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Mapping of adhesion forces on soil minerals in air and water by atomic force spectroscopy (AFS)

Leite, F. L.; Riul, A.; Herrmann, PSP
Fonte: Vsp Bv Publicador: Vsp Bv
Tipo: Artigo de Revista Científica Formato: 2141-2156
ENG
Relevância na Pesquisa
57.28209%
The adhesion force between an atomic force microscope (AFM) tip and sample surfaces, mica and quartz substrates, was measured in air and water. The force curves show that the adhesion has a strong dependence on both the surface roughness and the environmental conditions surrounding the sample. The variability of the adhesion force was examined in a series of measurements taken at the same point, as well as at different places on the sample surface. The adhesion maps obtained from the distribution of the measured forces indicated regions contaminated by either organic compounds or adsorbed water. Using simple mathematical expressions we could quantitatively predict the adhesion force behavior in both air and water. The experimental results are in good agreement with theoretical calculations, where the adhesion forces in air and water were mostly associated with capillary and van der Waals forces, respectively. A small long-range repulsive force is also observed in water due to the overlapping electrical double-layers formed on both the tip and sample surfaces.

Microscopia de força atômica em materiais biológicos : biossensores e nanoferramentas; Atomic force microscopy on biological materials : biosensors and nanotools

Alberto Luís Dario Moreau
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 14/01/2011 PT
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Na primeira parte deste trabalho, nós investigamos o processo de crescimento de um biofilme de bactérias (Xylella fastidiosa) inoculadas sobre lamínulas de vidro. O tamanho e a distância entre os biofilmes foram estudados por imagens de microscopia óptica; e uma análise fractal foi realizada usando conceitos de escala e imagens de AFM. Observamos que biofilmes diferentes mostram características fractais semelhantes, embora as variações na morfologia possam ser identificadas para diferentes estádios de crescimento do biofilme. Dois tipos de padrões estruturais são identificados através da dimensão fractal (Df) sugerindo que o crescimento do biofilme pode ser entendido como o modelo de Eden nos estágios de formação e no final, enquanto para o estágio de maturação aparecem evidências do modelo DLA (diffusion-limited aggregation). Estes resultados foram correlacionados à formação da matriz do biofilme que pode dificultar a difusão dos nutrientes e por isso criar condições para um crescimento DLA. Ainda com o AFM, fizemos medidas de espectroscopia de força para estudar a interação específica entre antígeno-anticorpo relacionados ao vírus CTV (citrus tristeza virus). Para tanto foi realizado o estudo da imobilização deste material biológico nas superfícies da ponta do AFM...

Single Molecule Recognition between Cytochrome C 551 and Gold-Immobilized Azurin by Force Spectroscopy

Bonanni, B.; Kamruzzahan, A. S. M.; Bizzarri, A. R.; Rankl, C.; Gruber, H. J.; Hinterdorfer, P.; Cannistraro, S.
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em /10/2005 EN
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57.30437%
Recent developments in single molecule force spectroscopy have allowed investigating the interaction between two redox partners, Azurin and Cytochrome C 551. Azurin has been directly chemisorbed on a gold electrode whereas cytochrome c has been linked to the atomic force microscopy tip by means of a heterobifunctional flexible cross-linker. When recording force-distance cycles, molecular recognition events could be observed, displaying unbinding forces of ∼95 pN for an applied loading rate of 10 nN/s. The specificity of molecular recognition was confirmed by the significant decrease of unbinding probability observed in control block experiments performed adding free azurin solution in the fluid cell. In addition, the complex dissociation kinetics has been here investigated by monitoring the unbinding forces as a function of the loading rate: the thermal off-rate was estimated to be ∼14 s−1, much higher than values commonly estimated for complexes more stable than electron transfer complexes. Results here discussed represent the first studies on molecular recognition between two redox partners by atomic force microscopy.

Effect of Temperature on the Nanomechanics of Lipid Bilayers Studied by Force Spectroscopy

Garcia-Manyes, Sergi; Oncins, Gerard; Sanz, Fausto
Fonte: Biophysical Society Publicador: Biophysical Society
Tipo: Artigo de Revista Científica
EN
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57.3448%
The effect of temperature on the nanomechanical response of supported lipid bilayers has been studied by force spectroscopy with atomic force microscopy. We have experimentally proved that the force needed to puncture the lipid bilayer (Fy) is temperature dependent. The quantitative measurement of the evolution of Fy with temperature has been related to the structural changes that the surface undergoes as observed through atomic force microscopy images. These studies were carried out with three different phosphatidylcholine bilayers with different main phase transition temperature (TM), namely, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, and 2-dilauroyl-sn-glycero-3-phosphocholine. The solid-like phase shows a much higher Fy than the liquid-like phase, which also exhibits a jump in the force curve. Within the solid-like phase, Fy decreases as temperature is increased and suddenly drops as it approaches TM. Interestingly, a “well” in the Fy versus temperature plot occurs around TM, thus proving an “anomalous mechanical softening” around TM. Such mechanical softening has been predicted by experimental techniques and also by molecular dynamics simulations and interpreted in terms of water ordering around the phospholipid headgroups. Ion binding has been demonstrated to increase Fy...

Energy Landscape of Chelated Uranyl: Antibody Interactions by Dynamic Force Spectroscopy

Odorico, Michael; Teulon, Jean-Marie; Bessou, Thérèse; Vidaud, Claude; Bellanger, Laurent; Chen, Shu-wen W.; Quéméneur, Éric; Parot, Pierre; Pellequer, Jean-Luc
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
57.30437%
We used dynamic force spectroscopy (DFS) to explore the energy landscape of interactions between a chelated uranyl compound and a monoclonal antibody raised against the uranyl-dicarboxy-phenanthroline complex. We estimated the potential energy barrier widths and the relevant thermodynamic rate constants along the dissociation coordinate. Using atomic force microscopy, four different experimental setups with or without the uranyl ion in the chelate ligand, we have distinguished specific and nonspecific binding in the binding affinity of the uranyl compound to the antibody. The force loading rates for our system were measured from 15 to 26,400 pN/s. The results showed two regimes in the plot of the most probable unbinding force versus the logarithm of the loading rate, revealing the presence of two (at least) activation barriers. Analyses of DFS suggest parallel multivalent binding present in either regime. We have also built a molecular model for the variable fragment of the antibody and used computational graphics to dock the chelated uranyl ion into the binding pocket. The structural analysis led us to hypothesize that the two regimes originate from two interaction modes: the first one corresponds to an energy barrier with a very narrow width of 0.5 ± 0.2 Å...

Subpiconewton Dynamic Force Spectroscopy Using Magnetic Tweezers

Kruithof, M.; Chien, F.; de Jager, M.; van Noort, J.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
EN
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57.48564%
We introduce a simple method for dynamic force spectroscopy with magnetic tweezers. This method allows application of subpiconewton force and twist control by calibration of the applied force from the height of the magnets. Initial dynamic force spectroscopy experiments on DNA molecules revealed a large hysteresis that is caused by viscous drag on the magnetic bead and will conceal weak interactions. When smaller beads are used, this hysteresis is sufficiently reduced to reveal intramolecular interactions at subpiconewton forces. Compared with typical quasistatic force spectroscopy, a significant reduction of measurement time is achieved, allowing the real-time study of transient structures and reaction intermediates. As a proof of principle, nucleosome-nucleosome interactions on a subsaturated chromatin fiber were analyzed.

Effects of Multiple-Bond Ruptures on Kinetic Parameters Extracted from Force Spectroscopy Measurements: Revisiting Biotin-Streptavidin Interactions

Guo, Senli; Ray, Chad; Kirkpatrick, Andrea; Lad, Nimit; Akhremitchev, Boris B.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
EN
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57.38833%
Force spectroscopy measurements of the rupture of the molecular bond between biotin and streptavidin often results in a wide distribution of rupture forces. We attribute the long tail of high rupture forces to the nearly simultaneous rupture of more than one molecular bond. To decrease the number of possible bonds, we employed hydrophilic polymeric tethers to attach biotin molecules to the atomic force microscope probe. It is shown that the measured distributions of rupture forces still contain high forces that cannot be described by the forced dissociation from a deep potential well. We employed a recently developed analytical model of simultaneous rupture of two bonds connected by polymer tethers with uneven length to fit the measured distributions. The resulting kinetic parameters agree with the energy landscape predicted by molecular dynamics simulations. It is demonstrated that when more than one molecular bond might rupture during the pulling measurements there is a noise-limited range of probe velocities where the kinetic parameters measured by force spectroscopy correspond to the true energy landscape. Outside this range of velocities, the kinetic parameters extracted by using the standard most probable force approach might be interpreted as artificial energy barriers that are not present in the actual energy landscape. Factors that affect the range of useful velocities are discussed.

Absolute Quantitation of Bacterial Biofilm Adhesion and Viscoelasticity by Microbead Force Spectroscopy

Lau, Peter C.Y.; Dutcher, John R.; Beveridge, Terry J.; Lam, Joseph S.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 08/04/2009 EN
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57.516206%
Bacterial biofilms are the most prevalent mode of bacterial growth in nature. Adhesive and viscoelastic properties of bacteria play important roles at different stages of biofilm development. Following irreversible attachment of bacterial cells onto a surface, a biofilm can grow in which its matrix viscoelasticity helps to maintain structural integrity, determine stress resistance, and control ease of dispersion. In this study, a novel application of force spectroscopy was developed to characterize the surface adhesion and viscoelasticity of bacterial cells in biofilms. By performing microbead force spectroscopy with a closed-loop atomic force microscope, we accurately quantified these properties over a defined contact area. Using the model gram-negative bacterium Pseudomonas aeruginosa, we observed that the adhesive and viscoelastic properties of an isogenic lipopolysaccharide mutant wapR biofilm were significantly different from those measured for the wild-type strain PAO1 biofilm. Moreover, biofilm maturation in either strain also led to prominent changes in adhesion and viscoelasticity. To minimize variability in force measurements resulting from experimental parameter changes, we developed standardized conditions for microbead force spectroscopy to enable meaningful comparison of data obtained in different experiments. Force plots measured under standard conditions showed that the adhesive pressures of PAO1 and wapR early biofilms were 34 ± 15 Pa and 332 ± 47 Pa...

Association Kinetics from Single Molecule Force Spectroscopy Measurements

Guo, Senli; Lad, Nimit; Ray, Chad; Akhremitchev, Boris B.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 22/04/2009 EN
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57.42876%
Single molecule force spectroscopy is often used to study the dissociation of single molecules by applying mechanical force to the intermolecular bond. These measurements provide the kinetic parameters of dissociation. We present what to our knowledge is a new atomic force microscopy-based approach to obtain the activation energy of the association reaction and approximate grafting density of reactive receptors using the dependence of the probability to form molecular bonds on probe velocity when one of the interacting molecules is tethered by a flexible polymeric linker to the atomic force microscopy probe. Possible errors in the activation energy measured with this approach are considered and resulting corrections are included in the data analysis. This new approach uses the same experimental setup as traditional force spectroscopy measurements that quantify dissociation kinetics. We apply the developed methodology to measure the activation energy of biotin-streptavidin association (including a contribution from the steric factor) and obtain a value of 8 ± 1 kT. This value is consistent with the association rate measured previously in solution. Comparison with the solution-derived activation energy indicates that kinetics of biotin-streptavidin binding is mainly controlled by the reaction step.

Single-Molecule Force Spectroscopy of Cartilage Aggrecan Self-Adhesion

Harder, Alexander; Walhorn, Volker; Dierks, Thomas; Fernàndez-Busquets, Xavier; Anselmetti, Dario
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 17/11/2010 EN
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57.3448%
We investigated self-adhesion between highly negatively charged aggrecan macromolecules extracted from bovine cartilage extracellular matrix by performing atomic force microscopy (AFM) imaging and single-molecule force spectroscopy (SMFS) in saline solutions. By controlling the density of aggrecan molecules on both the gold substrate and the gold-coated tip surface at submonolayer densities, we were able to detect and quantify the Ca2+-dependent homodimeric interaction between individual aggrecan molecules at the single-molecule level. We found a typical nonlinear sawtooth profile in the AFM force-versus-distance curves with a molecular persistence length of lp = 0.31 ± 0.04 nm. This is attributed to the stepwise dissociation of individual glycosaminoglycan (GAG) side chains in aggrecans, which is very similar to the known force fingerprints of other cell adhesion proteoglycan systems. After studying the GAG-GAG dissociation in a dynamic, loading-rate-dependent manner (dynamic SMFS) and analyzing the data according to the stochastic Bell-Evans model for a thermally activated decay of a metastable state under an external force, we estimated for the single glycan interaction a mean lifetime of τ = 7.9 ± 4.9 s and a reaction bond length of xβ = 0.31 ± 0.08 nm. Whereas the xβ-value compares well with values from other cell adhesion carbohydrate recognition motifs in evolutionary distant marine sponge proteoglycans...

Antibody-Unfolding and Metastable-State Binding in Force Spectroscopy and Recognition Imaging

Kaur, Parminder; Qiang-Fu; Fuhrmann, Alexander; Ros, Robert; Kutner, Linda Obenauer; Schneeweis, Lumelle A.; Navoa, Ryman; Steger, Kirby; Xie, Lei; Yonan, Christopher; Abraham, Ralph; Grace, Michael J.; Lindsay, Stuart
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 05/01/2011 EN
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57.526714%
Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance—possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images...

Characterization of Enhanced Monovalent and Bivalent Thrombin DNA Aptamer Binding Using Single Molecule Force Spectroscopy

Neundlinger, Isabel; Poturnayova, Alexandra; Karpisova, Ivana; Rankl, Christian; Hinterdorfer, Peter; Snejdarkova, Maja; Hianik, Tibor; Ebner, Andreas
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 05/10/2011 EN
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57.37537%
Thrombin aptamer binding strength and stability is dependent on sterical parameters when used for atomic force microscopy sensing applications. Sterical improvements on the linker chemistry were developed for high-affinity binding. For this we applied single molecule force spectroscopy using two enhanced biotinylated thrombin aptamers, BFF and BFA immobilized on the atomic force microscopy tip via streptavidin. BFF is a dimer composed of two single-stranded aptamers (aptabody) connected to each other by a complementary sequence close to the biotinylated end. In contrast, BFA consists of a single DNA strand and a complementary strand in the supporting biotinylated part. By varying the pulling velocity in force-distance cycles the formed thrombin-aptamer complexes were ruptured at different force loadings allowing determination of the energy landscape. As a result, BFA aptamer showed a higher binding force at the investigated loading rates and a significantly lower dissociation rate constant, koff, compared to BFF. Moreover, the potential of the aptabody BFF to form a bivalent complex could clearly be demonstrated.

Unequivocal Single-Molecule Force Spectroscopy of Proteins by AFM Using pFS Vectors

Oroz, Javier; Hervás, Rubén; Carrión-Vázquez, Mariano
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 08/02/2012 EN
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57.38833%
Nanomechanical analysis of proteins by single-molecule force spectroscopy based on atomic force microscopy is increasingly being used to investigate the inner workings of mechanical proteins and substrate proteins of unfoldase machines as well as to gain new insight into the process of protein folding. However, such studies are hindered by a number of technical problems, including the noise of the proximal region, ambiguous single-molecule identification, as well as difficulties in protein expression/folding and full-length purification. To overcome these major drawbacks in protein nanomechanics, we designed a family of cloning/expression vectors, termed pFS (plasmid for force spectroscopy), that essentially has an unstructured region to surmount the noisy proximal region, a homomeric polyprotein marker, a carrier to mechanically protect the protein of interest (only the pFS-2 version) that also acts as a reporter, and two purification tags. pFS-2 enables the unambiguous analysis of proteins with low mechanical stability or/and complex force spectra, such as the increasingly abundant class of intrinsically disordered proteins, which are hard to characterize by traditional bulk techniques and have important biological and clinical implications. The advantages...

Hidden Multiple Bond Effects in Dynamic Force Spectroscopy

Getfert, Sebastian; Reimann, Peter
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 07/03/2012 EN
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57.42876%
In dynamic force spectroscopy, a (bio-)molecular complex is subjected to a steadily increasing force until the chemical bond breaks. Repeating the same experiment many times results in a broad distribution of rupture forces, whose quantitative interpretation represents a formidable theoretical challenge. In this study we address the situation that more than a single molecular bond is involved in one experimental run, giving rise to multiple rupture events that are even more difficult to analyze and thus are usually eliminated as far as possible from the further evaluation of the experimental data. We develop and numerically solve a detailed model of a complete dynamic force spectroscopy experiment including a possible clustering of molecules on the substrate surface, the formation of bonds, their dissociation under load, and the postprocessing of the force extension curves. We show that the data, remaining after elimination of obvious multiple rupture events, may still contain a considerable number of hidden multiple bonds, which are experimentally indistinguishable from true single bonds, but which have considerable effects on the resulting rupture force statistics and its consistent theoretical interpretation.

Reference-Free Alignment and Sorting of Single-Molecule Force Spectroscopy Data

Bosshart, Patrick D.; Frederix, Patrick L.T.M.; Engel, Andreas
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 02/05/2012 EN
Relevância na Pesquisa
57.249717%
Single-molecule force spectroscopy has become a versatile tool for investigating the (un)folding of proteins and other polymeric molecules. Like other single-molecule techniques, single-molecule force spectroscopy requires recording and analysis of large data sets to extract statistically meaningful conclusions. Here, we present a data analysis tool that provides efficient filtering of heterogeneous data sets, brings spectra into register based on a reference-free alignment algorithm, and determines automatically the location of unfolding barriers. Furthermore, it groups spectra according to the number of unfolding events, subclassifies the spectra using cross correlation-based sorting, and extracts unfolding pathways by principal component analysis and clustering methods to extracted peak positions. Our approach has been tested on a data set obtained through mechanical unfolding of bacteriorhodopsin (bR), which contained a significant number of spectra that did not show the well-known bR fingerprint. In addition, we have tested the performance of the data analysis tool on unfolding data of the soluble multidomain (Ig27)8 protein.

Long Lifetime of Hydrogen-Bonded DNA Basepairs by Force Spectroscopy

Fuhrmann, Alexander; Getfert, Sebastian; Fu, Qiang; Reimann, Peter; Lindsay, Stuart; Ros, Robert
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
Publicado em 16/05/2012 EN
Relevância na Pesquisa
57.42876%
Electron-tunneling data suggest that a noncovalently-bonded complex of three molecules, two recognition molecules that present hydrogen-bond donor and acceptor sites via a carboxamide group, and a DNA base, remains bound for seconds. This is surprising, given that imino-proton exchange rates show that basepairs in a DNA double helix open on millisecond timescales. The long lifetime of the three-molecule complex was confirmed using force spectroscopy, but measurements on DNA basepairs are required to establish a comparison with the proton-exchange data. Here, we report on a dynamic force spectroscopy study of complexes between the bases adenine and thymine (A-T, two-hydrogen bonds) and 2-aminoadenine and thymine (2AA-T, three-hydrogen bonds). Bases were tethered to an AFM probe and mica substrate via long, covalently linked polymer tethers. Data for bond-survival probability versus force and the rupture-force distributions were well fitted by the Bell model. The resulting lifetime of the complexes at zero pulling force was ∼2 s for two-hydrogen bonds (A-T) and ∼4 s for three-hydrogen bonds (2AA-T). Thus, DNA basepairs in an AFM pulling experiment remain bonded for long times, even without the stabilizing influence of base-stacking in a double helix. This result suggests that the pathways for opening...

Multiscale sensing of antibody - antigen interactions by organic transistors and single-molecule force spectroscopy

Casalini, Stefano; Dumitru, Andra Cristina; Leonardi, Francesca; Bortolotti, Carlo Augusto; Herruzo, Elena Tomas; Campana, Alessandra; Oliveira, Rafael Furlan de; Cramer, Tobias; Garcia, Ricardo; Biscarini, Fabio
Fonte: Amer Chemical Soc Publicador: Amer Chemical Soc
Tipo: Artigo de Revista Científica Formato: 5051-5062
ENG
Relevância na Pesquisa
67.42876%
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Processo FAPESP: 2013/03857-0; Antibody-antigen (Ab-Ag) recognition is the primary event at the basis of many biosensing platforms. In label-free biosensors, these events occurring at solid liquid interfaces are complex and often difficult to control technologically across the smallest length scales down to the molecular scale. Here a molecular-scale technique, such as single-molecule force spectroscopy, is performed across areas of a real electrode functionalized for the immunodetection of an inflammatory cytokine, viz. interleukin-4 (114). The statistical analysis of force distance curves allows us to quantify the probability, the characteristic length scales, the adhesion energy, and the time scales of specific recognition. These results enable us to rationalize the response of an electrolyte-gated organic field-effect transistor (EGOFET) operated as an 114 immuno-sensor. Two different strategies for the immobilization of 114 antibodies on the Au gate electrode have been compared: antibodies are bound to (i) a smooth film of His-tagged protein G (PG)/Au; (ii) a 6-aminohexanethiol (HSC6NH2) self-assembled monolayer on Au through glutaraldehyde. The most sensitive EGOFET (concentration minimum detection level down to 5 nM of 114) is obtained with the first functionalization strategy. This result is correlated to the highest probability (30%) of specific binding events detected by force spectroscopy on Ab/PG/Au electrodes...

Minimizing Pulling Geometry Errors in Atomic Force Microscope Single Molecule Force Spectroscopy

Rivera, Monica; Lee, Whasil; Ke, Changhong; Marszalek, Piotr E.; Cole, Daniel G.; Clark, Robert L.
Fonte: The Biophysical Society Publicador: The Biophysical Society
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
57.418525%
In atomic force microscopy-based single molecule force spectroscopy (AFM-SMFS), it is assumed that the pulling angle is negligible and that the force applied to the molecule is equivalent to the force measured by the instrument. Recent studies, however, have indicated that the pulling geometry errors can drastically alter the measured force-extension relationship of molecules. Here we describe a software-based alignment method that repositions the cantilever such that it is located directly above the molecule's substrate attachment site. By aligning the applied force with the measurement axis, the molecule is no longer undergoing combined loading, and the full force can be measured by the cantilever. Simulations and experimental results verify the ability of the alignment program to minimize pulling geometry errors in AFM-SMFS studies.

Development of a State-of-the-Art Atomic Force Microscope for Improved Force Spectroscopy

Rivera, Monica
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação Formato: 17926839 bytes; application/pdf
Publicado em 19/11/2008 EN_US
Relevância na Pesquisa
67.58837%

This research describes the development of a state-of-the-art atomic force microscope (AFM) for improved force spectroscopy. Although the AFM has been used extensively in this field of research, the performance of the instrument has been limited by inefficient operation techniques, incorrect experimental assumptions, and inadequate controller design. This research focuses on overcoming these deficiencies by providing precise control over the instrument for specialized research in a manner that is conducive to the natural science researcher.

To facilitate this research, a custom, multi-axis AFM system was constructed. The instrument was designed primarily for AFM-based force spectroscopy and as a result a substantial amount of research focused on the development of a wide variety of approach/retraction methods for the instrument. Defining research in this area included the development of methods to minimize potentially damaging compressive forces, form polymer bridges at different tip-sample gap widths, produce clean, deconvoluted force-extension curves, and limit single molecule force spectroscopy pulling geometry errors. In an effort to increase the efficiency of the instrument, the programs developed during this research were fully automated...

Nanomechanics of Nucleic Acid Structures Investigated with AFM Based Force Spectroscopy

Rabbi, Mahir Haroon
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2010
Relevância na Pesquisa
57.59429%

Nucleic acids are subjected to many different mechanical loadings inside. These loadings could cause large deformations and conformational changes to these molecules. This is why the mechanical properties of nucleic acids are so important to their functions. Here we use a newly designed and built high-performance AFM force spectrometer, supplemented with molecular dynamics simulations and NMR spectroscopy to investigate the relationship between mechanical properties and structure of different nucleic acids.

To test the mechanical properties of nucleic acids, we successfully designed and purpose-built a single molecule puller, an instrument to physically stretch single molecules, at a fraction of the cost of a commercial AFM instrument. This instrument has similar force noise to hybrid instruments, while also exhibiting significantly lower drift, on the order of five times lower. This instrument allows the measurement of subtle transitions as a molecule is stretched. With the addition of a lock-in amplifier, we possibly could obtain better force resolution, the order of femtonewtons.

We find that helical structure does indeed have an effect on the mechanical properties of double-stranded DNA. As the A-form double helix has a shorter...