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1.  Molecular Self-Assembly into One-Dimensional Nanostructures 
Accounts of chemical research  2008;41(12):1674-1684.
CONSPECTUS
Self-assembly of small molecules into one-dimensional nanostructures offers many potential applications in electronically and biologically active materials. The recent advances discussed in this Account demonstrate how researchers can use the fundamental principles of supramolecular chemistry to craft the size, shape, and internal structure of nanoscale objects. In each system described here, we used atomic force microscopy (AFM) and transmission electron microscopy (TEM) to study the assembly morphology. Circular dichroism, nuclear magnetic resonance, infrared, and optical spectroscopy provided additional information about the self-assembly behavior in solution at the molecular level.
Dendron rod–coil molecules self-assemble into flat or helical ribbons. They can incorporate electronically conductive groups and can be mineralized with inorganic semiconductors. To understand the relative importance of each segment in forming the supramolecular structure, we synthetically modified the dendron, rod, and coil portions. The self-assembly depended on the generation number of the dendron, the number of hydrogen-bonding functions, and the length of the rod and coil segments. We formed chiral helices using a dendron–rod–coil molecule prepared from an enantiomerically enriched coil.
Because helical nanostructures are important targets for use in biomaterials, nonlinear optics, and stereoselective catalysis, researchers would like to precisely control their shape and size. Tripeptide-containing peptide lipid molecules assemble into straight or twisted nanofibers in organic solvents. As seen by AFM, the sterics of bulky end groups can tune the helical pitch of these peptide lipid nanofibers in organic solvents. Furthermore, we demonstrated the potential for pitch control using trans-to-cis photoisomerization of a terminal azobenzene group. Other molecules called peptide amphiphiles (PAs) are known to assemble in water into cylindrical nanostructures that appear as nanofiber bundles. Surprisingly, TEM of a PA substituted by a nitrobenzyl group revealed assembly into quadruple helical fibers with a braided morphology. Upon photocleavage of this the nitrobenzyl group, the helices transform into single cylindrical nanofibers.
Finally, inspired by the tobacco mosaic virus, we used a dumbbell-shaped, oligo(phenylene ethynylene) template to control the length of a PA nanofiber self-assembly (<10 nm). AFM showed complete disappearance of long nanofibers in the presence of this rigid-rod template. Results from quick-freeze/deep-etch TEM and dynamic light scattering demonstrated the templating behavior in aqueous solution. This strategy could provide a general method to control size the length of non-spherical supramolecular nanostructures.
doi:10.1021/ar8000926
PMCID: PMC2645948  PMID: 18754628
2.  Organic Nanofibers Embedding Stimuli-Responsive Threaded Molecular Components 
Journal of the American Chemical Society  2014;136(40):14245-14254.
While most of the studies on molecular machines have been performed in solution, interfacing these supramolecular systems with solid-state nanostructures and materials is very important in view of their utilization in sensing components working by chemical and photonic actuation. Host polymeric materials, and particularly polymer nanofibers, enable the manipulation of the functional molecules constituting molecular machines and provide a way to induce and control the supramolecular organization. Here, we present electrospun nanocomposites embedding a self-assembling rotaxane-type system that is responsive to both optical (UV–vis light) and chemical (acid/base) stimuli. The system includes a molecular axle comprised of a dibenzylammonium recognition site and two azobenzene end groups and a dibenzo[24]crown-8 molecular ring. The dethreading and rethreading of the molecular components in nanofibers induced by exposure to base and acid vapors, as well as the photoisomerization of the azobenzene end groups, occur in a similar manner to what observed in solution. Importantly, however, the nanoscale mechanical function following external chemical stimuli induces a measurable variation of the macroscopic mechanical properties of nanofibers aligned in arrays, whose Young’s modulus is significantly enhanced upon dethreading of the axles from the rings. These composite nanosystems show therefore great potential for application in chemical sensors, photonic actuators, and environmentally responsive materials.
doi:10.1021/ja5080322
PMCID: PMC4195382  PMID: 25264943
3.  The interfacial structure and Young's modulus of peptide films having switchable mechanical properties 
We report the structure and Young's modulus of switchable films formed by peptide self-assembly at the air–water interface. Peptide surfactant AM1 forms an interfacial film that can be switched, reversibly, from a high- to low-elasticity state, with rapid loss of emulsion and foam stability. Using neutron reflectometry, we find that the AM1 film comprises a thin (approx. 15 Å) layer of ordered peptide in both states, confirming that it is possible to drastically alter the mechanical properties of an interfacial ensemble without significantly altering its concentration or macromolecular organization. We also report the first experimentally determined Young's modulus of a peptide film self-assembled at the air–water interface (E=80 MPa for AM1, switching to E<20 MPa). These findings suggest a fundamental link between E and the macroscopic stability of peptide-containing foam. Finally, we report studies of a designed peptide surfactant, Lac21E, which we find forms a stronger switchable film than AM1 (E=335 MPa switching to E<4 MPa). In contrast to AM1, Lac21E switching is caused by peptide dissociation from the interface (i.e. by self-disassembly). This research confirms that small changes in molecular design can lead to similar macroscopic behaviour via surprisingly different mechanisms.
doi:10.1098/rsif.2007.1063
PMCID: PMC2605502  PMID: 17550885
peptide; Young's modulus; interface; film; neutron reflectometry
4.  Fast photodynamics of azobenzene probed by scanning excited-state potential energy surfaces using slow spectroscopy 
Nature Communications  2015;6:5860.
Azobenzene, a versatile and polymorphic molecule, has been extensively and successfully used for photoswitching applications. The debate over its photoisomerization mechanism leveraged on the computational scrutiny with ever-increasing levels of theory. However, the most resolved absorption spectrum for the transition to S1(nπ*) has not followed the computational advances and is more than half a century old. Here, using jet-cooled molecular beam and multiphoton ionization techniques we report the first high-resolution spectra of S1(nπ*) and S2(ππ*). The photophysical characterization reveals directly the structural changes upon excitation and the timescales of dynamical processes. For S1(nπ*), we find that changes in the hybridization of the nitrogen atoms are the driving force that triggers isomerization. In combination with quantum chemical calculations we conclude that photoisomerization occurs along an inversion-assisted torsional pathway with a barrier of ~2 kcal mol−1. This methodology can be extended to photoresponsive molecular systems so far deemed non-accessible to high-resolution spectroscopy.
Azobenzene is perhaps the archetypal light-activated molecule, widely used for photoswitching applications, but the mechanism of isomerisation remains in doubt. Here, the authors provide high-resolution excitation spectra of trans-azobenzene, identifying the structural changes accompanying photoisomerisation.
doi:10.1038/ncomms6860
PMCID: PMC4308720  PMID: 25562840
5.  Photon-Regulated DNA-Enzymatic Nanostructures by Molecular Assembly 
ACS nano  2011;5(12):10090-10095.
Future smart nanostructures will have to rely on molecular assembly for unique or advanced desired functions. For example, the evolved ribosome in nature is one example of functional self-assembly of nucleic acids and proteins employed in nature to perform specific tasks. Artificial self-assembled nanodevices have also been developed to mimic key biofunctions, and various nucleic acid- and protein-based functional nanoassemblies have been reported. However, functionally regulating these nanostructures is still a major challenge. Here we report a general approach to fine-tune the catalytic function of DNA-enzymatic nanosized assemblies by taking advantage of the trans-cis isomerization of azobenzene molecules. To the best of our knowledge, this is the first study to precisely modulate the structures and functions of an enzymatic assembly based on light-induced DNA scaffold switching. Via photocontrolled DNA conformational switching, the proximity of multiple enzyme catalytic centers can be adjusted, as well as the catalytic efficiency of cofactor-mediated DNAzymes. We expect that this approach will lead to the advancement of DNA-enzymatic functional nanostructures in future biomedical and analytical applications.
doi:10.1021/nn204007y
PMCID: PMC3246559  PMID: 22098552
azobenzene; DNA-enzyme conjugation; enzyme cascade; photo controllable
6.  Surface Chemical and Mechanical Properties of Plasma Polymerized N-isopropylacrylamide 
Surface immobilized poly(N-isopropyl acrylamide) (pNIPAM) is currently used for a wide variety of biosensor and biomaterial applications. A thorough characterization of the surface properties of pNIPAM thin films will benefit those applications. In this work, we present analysis of a plasma polymerized NIPAM (ppNIPAM) coating by multiple surface analytical techniques, including time-of-flight secondary ion mass spectrometry (ToF-SIMS), contact angle measurement, atomic force microscopy (AFM) and sum frequency generation (SFG) vibrational spectroscopy. ToF-SIMS data show that the plasma-deposited NIPAM polymer on the substrate is crosslinked with a good retention of the monomer integrity. Contact angle results confirm the thermoresponse of the film as observed by a change of surface wettability as a function of temperature. Topographic and force distance curve measurements by AFM further demonstrate that the grafted film shrinks or swells depending on the temperature of the aqueous environment. A clear transition of the elastic modulus is observed at 31-32°C. The change of the surface wettability and mechanical properties vs. temperature are attributed to different conformations taken by the polymer, which is reflected on the outmost surface as distinct side chain groups orienting outwards at different temperatures as measured by SFG. The results suggest that a ppNIPAM thin film on a substrate experiences similar mechanical and chemical changes to pNIPAM bulk polymers in solution. The SFG result provides evidence supporting the current theory of the lower critical solution temperature (LCST) behavior of pNIPAM.
doi:10.1021/la050417o
PMCID: PMC2527476  PMID: 16089389
N-isopropylacrylamide; plasma polymerized NIPAM (ppNIPAM); thermoresponsive polymer; surface analysis; elastic modulus; ToF-SIMS; AFM; SFG
7.  Photoswitchable gel assembly based on molecular recognition 
Nature Communications  2012;3:603-.
The formation of effective and precise linkages in bottom-up or top-down processes is important for the development of self-assembled materials. Self-assembly through molecular recognition events is a powerful tool for producing functionalized materials. Photoresponsive molecular recognition systems can permit the creation of photoregulated self-assembled macroscopic objects. Here we demonstrate that macroscopic gel assembly can be highly regulated through photoisomerization of an azobenzene moiety that interacts differently with two host molecules. A photoregulated gel assembly system is developed using polyacrylamide-based hydrogels functionalized with azobenzene (guest) or cyclodextrin (host) moieties. Reversible adhesion and dissociation of the host gel from the guest gel may be controlled by photoirradiation. The differential affinities of α-cyclodextrin or β-cyclodextrin for the trans-azobenzene and cis-azobenzene are employed in the construction of a photoswitchable gel assembly system.
Self-assembly through molecular recognition events is used in the production of functionalized materials. This study shows that macroscopic gel assembly can be regulated through photoisomerization of an azobenzene moiety that interacts differently with two host molecules.
doi:10.1038/ncomms1617
PMCID: PMC3272571  PMID: 22215078
8.  Reactive Polymer Multilayers Fabricated by Covalent Layer-by-Layer Assembly: 1,4-Conjugate Addition-Based Approaches to the Design of Functional Biointerfaces 
Biomacromolecules  2012;13(5):1523-1532.
We report on conjugate addition-based approaches to the covalent layer-by-layer assembly of thin films and the post-fabrication functionalization of biointerfaces. Our approach is based on a recently reported approach to the ‘reactive’ assembly of covalently-crosslinked polymer multilayers driven by the 1,4-conjugate addition of amine functionality in poly(ethyleneimine) (PEI) to the acrylate groups in a small-molecule pentacrylate species (5-Ac). This process results in films containing degradable β-amino ester crosslinks and residual acrylate and amine functionality that can be used as reactive handles for the subsequent immobilization of new functionality. Layer-by-layer growth of films fabricated on silicon substrates occurred in a supra-linear manner to yield films ~750 nm thick after the deposition of 80 PEI/5-Ac layers. Characterization by AFM suggested a mechanism of growth that involves the reactive deposition of nanometer-scale aggregates of PEI and 5-Ac during assembly. IR spectroscopy studies revealed covalent assembly to occur by 1,4-conjugate addition without formation of amide functionality. Additional experiments demonstrated that acrylate-containing films could be post-functionalized via conjugate addition reactions with small-molecule amines that influence important bio-interfacial properties, including water contact angles and the ability of film-coated surfaces to prevent or promote the attachment of cells in vitro. For example, whereas conjugation of the hydrophobic molecule decylamine resulted in films that supported cell adhesion and growth, films treated with the carbohydrate-based motif D-glucamine resisted cell attachment and growth almost completely for up to 7 days in serum-containing media. We demonstrate that this conjugate addition-based approach also provides a means of immobilizing functionality through labile ester linkages that can be used to promote the long-term, surface-mediated release of conjugated species and promote gradual changes in interfacial properties upon incubation in physiological media (e.g., over a period of at least one month). These covalently-crosslinked films are relatively stable in biological media for prolonged periods, but they begin to physically disintegrate after ~30 days, suggesting opportunities to use this covalent layer-by-layer approach to design functional biointerfaces that ultimately erode or degrade to facilitate elimination.
doi:10.1021/bm300234q
PMCID: PMC3351563  PMID: 22468967
9.  Manipulation and assembly of small objects in liquid crystals by dynamical disorganizing effect of push-pull-azobenzene-dye 
Scientific Reports  2013;3:2167.
The phase transition of a nematic liquid crystal containing a push-pull azobenzene dye could be induced efficiently during irradiation with visible light. The dynamical disorganizing effect of the push-pull azobenzene dye on the liquid crystalline order through its trans-cis-trans photoisomerizaion cycle under visible light was contributed to the efficient phase transition. Then, the effects of light irradiation on the motion of small objects dispersed in the liquid crystals containing the push-pull azobenzene were explored, and the manipulation and assembly of those objects were successfully achieved in the nematic phase but also in the smectic phase. The combination of the photo-controlled dynamical change in the liquid crystalline order and the intrinsic self-assembly property of a liquid crystal is promising for use in technologies that require not only the organization of small objects but also the photo-driving of nano- and micro-sized mechanical materials.
doi:10.1038/srep02167
PMCID: PMC3705281  PMID: 23835605
10.  Tuning the mechanical properties of bioreducible multilayer films for improved cell adhesion and transfection activity 
Biomaterials  2010;31(27):7167-7174.
A simple approach to the mechanical modulation of layer-by-layer (LbL) films is through manipulation of the film assembly. Here, we report results based on altering the salt concentration during film assembly and its effect on film rigidity. Based on changes in film rigidity, cell adhesion characteristics and transfection activity were investigated in vitro. LbL films consisting of reducible hyperbranched poly(amide amine) (RHB) have been implemented along with DNA for investigating fibroblast adhesion on [RHB/DNA]n/2 films with varying rigidities. The rigidity was varied by changing the ionic concentration of the deposition solution between 0.01 M NaCl and 1.0 M NaCl. Molecular force probe (MFP) measurements were performed to measure the apparent Young’s modulus, EAPP, of the films in situ. Cell adhesion and stress-fiber characteristics were investigated using total internal reflectance microscopy (TIRF-M). The average cell peripheral area, fiber density and average fiber length during 5 days of cell growth on films with either low (below 2.0 MPa) or high (above 2.0 MPa) film elastic modulus were investigated. Transfection studies were performed using gfpDNA and SEAP-DNA to investigate if changes in cell adhesion affect transfection activity. Furthermore, cell proliferation and cytotoxicity studies were used to investigate cellular viability over a week. The results have shown that surface modification of bioreducible LbL films of controlled thickness and roughness promotes cellular adhesion, stress-fiber growth and increased transfection activity without the need for an additional adhesive protein pre-coating of the surface or chemical cross-linking of the film.
doi:10.1016/j.biomaterials.2010.06.002
PMCID: PMC2904610  PMID: 20580430
molecular force probe; MFP; cell adhesion; transfection; TIRF-M; stress fiber orientation; layer-by-layer; LbL; mechanical properties; thin films; elastic modulus; film rigidity
11.  Measurement of Mechanical Properties of Cantilever Shaped Materials 
Sensors (Basel, Switzerland)  2008;8(5):3497-3541.
Microcantilevers were first introduced as imaging probes in Atomic Force Microscopy (AFM) due to their extremely high sensitivity in measuring surface forces. The versatility of these probes, however, allows the sensing and measurement of a host of mechanical properties of various materials. Sensor parameters such as resonance frequency, quality factor, amplitude of vibration and bending due to a differential stress can all be simultaneously determined for a cantilever. When measuring the mechanical properties of materials, identifying and discerning the most influential parameters responsible for the observed changes in the cantilever response are important. We will, therefore, discuss the effects of various force fields such as those induced by mass loading, residual stress, internal friction of the material, and other changes in the mechanical properties of the microcantilevers. Methods to measure variations in temperature, pressure, or molecular adsorption of water molecules are also discussed. Often these effects occur simultaneously, increasing the number of parameters that need to be concurrently measured to ensure the reliability of the sensors. We therefore systematically investigate the geometric and environmental effects on cantilever measurements including the chemical nature of the underlying interactions. To address the geometric effects we have considered cantilevers with a rectangular or circular cross section. The chemical nature is addressed by using cantilevers fabricated with metals and/or dielectrics. Selective chemical etching, swelling or changes in Young's modulus of the surface were investigated by means of polymeric and inorganic coatings. Finally to address the effect of the environment in which the cantilever operates, the Knudsen number was determined to characterize the molecule-cantilever collisions. Also bimaterial cantilevers with high thermal sensitivity were used to discern the effect of temperature variations. When appropriate, we use continuum mechanics, which is justified according to the ratio between the cantilever thickness and the grain size of the materials. We will also address other potential applications such as the ageing process of nuclear materials, building materials, and optical fibers, which can be investigated by monitoring their mechanical changes with time. In summary, by virtue of the dynamic response of a miniaturized cantilever shaped material, we present useful measurements of the associated elastic properties.
doi:10.3390/s8053497
PMCID: PMC3675557
Microcantilever; mechanics; ageing; environment; stress; gas; materials; sensor; pressure; temperature
12.  The effect of standard and low-modulus cement augmentation on the stiffness, strength, and endplate pressure distribution in vertebroplasty 
European Spine Journal  2011;21(5):920-929.
Purpose
Vertebroplasty restores stiffness and strength of fractured vertebral bodies, but alters their stress transfer. This unwanted effect may be reduced by using more compliant cements. However, systematic experimental comparison of structural properties between standard and low-modulus augmentation needs to be done. This study investigated how standard and low-modulus cement augmentation affects apparent stiffness, strength, and endplate pressure distribution of vertebral body sections.
Methods
Thirty-nine human thoracolumbar vertebral body sections were prepared by removing cortical endplates and posterior elements. The specimens were scanned with a HR-pQCT system and loaded in the elastic range. After augmentation with standard or low-modulus cement they were scanned again and tested in two steps. First, the contact pressure distribution between specimen and loading plates was measured with pressure-sensitive films. Then, they were loaded again in the elastic range and compressed until failure. Apparent stiffness was compared before and after augmentation, whereas apparent strength of augmented specimens was compared to a non-augmented reference group.
Results
Vertebral body sections with fillings connecting both endplates were on average 33% stiffer and 47% stronger with standard cement, and 27% stiffer and 30% stronger with low-modulus cement. In contrast, partial fillings showed no significant strengthening for both cements and only a slight stiffness increase (<16%). The averaged endplate pressure above/below the cement was on average 15% lower with low-modulus cement compared to standard cement.
Conclusion
Augmentation connecting both endplates significantly strengthened and stiffened vertebral body sections also with low-modulus cement. A trend of reduced pressure concentrations above/below the cement was observed with low-modulus cement.
doi:10.1007/s00586-011-2119-5
PMCID: PMC3337907  PMID: 22170449
Vertebroplasty; Low-modulus; PMMA; Cement; Vertebral body; Mechanical properties
13.  In vitro characterization of polycaprolactone matrices generated in aqueous media 
Acta biomaterialia  2009;6(3):1061-1068.
In this study, a novel process of dissolving polycaprolactone (PCL) matrices in glacial acetic acid was explored in which matrices spontaneously formed upon contact with water. Scanning electron microscopy analysis showed rough architecture and holes on the self-assembled matrix relative to matrices formed after dissolving in chloroform. Immersion in the gelatin solution reduced its roughness and number of micropores. Atomic force microscopy (AFM) analysis confirmed the increased roughness of the self-assembled matrices. The roughness of the matrices decreased after incubation in 1 N NaOH for 10 min. AFM analysis also revealed that the self-assembled matrix had a net positive surface charge, whereas chloroform–cast matrix had a negative surface charge. The surface charge of self-assembled matrix after immersion in gelatin changed to negative. However, incubation in NaOH did not affect the surface charge. The tensile properties were tested in both the dry state (25 °C) and the wet state (37 °C) by immersion in phosphate-buffered saline. Self-assembled matrix had lower elastic modulus, break stress and break strain than chloroform–cast matrix in both states. The elastic modulus in the wet condition was reduced by half in self-assembled matrix but tensile strain increased. Samples were further analyzed by ramp-hold test for assessing stress relaxation behavior. Both self-assembled and chloroform–cast matrices had similar trends in stress relaxation behavior. However, stress accumulation in self-assembled matrix was half that of chloroform–cast matrix. In vitro cell cultures were conducted using human foreskin fibroblast (HFF-1) in serum-free medium. Cytoskeletal actin staining showed cell adhesion and spreading on all matrices. Cell retention was significantly increased in self-assembled matrix compared to chloroform–cast matrix. Addition of gelatin improved the retention of seeded cells on the surface. In summary, PCL matrices generated using this novel technique show significant promise in biomedical applications.
doi:10.1016/j.actbio.2009.08.002
PMCID: PMC2997440  PMID: 19664731
Matrix; PCL; Stress relaxation; Roughness; Surface property
14.  CIS-TRANS ISOMERS OF VITAMIN A AND RETINENE IN THE RHODOPSIN SYSTEM 
The Journal of General Physiology  1952;36(2):269-315.
Vitamin A and retinene, the carotenoid precursors of rhodopsin, occur in a variety of molecular shapes, cis-trans isomers of one another. For the synthesis of rhodopsin a specific cis isomer of vitamin A is needed. Ordinary crystalline vitamin A, as also the commercial synthetic product, both primarily all-trans, are ineffective. The main site of isomer specificity is the coupling of retinene with opsin. It is this reaction that requires a specific cis isomer of retinene. The oxidation of vitamin A to retinene by the alcohol dehydrogenase-cozymase system displays only a low degree of isomer specificity. Five isomers of retinene have been isolated in crystalline condition: all-trans; three apparently mono-cis forms, neoretinenes a and b and isoretinene a; and one apparently di-cis isomer, isoretinene b. Neoretinenes a and b were first isolated in our laboratory, and isoretinenes a and b in the Organic Research Laboratory of Distillation Products Industries. Each of these substances is converted to an equilibrium mixture of stereoisomers on simple exposure to light. For this reaction, light is required which retinene can absorb; i.e., blue, violet, or ultraviolet light. Yellow, orange, or red light has little effect. The single geometrical isomers of retinene must therefore be protected from low wave length radiation if their isomerization is to be avoided. By incubation with opsin in the dark, the capacity of each of the retinene isomers to synthesize rhodopsin was examined. All-trans retinene and neoretinene a are inactive. Neoretinene b yields rhodopsin indistinguishable from that extracted from the dark-adapted retina (λmax· 500 mµ). Isoretinene a yields a similar light-sensitive pigment, isorhodopsin, the absorption spectrum of which is displaced toward shorter wave lengths (λmax· 487 mµ). Isoretinene b appears to be inactive, but isomerizes preferentially to isoretinene a, which in the presence of opsin is removed to form isorhodopsin before the isomerization can go further. The synthesis of rhodopsin in solution follows the course of a bimolecular reaction, as though one molecule of neoretinene b combines with one of opsin. The synthesis of isorhodopsin displays similar kinetics. The bleaching of rhodopsin, whether by chemical means or by exposure to yellow or orange (i.e., non-isomerizing) light, yields primarily or exclusively all-trans retinene. The same appears to be true of isorhodopsin. The process of bleaching is therefore intrinsically irreversible. The all-trans retinene which results must be isomerized to active configurations before rhodopsin or isorhodopsin can be regenerated. A cycle of isomerization is therefore an integral part of the rhodopsin system. The all-trans retinene which emerges from the bleaching of rhodopsin must be isomerized to neoretinene b before it can go back; or if first reduced to all-trans vitamin A, this must be isomerized to neovitamin Ab before it can regenerate rhodopsin. The retina obtains new supplies of the neo-b isomer: (a) by the isomerization of all-trans retinene in the eye by blue or violet light; (b) by exchanging all-trans vitamin A for new neovitamin Ab from the blood circulation; and (c) the eye tissues may contain enzymes which catalyze the isomerization of retinene and vitamin A in situ. When the all-trans retinene which results from bleaching rhodopsin in orange or yellow light is exposed to blue or violet light, its isomerization is accompanied by a fall in extinction and a shift of absorption spectrum about 5 mµ toward shorter wave lengths. This is a second photochemical step in the bleaching of rhodopsin. It converts the inactive, all-trans isomer of retinene into a mixture of isomers, from which mixtures of rhodopsin and isorhodopsin can be regenerated. Isorhodopsin, however, is an artefact. There is no evidence that it occurs in the retina; nor has isovitamin Aa or b yet been identified in vivo. In rhodopsin and isorhodopsin, the prosthetic groups appear to retain the cis configurations characteristic of their retinene precursors. In accord with this view, the β-bands in the absorption spectra of both pigments appear to be cis peaks. The conversion to the all-trans configuration occurs during the process of bleaching. The possibility is discussed that rhodopsin may represent a halochromic complex of a retinyl ion with opsin. The increased resonance associated with the ionic state of retinene might then be responsible both for the color of rhodopsin and for the tendency of retinene to assume the all-trans configuration on its release from the complex. A distinction must be made between the immediate precursor of rhodopsin, neovitamin Ab, and the vitamin A which must be fed in order that rhodopsin be synthesized in vivo. Since vitamin A isomerizes in the body, it is probable that any geometrical isomer can fulfill all the nutritional needs for this vitamin.
PMCID: PMC2147363  PMID: 13011282
15.  Reprogramming an ATP-driven protein machine into a light-gated nanocage 
Nature nanotechnology  2013;8(12):10.1038/nnano.2013.242.
Natural protein assemblies have many sophisticated architectures and functions, creating nanoscale storage containers, motors and pumps1–3. Inspired by these systems, protein monomers have been engineered to self-assemble into supramolecular architectures4 including symmetrical5,6, metal-templated7,8 and cage-like structures8–10. The complexity of protein machines, however, has made it difficult to create assemblies with both defined structures and controllable functions. Here we report protein assemblies that have been engineered to function as light-controlled nanocontainers. We show that an adenosine-5′-triphosphate (ATP)-driven group II chaperonin11,12, which resembles a barrel with a builtin lid, can be reprogrammed to open and close on illumination with different frequencies of light. By engineering photoswitchable azobenzene-based molecules into the structure, light-triggered changes in interatomic distances in the azobenzene moiety are able to drive large-scale conformational changes of the protein assembly. The different states of the assembly can be visualized with single particle cryo-electron microscopy, and the nanocages can be used to capture and release non-native cargos. Similar strategies switching atomic distances with light could be used to build other controllable nanoscale machines.
doi:10.1038/nnano.2013.242
PMCID: PMC3859876  PMID: 24270642
16.  In Situ Mechanical Interferometry of Matrigel™ Films 
Many biological materials and cell substrates are very soft (Young's modulus < 500 Pa) and it is difficult to characterize their mechanical properties. Here we report local elasticity of the surface layers of Matrigel™ films used for cell culture. We used a new measurement technology, Mechanical Imaging Interferometry, to obtain point mechanical measurements over micron-sized areas. The median values of 650 Pa +/- 400 Pa (# measurements, n=50), determined by the Hertz contact model, agree well with bulk measurements, however on the micro-scale the films were heterogeneous and contained regions distinctly stiffer than average (1-2 kPa). The first measurement of yield strengths of 170 Pa +/- 100 Pa (n=43) indicate that Matrigel™ films deform plastically at stress levels of similar scale to cell tractional forces.
doi:10.1021/la8033098
PMCID: PMC3437936  PMID: 19049394
17.  The mechanical properties of a surface-modified layer on poly(dimethylsiloxane) 
Journal of materials research  2008;23(1):37-48.
Surface-modification of the elastomer poly(dimethylsiloxane) by exposure to oxygen plasma for four minutes creates a thin, stiff film. In this study, the thickness and mechanical properties of this surface-modified layer were determined. Using the phase image capabilities of a tapping-mode atomic-force microscope, the surface-modified region was distinguished from the bulk PDMS; specifically, it suggested a graded surface layer to a depth of about 200 nm. Load-displacement data for elastic indentation using a compliant AFM cantilever was analyzed as a plate bending on an elastic foundation to determine the elastic modulus of the surface (37 MPa). An applied uniaxial strain generated a series of parallel nano-cracks with spacing on the order of a few microns. Numerical analyses of this cracking phenomenon showed that the depth of these cracks was in the range of 300–600 nm and that the surface layer was extremely brittle, with its toughness in the range of 0.1–0.3 J/m2.
doi:10.1557/JMR.2008.0029
PMCID: PMC2749279  PMID: 19779588
18.  Local Rheology of Human Neutrophils Investigated Using Atomic Force Microscopy 
During the immune response, neutrophils display localized mechanical events by interacting with their environment through the micro-vascular transit, trans-endothelial, and trans-epithelial migration. Nano-mechanical studies of human neutrophils on localized nano-domains could provide the essential information for understanding their immune responsive functions. Using the Atomic Force Microscopy (AFM) - based micro-rheology, we have investigated rheological properties of the adherent human neutrophils on local nano-domains. We have applied the modified Hertz model to obtain the viscoelastic moduli from the relatively thick body regions of the neutrophils. In addition, by using more advanced models to account for the substrate effects, we have successfully characterized the rheological properties of the thin leading and tail regions as well. We found a regional difference in the mechanical compliances of the adherent neutrophils. The central regions of neutrophils were significantly stiffer (1,548 ± 871 Pa) than the regions closer to the leading edge (686 ± 801 Pa), while the leading edge and the tail (494 ± 537 Pa) regions were mechanically indistinguishable. The frequency-dependent elastic and viscous moduli also display a similar regional difference. Over the studied frequency range (100 to 300 Hz), the complex viscoelastic moduli display the partial rubber plateau behavior where the elastic moduli are greater than the viscous moduli for a given frequency. The non-disparaging viscous modulus indicates that the neutrophils display a viscoelastic dynamic behavior rather than a perfect elastic behavior like polymer gels. In addition, we found no regional difference in the structural damping coefficient between the leading edge and the cell body. Thus, we conclude that despite the lower loss and storage moduli, the leading edges of the human neutrophils display partially elastic properties similar to the cell body. These results suggest that the lower elastic moduli in the leading edges are more favorable for the elastic fluctuation of actin filaments, which supports the polymerization of the actin filaments leading to the active protrusion during the immune response.
PMCID: PMC3030146  PMID: 21278920
Atomic force microscopy; neutrophils; micro-rheology; viscoelasticity; nano-indentation; Young's modulus
19.  Time-harmonic magnetic resonance elastography of the normal feline brain 
Journal of biomechanics  2010;43(14):2747-2752.
Imaging of the mechanical properties of in vivo brain tissue could eventually lead to non-invasive diagnosis of hydrocephalus, Alzheimer’s disease and other pathologies known to alter the intracranial environment. The purpose of this work is to (1) use time-harmonic magnetic resonance elastography (MRE) to estimate the mechanical property distribution of cerebral tissue in the normal feline brain and (2) compare the recovered properties of grey and white matter. Various in vivo and ex vivo brain tissue property measurement strategies have led to the highly variable results that have been reported in the literature. MR elastography is an imaging technique that can estimate mechanical properties of tissue non-invasively and in vivo. Data was acquired in 14 felines and elastic parameters were estimated using a globo-regional nonlinear image reconstruction algorithm. Results fell within the range of values reported in the literature and showed a mean shear modulus across the subject group of 7–8 kPa with all but one animal falling within 5–15 kPa. White matter was statistically stiffer (p < 0.01) than grey matter by about 1 kPa on a per subject basis. To the best of our knowledge, the results reported represent the most extensive set of estimates in the in vivo brain which have been based on MRE acquisition of the three-dimensional displacement field coupled to volumetric shear modulus image reconstruction achieved through nonlinear parameter estimation. However, the inter-subject variation in mean shear modulus indicates the need for further study, including the possibility of applying more advanced models to estimate the relevant tissue mechanical properties from the data.
doi:10.1016/j.jbiomech.2010.06.008
PMCID: PMC2963725  PMID: 20655045
Magnetic resonance elastography; brain; grey matter; white matter; finite element method
20.  The Emergence of AFM Applications to Cell Biology: How new technologies are facilitating investigation of human cells in health and disease at the nanoscale 
Journal of nanoscience letters  2011;1`(2):87-101.
Atomic Force Microscopy (AFM) based nanorobotics has been used for building nano devices in semiconductors for almost a decade. Leveraging the unparallel precision localization capabilities of this technology, high resolution imaging and mechanical property characterization is now increasingly being performed in biological settings. AFM also offers the prospect for handling and manipulating biological materials at nanometer scale. It has unique advantages over other methods, permitting experiments in the liquid phase where physiological conditions can be maintained. Taking advantage of these properties, our group has visualized membrane and cytoskeletal structures of live cells by controlling the interaction force of the AFM tip with cellular components at the nN or sub-nN range. Cell stiffness changes were observed by statistically analyzing the Young’s modulus values of human keratinocytes before and after specific antibody treatment. Furthermore, we used the AFM cantilever as a robotic arm for mechanical pushing, pulling and cutting to perform nanoscale manipulations of cell-associated structures. AFM guided nano-dissection, or nanosurgery was enacted on the cell in order to sever intermediate filaments connecting neighboring keratinocytes via sub 100 nm resolution cuts. Finally, we have used a functionalized AFM tip to probe cell surface receptors to obtain binding force measurements. This technique formed the basis for Single Molecule Force Spectroscopy (SMFS). In addition to enhancing our basic understanding of dynamic signaling events in cell biology, these advancements in AFM based biomedical investigations can be expected to facilitate the search for biomarkers related to disease diagnosis progress and treatment.
PMCID: PMC3886279  PMID: 24416719
21.  Photoswitching of Site-Selective RNA Scission by Sequential Incorporation of Azobenzene and Acridine Residues in a DNA Oligomer 
Journal of Nucleic Acids  2011;2011:162452.
Photoresponsive systems for site-selective RNA scission have been prepared by combining Lu(III) ions with acridine/azobenzene dual-modified DNA. The modified DNA forms a heteroduplex with substrate RNA, and the target phosphodiester linkages in front of the acridine residue is selectively activated so that Lu(III) ion rapidly cleaves the linkage. Azobenzene residue introduced adjacent to the acridine residue acts as a photoresponsive switch, which triggers the site-selective scission upon UV irradiation. A trans isomer of azobenzene efficiently suppresses the scission, whereas the cis isomer formed by UV irradiation hardly affects the scission. As a result, 1.7–2.4-fold acceleration of the cleavage was achieved simply by irradiating UV for 3 min to the mixture prior to the reaction. Considering the yield of photoisomerization, the intrinsic activity of a cis isomer is up to 14.5-fold higher than that of the trans isomer.
doi:10.4061/2011/162452
PMCID: PMC3177363  PMID: 21941627
22.  Atomic force microscopy measurements of lens elasticity in monkey eyes 
Molecular Vision  2007;13:504-510.
Purpose
To demonstrate the feasibility of measuring the elasticity of intact crystalline lenses using atomic force microscopy (AFM).
Methods
AFM elasticity measurements were performed on intact lenses from 18 fresh cynomolgus monkey cadaver eyes (4-10 years old, <1 day postmortem) that had been left attached to their zonule-ciliary body-sclera framework. The eyes were prepared by bonding a plastic ring on the sclera after removal of the conjunctival, adipose, and muscle tissues. The posterior pole was sectioned, with the excess vitreous removed, and the eye's anterior section was placed on a Teflon slide to protect the posterior pole of the lens. The cornea and iris were then sectioned. The lens-zonule-ciliary body-sclera section was then placed in a Petri dish filled with balanced salt solution in an AFM system designed for force measurements. Next, the central pole of the anterior surface of the intact lens was probed with the AFM cantilever tip. The recorded AFM cantilever deflection-indentation curves were used to derive force-indentation curves for the lens after factoring out the deflection of the cantilever on a hard surface. Young's modulus of the lens was calculated from the force-indentation relation using the Hertz model.
Results
Young's modulus was 1,720±880 Pa (range: 409-3,210 Pa) in the 18 cynomolgus monkey lenses.
Conclusions
AFM can be used to provide measurements of the elasticity of the whole lens including the capsule. Values obtained using AFM on cynomolgus monkey lenses are similar to published values obtained using dynamic mechanical analysis on young human lenses.
PMCID: PMC2649306  PMID: 17417612
23.  Antigen structural requirements for immunoglobulin isotype switching in mice 
The Journal of Experimental Medicine  1980;152(6):1670-1683.
L-Tyrosine-p-azobenzene-p-arsonate (RAT) is immunogenic and serves as a carrier for anti-hapten antibody responses in guinea pigs, rats, and mice. However, the murine anti-N-2,4-dinitrophenyl (DNP) plaque-forming cell (PFC) response to the bifunctional antigen 2,4-dinitrophenyl-6-amino-caproyl-L- tyrosine-p-azobenzene-p-arsonate (DNP-SAC-RAT; or BI-1) is extremely weak (2,000-4,000 PFC/spleen) and exclusively IgM in both primary and secondary responses. The 6-amino-caproyl group serves as a spacer in this antigen between the DNP haptenic and RAT carrier epitopes. In view of recent evidence indicating that different T helper cells synergize for optimal antibody responses, a trifunctional antigen, N-2,4- dinitrophenyl-6-amino-caproyl-L-tyrosine-p-azobenze-p-arsonate-(proline)9-L- tyrosine-p-azobenzene-p-arsonate (DNP-SAC-RAT-PRO(9)-RAT; or TRI), was prepared to investigate the effect of adding a second RAT epitope to BI-1. The nonaproline spacer between the two RAT epitopes in TRI is assumed to be a rigid rod of approximately 28 A. TRI induced about twice as many PFC as BI-1 in primary responses of A/J mice, and induced both IgM and IgG PFC in secondary responses. Furthermore, TRI induced IgG PFC responses in mice primed with p-azobenzene-p-arsonate-keyhole limpet hemocyanin, BI-1, or RAT, whereas boosting with BI-1 failed to induce IgG PFC, even in mice primed with TRI. These findings indicate that the minimum antigen structural requirements for inducing IgG PFC in mice are two carrier epitopes and one haptenic epitope. In addition, priming with a mono-epitope carrier (RAT) is sufficient preparation for IgG responses to a trifunctional immunogen. Because TRI differs from BI-1 by the (proline)(9) spacer as well as the additional RAT epitope, two other compounds, N-2,4-dinitrophenyl-6-amino- caproyl-(proline)(9)-L-tyrosine-p-azobenzene-p-arsonate (DNP-SAC-PRO(9)-RAT; or BI-2) and N-2,4-dinitrophenyl-6-amino-caproyl-(proline)(9)-L-tyrosine-p- azobenzene-arsonate (DNP-SAC-RAT-PRO(10); or BI-3), were prepared to evaluate the possible role of the spacer in the observed responses. BI-2, but not BI-3, induced IgG as well as IgM PFC in TRI-primed mice. However, BI-2 failed to induce IgG responses in RAT-primed mice, indicating that TRI and BI-2 were not equivalent immunogens. Because anti-prolyl antibodies had been found in guinea pigs immunized with N-2,4-dinitrophenyl-(proline)10-L-tyrosine-p- azobenzene-p-arsonate (DNP-PRO(10)-RAT), it seemed possible that priming with TRI might induce anti-prolyl antibodies, which, in turn, could cross-link BI-2 molecules into aggregates containing at least two carrier epitopes. To help resolve this question, mice were immunized with acetyl-(proline)10-L- tyrosine-p-azobenzene-p-arsonate and boosted with BI-2. IgG PFC responses were detected, suggesting that anti-prolyl antibodies were indeed responsible, because priming with RAT and boosting with BI-2 did not induce IgG formation. Accordingly, the observations that IgG responses in RAT-primed mice were induced only by TRI and not by any of the bifunctional antigens indicate that two carrier epitopes per antigen molecule are indeed required for IgG induction. They also provide indirect evidence for synergistic help in the switching of immunoglobulin isotypes.
PMCID: PMC2186037  PMID: 6161201
24.  Bis-azobenzene cross-linkers for photo-control of peptide structure 
Cross-linkers that undergo large changes in length upon photoisomerization may produce large conformational changes, and thereby functional changes, in biomolecules. We have designed and synthesized extended and rigid bis-azobenzene cross-linkers, 4,4’-bis(4-(2-chloroacetamido) phenyl)diazenylbiphenyl (BPDB) and the water-soluble sulfonated analog 4,4’-bis(4-(2-chloroacetamido) phenyl)diazenylbiphenyl-2,2’-disulfonate (BPDBS). These photo-switches can produce a minimum ~ 5 Å and a maximum ~ 23 Å end-to-end distance change upon trans/cis isomerization. They have high absorption coefficients (45-60,000 M-1cm-1) and can produce up to ~80% cis isomers under favorable conditions. The photo-switching behavior of BPDBS cross-linked peptides was found to be highly dependent on the cross-linker attachment site. Upon UV (365 nm) irradiation, a significant decrease in α-helix content was observed for peptides that were cross-linked with BPDBS via Cys residues at i, i+19, and i, i+21 positions. In contrast, a large increase in α-helix content was exhibited by i, i+11 cross-linked peptides. BPDBS thus constitutes a particularly bright and effective photoswitch for biomolecule photo-control.
doi:10.1002/cbic.201100204
PMCID: PMC3517121  PMID: 21698738
azobenzene; peptide; conformation; photo; control
25.  Thermally Driven Stability of Octadecylphosphonic Acid Thin Films Grown on SS316L 
Scanning  2010;32(5):304-311.
Stainless steel 316L is widely used as a biomedical implant material; however, there is concern about the corrosion of metallic implants in the physiological environment. The corrosion process can cause mechanical failure due to resulting cracks and cavities in the implant. Alkyl phosphonic acid forms a thin film by self-assembly on the stainless steel surface and this report conclusively shows that thermal treatment of the octadecylphosphonic acid (ODPA) film greatly enhances the stability of the ODPA molecules on the substrate surface. AFM images taken from the modified substrates revealed that thermally treated films remain intact after methanol, THF and water flushes while untreated films suffer substantial loss. Water contact angles also show that the hydrophobicity of thermally treated films does not diminish after being incubated in a dynamic flow of water for a three hour period while the untreated film becomes increasingly hydrophilic due to loss of ODPA. IR spectra taken of both treated and untreated films after water and THF flushes show that the remaining film retains its initial crystallinity. A model is suggested to explain the stability of ODPA film enhanced by thermal treatment. An ODPA molecule is physisorbed to the surface weakly by hydrogen bonding. Heating drives away water molecules leading to the formation of strong monodentate or mixed mono/bi-dentate bonds of ODPA molecule to the surface.
doi:10.1002/sca.20192
PMCID: PMC2962881  PMID: 20648546
Octadecylphosphonic acid; Stainless steel (SS316L); biomaterials; self-assembly; stability; contact angles; atomic force microscopy (AFM); IR spectroscopy

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