriminate against intrinsic and extrinsic sources of short-lived emission in cultured cells, which enables long-lived, extrinsic luminescence to be selectively detected. Potential applications of this technology are discussed. © 1995 Elsevier Science B.V. All rights reserved.
- Yang, L., & Saavedra, S. S. (1995). Chemical Sensing Using Sol-Gel Derived Planar Waveguides and Indicator Phases. Analytical Chemistry, 67(8), 1307-1314.More infoAbstract: A new optical sensing platform based on a combination of planar waveguiding and sol-gel processing technologies is described. The sensing element consists of two, submicrometer thick glass layers supported on an optically thick glass substrate; both layers were fabricated using a sol-gel coating method. The lower layer is a densified glass that functions as a planar integrated optical waveguide (IOW). The upper layer is an undensified glass of lower index doped with an optical indicator that is immobilized, yet remains sterically accessible to analytes that diffuse into the pore network. Formation of a complex between the analyte and indicator is detected via attenuated total reflection (ATR) of light guided in the IOW. Feasibility was evaluated by constructing IOW-ATR sensors for Pb2+ and pH, based on immobilized xylenol orange and bromocresol purple, respectively. The response of both sensors was sensitive and rapid, features that are difficult to achieve simultaneously in monolithic sol-gel glass sensors. In the IOW-ATR geometry, these features are realized simultaneously because the primary axes of light propagation and analyte diffusion are orthogonal. The overall approach is technically simple, inexpensive, and applicable to a wide variety of indicator chemistries.
- Banovac, F., Saavedra, S. S., & Truskey, G. A. (1994). Local conformational changes of vitronectin upon adsorption on glass and silane surfaces. Journal of Colloid and Interface Science, 165(1), 31-40.More infoAbstract: The conformation of vitronectin (Vn) in solution and adsorbed to hydrophilic and hydrophobic glass was studied using the polarity-sensitive fluor acrylodan. Free sulfhydryl groups of vitronectin were labeled with acrylodan under nondenaturing conditions. The number and location of the free sulfhydryl groups were determined by cleavage with either formic acid or cyanogen bromide followed by polyacrylamide gel electrophoresis. Based upon published amino acid sequences of Vn, the fluorescence of acrylodan-labeled Vn (Ac-Vn) fragments is consistent with free sulfhydryls being located at cysteine residues 196 and 274. Conformational changes upon adsorption on glass and silanized glass surfaces placed the free sulfhydryl residues in a more hydrophobic environment than was observed when Vn was in PBS solution. Small but statistically significant quenching of Ac-Vn fluorescence with iodide could be explained in terms of a two-site model. Results of this study suggest that the domains surrounding the free sulfhydryl groups of vitronectin experience different environments in solution and adsorbed to surfaces.
- Edmiston, P. L., Wambolt, C. L., Smith, M. K., & Saavedra, S. (1994). Spectroscopic characterization of albumin and myoglobin entrapped in bulk sol-gel glasses. Journal of Colloid and Interface Science, 163(2), 395-406.More infoAbstract: The immobilization of proteins by entrapment in optically clear, porous glasses prepared by sol-gel techniques appears to be a promising approach to optical biosensor development. However, little is known about the physical environment of the immobilized protein or the mechanism(s) of entrapment. In this study, absorbance and fluorescence spectroscopies have been used to characterize the properties of two model proteins, bovine serum albumin (BSA) and horse heart myoglobin (Mb), entrapped in wet sol-gel glass bulks. The fluorescence behavior of dissolved and entrapped BSA in the presence of acid, a chemical denaturant, and a collisional quencher was examined. The results show that a large fraction of the BSA added to the sol is entrapped within the gelled glass in a native conformation. However, the reversible conformational transitions that BSA undergoes in solution are sterically restricted in the gel. In contrast, the native properties of Mb are largely lost upon entrapment, as judged by the changes in the visible absorbance spectra of dissolved and entrapped Mb in acidic solutions. Fluorescence studies of dissolved and entrapped apomyoglobin support this conclusion.
- Lee, J. E., & Saavedra, S. (1994). Evanescent sensing in doped sol-gel glass films. Analytica Chimica Acta, 285(3), 265-269.More infoAbstract: Bromophenol blue, a pH indicator, was entrapped in porous glass films prepared by the sol-gel method. The colorimetric response of the films to pH was assessed visibly and by attenuated total reflectance spectrometry. The entrapped indicator was nonleachable and responded to solution pH changes in a reversible manner. The immobilization of indicator molecules in porous sol-gel films appears to be a particulary promising approach to optical waveguide sensor development. © 1994.
- Edmiston, P. L., Kölchens, S., & Saavedra, S. S. (1993). Temporally gating a slow-scan CCD with a liquid crystal shutter. Applied Spectroscopy, 47(2), 250-253.
- Iuliano, D. J., Saavedra, S. S., & Truskey, G. A. (1993). Effect of the conformation and orientation of adsorbed fibronectin on endothelial cell spreading and the strength of adhesion. Journal of Biomedical Materials Research, 27(8), 1103-1113.More infoPMID: 8408123;Abstract: The effect of surface hydrophobicity upon the conformation of the cell binding domain of fibronectin (Fn) and the influence of Fn conformation on bovine aortic endothelial cell (BAEC) adhesion were examined. The free sulfhydryl group of Fn located near the cell binding domain was selectively labeled with acrylodan, a polarity sensitive fluor. Fluorescence emission was monitored in solution and upon adsorption to hydrophilic glass and hydrophobic silanized glass. The acrylodan-labeled Fn emission maximum shifted to longer wavelengths upon adsorption and the shift was greater for acrylodan-labeled Fn adsorbed to hydrophilic glass than hydrophobic silane, suggesting that the acrylodan was in a more solvent accessible environment on glass than silane. BAEC, suspended in serum-free medium, attached for 15 or 120 min onto glass or silane surface containing pre-adsorbed Fn, after which cell spreading and the strength of adhesion in a parallel plate flow chamber were measured. Cell spreading was similar on both surfaces after 15 min attachment, but BAECs were more spread on glass than silane after 120 min. At low surface concentrations of Fn, BAECs were more adherent on glass than silane. At higher surfaces concentrations, adhesion was similar. After a 2-h incubation in serum-free medium, cells on glass showed more extensive development of focal contacts as determined by immunofluorescent staining for vinculin. Cell adhesion under flow was reduced on silane by inhibition of protein synthesis with cycloheximide, suggesting that cell attachment to silane was promoted by cellular synthesis of Fn. The results indicate that changes in the conformation of the Fn cell binding domain affect Fn affinity for its cell surface receptor.
- Walker, D. S., Hellinga, H. W., Saavedra, S. S., & Reichert, W. M. (1993). Integrated optical waveguide attenuated total reflection spectrometry and resonance Raman spectroscopy of adsorbed cytochrome c. Journal of Physical Chemistry, 97(39), 10217-10222.More infoAbstract: The heme group of cytochrome c (Cyt C) has resonance absorptions at 520 and 550 nm that arise from x-y degenerate in-plane electronic transitions of the heme moiety. In the present paper, horse heart Cyt C was adsorbed to the surface of a micron-thick silicon oxynitride integrated optical waveguide configurated into a liquid flow cell and prism-coupled with 514.5-nm laser light that was polarized horizontal (TE) and nearly vertical (TM) with respect to the waveguide surface. The adsorbed protein film absorbed light from the evanescent field at the waveguide surface, resulting in two measurable quantities: (1) an increased attenuation of the guided mode intensity in the waveguide and (2) excitation of Cyt C resonance Raman emission. Propagation losses owing to the adsorbed film revealed a Cyt C surface density indicating submonolayer coverage (50-75 ng/cm2). The dichroic ratio measured by guided mode attenuation was 1.23 ± 0.37, indicating the heme plane of Cyt C had an ensemble-averaged orientation angle of 48° with respect to the surface normal. The TE and TM polarized resonance Raman bands at 1578 cm-1 of the adsorbed Cty C had an intensity ratio of 1.49 that is statistically indistinguishable from the dichroic ratio measured by attenuated total reflection spectrometry. The integrated optical waveguide resonance Raman results are believed to be the first reported Raman spectra of a protein film bound to a dielectric substrate. © 1993 American Chemical Society.
- Garrison, M., Iuliano, D., Saavedra, S., Truskey, G., & Reichert, W. (1992). Postadsorption changes in the emission maximum of acrylodan-labeled bovine serum albumin using total internal reflection fluorescence. Journal of Colloid And Interface Science, 148(2), 415-424.More infoAbstract: Acrylodan (Ac), the thiol reactive analog of the polarity-sensitive fluorescent dye Prodan, was investigated as a site-specific probe for postadsorption conformational changes of bovine serum albumin (BSA) adsorbed to bare and alkyl silane-treated glass coverslips. A commercial fluorometer, adapted with a custom-built total internal reflection fluorescence (TIRF) cell, was utilized to examine the adsorbed Ac-BSA fluorescence as a function of substrate surface wettability as measured by the Wilhelmy plate balance technique. Fluorescence emission of adsorbed Ac-BSA displayed a blue shift and edge excited red shift upon adsorption to all substrates that increased with increasing substrate hydrophobicity. Additionally, center excited emission spectra from unconjugated Ac adsorbed to bare and hydrophobic glass were similar to those for the adsorbed Ac-BSA. These results are consistent with the hypothesis of postadsorption conformational changes, but also suggest a direct probe-surface contact for adsorbed Ac-BSA. © 1992.
- Iuliano, D. J., Proulx, T. L., Saavedra, S. S., & Truskey, G. A. (1991). Role of adhesion proteins and cytoskeleton in the short-term response of fibroblasts and endothelial cells to steady laminar flow. American Society of Mechanical Engineers, Bioengineering Division (Publication) BED, 21, 23-25.More infoAbstract: Forces exerted on adherent calls by fluid shear stresses can cause cell detachment. In this study, we examined the effect of cell spreading, actin fiber density, and the conformation of adsorbed adhesion proteins in promoting cell adhesion after a brief exposure to shear stresses between 5-80 dyne/cm2 in a parallel plate flow channel. Cell spreading led to increased adhesion. Cell spreading and adhesion were greater when the surface contained preadsorbed fibronectin. Spreading was accompanied by formation of cytoskeleton which strengthened the adhesive contact between the cell and surface. Similar numbers of cells were detached from hydrophobic and hydrophilic glass containing preadsorbed in even though the cell binding domain was in different conformations.
- Potter, B. L., Walker, D. S., Greer, L., Saavedra, S. S., & Reichert, W. M. (1991). Multiple mode and multiple source coupling into polymer thin film waveguides. Proceedings of SPIE - The International Society for Optical Engineering, 1368, 251-257.More infoAbstract: A broadly focused laser beam was used to investigate simultaneous excitation of multiple modes in prism and grating coupled polymer thin film optical waveguides. Concurrent coupling of two lines from different lasers through a fiber bundle was also examined. Results indicate that simultaneous multiple pathlength and multiple source sensing at the surface of thin film waveguides is feasible.
- Saavedra, S. S., & Reichert, W. M. (1991). In situ quantitation of protein adsorption density by integrated optical waveguide attenuated total reflection spectrometry. Langmuir, 7(5), 995-999.More infoAbstract: The adsorption of hemoglobin to a polystyrene (PS) thin film has been examined by integrated optical waveguide attenuated total reflection (IOW-ATR) spectrometry. Protein adsorption densities were determined by measuring the evanescent attenuation of propagating modes that were prism coupled into and out of the PS film. Knowledge of the electric field intensity distribution of the waveguide structure permitted adsorbed protein to be quantitated in the presence of bulk dissolved protein. The Langmuir adsorption model was used to fit the isotherm data and indicates that Hb binds with high affinity (Ka > 106 M-1) to PS and forms a complete monolayer at bulk concentrations greater than 3 μM. The results demonstrate that the IOW-ATR technique is particularly well suited to in situ measurements of protein adsorption density at the polymer/solution interface. © 1991 American Chemical Society.
- Saavedra, S. S., & Reichert, W. M. (1990). Flow cell for mode-specific, integrated optical waveguide spectroscopy in aqueous superstrates. Applied Spectroscopy, 44(8), 1420-1423.More infoAbstract: In this communication, we describe the design, construction, and operation of a flow cell that allows for proper waveguide coupling in liquid media. Preliminary results showing mode-dependent attenuation of evanescent energy by surface-bound molecules are presented which demonstrate the feasibility of mode-specific IOW spectroscopy at the solid/liquid interface.
- Saavedra, S. S., & Reichert, W. M. (1990). Integrated optical attenuated total reflection spectrometry of aqueous superstrates using prism-coupled polymer waveguides. Analytical Chemistry, 62(20), 2251-2256.More infoPMID: 2268054;Abstract: Attenuated total reflection (ATR) spectrometry of aqueous solutions in contact with polystyrene integrated optical waveguides has been Investigated. The mode-dependent absorption of evanescent energy by fluorescein solutions adjacent to the waveguide surface was measured and compared to theoretical predictions based on a ray optics approach. Although enhanced sensitivity was observed with increasing mode number, the sensitivity for the highest order mode was less than that predicted by theory.
- Saavedra, S. S., & Reichert, W. M. (1990). Prism coupling into polymer integrated optical waveguides with liquid superstrates. Applied Spectroscopy, 44(7), 1210-1217.More infoAbstract: We have investigated the light coupling behavior of prism-coupled waveguides adjacent to liquid superstrates. A flow cell was used for delivering the liquid superstrates to the waveguide surface. The incoupled angles and outcoupled mode patterns were collected from polystyrene waveguides subjected to superstrates of air, water, and dimethylsulfoxide. Results showed that poor coupling and significant mode scrambling were observed when liquids were infused into the flow cell volume. The poor waveguiding characteristics were found to be caused by the presence of air in the prism coupling gaps. Poor coupling was eliminated for a water superstrate by ensuring that water also occupied the prism coupling gaps. The mode coupled into the water-coupled waveguide with a water superstrate was always the most intense mode in the outcoupled pattern.
- Walker, D. S., Putegnat, S., Saavedra, S. S., & Reichert, W. M. (1990). Apparent inequivalence between the in- and outcoupling angles of prism coupled IO waveguides. Optics Communications, 78(2), 128-132.More infoAbstract: The incoupling and outcoupling angles of a thin film waveguide were determined using the conventional prism coupling configuration. The consistent intermodal spacing observed between incoupling and outcoupling angles indicated that these angles contained equivalent information on the waveguide structure. However, a geometry mismatch between the rotation goniometer and the triangular coupling prisms produced a systematic error in the determination of the outcoupling angles that increased with decreasing mode number. © 1990.
- Saavedra, S. S., & Picozza, E. G. (1989). Time-resolved fluorimetric detection of terbium-labelled deoxyribonucleic acid separated by gel electrophoresis. The Analyst, 114, 835-838.More infoPMID: 2802165;Abstract: Deoxyribonucleic acid (DNA) was reacted with a strong chelating agent and labelled with terbium, yielding a highly fluorescent conjugate with a lifetime of 1.5 ms. When a pulsed source and gated detection electronics were employed, the long-lived decay allowed effective discrimination against background fluorescence and scattered excitation. Detection limits should therefore be signifcantly improved in comparison with covalent labels with fluorescence lifetimes in the nanosecond regime or stains such as ethidium bromide. The conjugate is very stable, remaining fluorescent on dilution and in an electric field at elevated temperatures (60 °C), conditions typically encountered during polyacrylamide gel electrophoresis. As an enhancement solution is not required to develop the fluorescence, this system could be utilised in situations where on-line detection is desirable. Although only DNA was labelled, the method is equally applicable to ribonucleic acid.
- Saavedra, S. S., Grobin, A. W., & Lochmüller, C. (1988). Fluorescence of chemically modified papain adsorbed to silica gel. Analytical Chemistry, 60(19), 2156-2158.
- Lochmüller, C., & Saavedra, S. S. (1987). Interconversion of conformation of apomyoglobin adsorbed on hydrophobic silica gel. Journal of the American Chemical Society, 109(4), 1244-1245.
- Lochmüller, C., & Saavedra, S. S. (1987). Intrinsic fluorescence characteristics of apomyoglobin adsorbed to microparticulate silica. Langmuir, 3(3), 433-438.More infoAbstract: The intrinsic fluorescence of sperm whale apomyoglobin was utilized as the probe in a study of protein adsorption, desorption, and conformational behavior on a series of microparticulate silica gels. The results indicate that adsorption is rapid and largely irreversible. The degree of protein unfolding that takes place upon adsorption is dependent on the pH of the contact buffer. The tryptophans in the pH 4.0 surface conformer are more exposed and interact to a greater extent with the interfacial environment than in the pH 7.5 surface conformer. Partial, reversible refolding of the protein in the sorbed state is observed with a change in the pH of the contact buffer. © 1987 American Chemical Society.
- Lochmuller, C. H., & Saavedra, S. S. (1986). CONFORMATIONAL CHANGES IN A SOIL FULVIC ACID MEASURED BY TIME-DEPENDENT FLUORESCENCE DEPOLARIZATION.. Analytical Chemistry, 58(9), 1978-1981.More infoAbstract: The dependence of the aqueous conformation of a soil-derived fulvic acid on the independent parameters specifying the solution composition has been studied by time-dependent fluorescence depolarization spectroscopy. The molecular size and shape of the fluorescent components in fulvic acid were found to vary with solution pH, ionic strength, and sample concentration. These results indicate that this material behaves like a flexible polyelectrolyte in solutions approximating naturally occurring concentrations.
- Cai, M., Zhang, H., Dedek, M., Saavedra, S., & Hruby, V. (2012, Fall). Selective cell signaling study of GPCR via PWR spectroscopy. In J. Peptide Science, 18.
- Ehamparam, R., Pavlopoulos, N., Liao, M. W., Hill, L. J., Armstrong, N. R., Pyun, J., & Saavedra, S. S. (2014, AUG 10). Waveguide spectroelectrochemical and photoemission spectroscopic characterization of frontier orbital energies and charge injection processes in Au-CdSe hybrid nanostructures. 248th ACS National Meeting.
- Saavedra, S. (2014, January). Mass spectrometry on planar supported lipid bilayers. Society of Western Analytical Professors Annual Meeting. Tempe, AZ.
- Zheng, Y., Giordano, A. J., Cowan, S. R., Fleming, S. R., Shallcross, R. C., Gliboff, M., Huang, Y., Ginger, D. S., McGrath, D. V., Armstrong, N. R., Olson, D. C., Marder, S. R., & Saavedra, S. S. (2014, AUG 10). Surface modification of transparent conducting oxides with functionalized perylene diimide dyes: Characterization of orientation, charge-transfer kinetics, and related OPV performance. 248th ACS National Meeting.
- Ehamparam, R., Hill, L., Richey, N., Pavlopoulos, N., Pyun, J., Saavedra, S., & Armstrong, N. (2014, April). "Electronically Wired” Semiconductor Nanoparticles: Toward Vectoral Electron Transport in Hybrid Materials. Arizona Student Energy Conference. Tucson, AZ.
- Mendoza, M., Agasid, M., Bright, K., Saavedra, S., & Aspinwall, C. (2013, June). Evaluation of Membrane Composition for Fabrication of Ion Channel-Functionalized Sensors. 87th ACS Colloids and Surface Science Symposium. Riverside, CA.
- Saavedra, S. (2013, January). Correlating structure and charge transfer kinetics in molecular films using waveguide spectroelectrochemistry in the frequency domain. Society of Western Analytical Professors Annual Meeting. Fort Collins, CO.
- Saavedra, S. (2013, September). Sensors Based on Poly(lipid) Membranes and Ion Channel Receptors. JSAP-MRS Joint Symposia. Kyoto, Japan.
- Saavedra, S. S. (2013, September). Waveguide spectroelectrochemistry in the frequency and time-domains: Development and application to photochemical and electrochemical reactions in molecular films. Nagasaki University. Japan.
- Armstrong, N., Pyun, J., & Saavedra, S. (2012, June). Formation and Characterization of Semiconductor Nanorod/Oxide Nanoparticle Hybrid Materials: Toward Vectoral Electron Transport in Hybrid Materials. 34th Solar Photochemistry Program Research Meeting. Annapolis, MD.
- Liang, B., Joubert JR, ., Ju, Y., Kaleta, E., Jones, I., Wysocki, V., & Saavedra, S. (2012, March). MALDI-MS detection and identification of bacterial toxins captured by cell-surface receptors on a stable yet fluid lipid bilayer. 243nd ACS National Meeting. San Diego, CA.
- Lin, H., Polaske, N., Shi, Y., Oquendo, L., Gliboff, M., Knesting, K., Nordlund, D., Ginger, D., Ratcliff, E., Beam, B., Armstrong, N., Marder SR, ., McGrath, D., & Saavedra, S. (2012, July). Electron transfer processes in monolayer-tethered phosphonic acid functionalized zinc phthalocyanines on indium tin oxide. ICSM 2012. Atlanta, GA.
- Saavedra, S. (2012, September). Poly(Lipid) Supramolecular Assemblies: Structure, Receptor Functionalization, and Bioanalytical Applications. International Conference on Electronic Materials. Yokohama, Japan.
- Saavedra, S. S. (2012, April). Poly(Lipid) Supramolecular Assemblies: Structure, Receptor Functionalization, and Bioanalytical Applications. Colorado State University.
- Saavedra, S. S. (2012, October). Poly(Lipid) Supramolecular Assemblies: Structure, Receptor Functionalization, and Bioanalytical Applications. Kobe University. Japan.
- Saavedra, S. S. (2012, September). Poly(Lipid) Supramolecular Assemblies: Structure, Receptor Functionalization, and Bioanalytical Applications. Tokyo University of Science. Japan.
- Saavedra, S. S. (2012, September). Waveguide spectroelectrochemistry in the frequency and time-domains: Development and application to photochemical and electrochemical reactions in molecular films. Gunma University. Japan.
- Leight, K. R., Lin, H., Oquendo, L. E., Saavedra, S. S., & McGrath, D. V. (2014, AUG 10). Investigation of distance and orientation dependence on charge transfer kinetics at indium tin oxide/zinc phthalocyanine interface using waveguide spectroelectrochemistry. 248th ACS National Meeting.
- Jenkins, J., Malfavon, M., Hernandez, E., Armstrong, N., McGrath, D., & Saavedra, S. (2013, September). Photochemistry of chromophore/transparent conductive oxide interfaces measured using transient absorbance spectroscopy in attenuated total reflectance geometry. 246th ACS National Meeting. Indianapolis, IN.
- Liang, B., Ju, Y., Orosx, K., Wysocki, V., & Saavedra, S. (2013, March). Utilizing Polymerizable Lipid Membranes in Characterization of Ligand-Receptor Interactions by MALDI-MS, AFM, and TIRF-M. Pittsburgh Conference. Philadelphia, PA.
- Huang, Y., Lu, C., Zheng, Y., Jenkins, J. L., Oquendo, L. E., Leight, K. R., Saavedra, S. S., & McGrath, D. V. (2014, MAR 16). Synthesis of Pc-PDI dyads as a molecular heterojunction for studies of electron injection at organic-oxide interfaces. ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY.
- Oquendo, L. E., Lin, H., Ehamparam, R., Polaske, N. W., Leight, K. R., Gliboff, M., Knesting, K. M., Nordlund, D., Ginger, D. S., Ratcliff, E. L., Beam, B. M., Lee, P., Armstrong, N. R., Saavedra, S. S., & McGrath, D. V. (2013, APR 7). Modification of indium tin oxide surface with phosphonic acids functionalized asymmetric zinc phthalocyanines. ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY.