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Huong Nguyen

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Leader of the Junior Research Group at the Institute for Bioprocessing and Analytical Measurement Techniques (iba), Heiligenstadt, Germany.Research interest: 1) characterization of interactions among thrombotic proteins that trigger thrombocytopenia in various diseases, 2) development of nanostructures and hydrogel surfaces to improve platelet contacting devices, and 3) development of cell-based ELISA and biosensors for better detection of pathogenic antibodies.Methodologies: Single-molecule force spectroscopy- based Atomic force microscopy, FluidFM, Confocal laser scanning microscopy, Isothermal titration calorimetry, dynamic light scattering, Circular dichroism spectroscopy, Spectra shift, Antibody purification, Enzyme-linked immunosorbent assays (ELISA), Cell culture/ Cancer cell spheroids, Isolation of platelets from whole blood, Substrate/nanoparticle functionalization, Platelet labeling, Atomic absorption spectroscopy, and Flow cytometry.

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The George Washington University

The Cyprus Institute

Kyrgyz State Medical Academy

University of Cape Town

Marek Titien Olszewski (Marek Tycjan Olszewski)

University of Warsaw

Jammu University

The University of Newcastle

Manoj Kumar Narasimhan

SRM UNIVERSITY

Jagannath University, Dhaka

Istanbul University

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Papers by Huong Nguyen

SARS-CoV-2 spike protein-induced platelet activation: Mechanism for virus and vaccine-induced thrombotic thrombocytopenia

Covid-19 pandemic stimulated an extremely fast development of effective vaccines. Recent studies ... more Covid-19 pandemic stimulated an extremely fast development of effective vaccines. Recent studies found platelet-activating antibodies against platelet factor 4 (PF4) in both clinically ill Covid-19 patients and vaccine-induced thrombotic thrombocytopenia (VITT) patients. Here, we use various tools to identify the binding reaction of the SARS-CoV-2 spike glycoprotein (SP) with PF4 that results in immunogenic platelet-activating PF4/SP complexes. This binding is evidenced by an increase in mass, optical intensity, and stable binding force observed by quartz crystal microbalance, enzyme immune assay, and force spectroscopy, respectively. The SP induced an increase in the size of PF4 and switched the surface zeta potential of the PF4 from positive to negative values as evaluated by dynamic light scattering. The SP-induced platelet aggregation was identified by functional assay and flow cytometry but in a concentration-dependent manner. Our results indicated that the formed PF4/SP comple...

Investigation of Structural Transition of dsDNA on Various Substrates Studied by Atomic Force Microscopy

Structural transition of single dsDNA molecule which is immobilized on 3-aminopropyltriethoxysila... more Structural transition of single dsDNA molecule which is immobilized on 3-aminopropyltriethoxysilane (APTES) treated substrate (APTES/substrate) or alkylthiol treated substrate (alkylthiol/substrate) has been investigated by atomic force microscopy (AFM). The obtained force versus distance (F –D) curves are used to dissect the transition from B-form to S-form, the melting from double stranded (ds) to single stranded (ss) DNA, and its Young's modulus as well as persistence length. The melt from dsDNA to ssDNA is evidenced by fitting with freely jointed chain (FJC) model. FJC fit and Young's modulus or persistence length values when the molecules are fixed on alkylthiol/substrate are more agreeable with other studies than those on APTES. We have clarified the different results of those experiments by analyzing the binding force between DNA molecules and APTES or alkylth-iol linkers on the substrate. The DNA binding to APTES linker is much stronger than that on alkylthiol/substrate.

Tip Functionalization by Biotin Linkers: A Technique for DNA Elasticity Measurement Studied by Atomic Force Microscopy

We have investigated the mechanical properties of a double strands deoxyribonucleic acid (dsDNA) ... more We have investigated the mechanical properties of a double strands deoxyribonucleic acid (dsDNA) in deionized water and in 10 mM NaCl solution by using atomic force microscopy (AFM). In order to do this, a number of the force vs. displacement (F-D) curves between the AFM tip functionalized by biotin linkers and the dsDNA immobilized on 3-aminopropyltriethoxysilane treated glass substrate has been measured. These force curves will be compared to those measured by untreated tip to compare the difference of the adhesion forces between the two tip types. The retraction parts of the force curves reveal that the binding force between the DNA and the biotin treated tip is increased up to four times compared to those measured by the untreated one. Analysis of the force curves measured by biotin treated tip shows that the dissipated force for the molecular stretching in salt solution (702 ± 19 pN) is greater than that in DI water (558 ± 21 pN) due to the effect of electrostatic attraction generated by the Na + ions around the DNA segments. It also shows that the Young's modulus and the persistence length of the dsDNA measured in DI water are smaller than those measured in NaCl medium which implies that the salt solution increased the rigidity of the biological system.

Nanoscience and Nanotechnology CONFOCAL MICROSCOPY AND ATOMIC FORCE MICROSCOPY OF SACCHAROMYCES CEREVISIAE

We examined the physical properties of the surrounding yeast cell walls by using atomic force mic... more We examined the physical properties of the surrounding yeast cell walls by using atomic force microscopy (AFM). The yeast cells were prepared on a cleaned glass substrate for CM observation and were mechanically trapped into a porous membrane for AFM measurement. The confocal image of the yeast cells was measured in air meanwhile the AFM topography images of the cells were measured in both de-ionized (DI) water (pH = 6.9) and phosphate-buffered saline (PBS) solution (pH = 7.4). No significant differences between the AFM topography images of the yeast cells measured in DI water and in phosphate buffered saline (PBS) solution could be inferred. In order to get the quantitative information on the sample elasticity, the force curves between an AFM tip and the yeast cell have been measured. These curves were measured in both DI water and in PBS solution on the same yeast cell using the same AFM cantilever to get the reliable result. The contact region of these force curves in approach mode was then converted into force versus indentation curves. These curves would be fitted with Hertz-Sneddon model for the calculation of the elasticity. Analysis of the curves indicates that there is a difference of the Young's modulus values of the yeast cell in various environments. These data show that the salt buffer solution increases the rigidity of the biological system.

Measuring contact angle and meniscus shape with a reflected laser beam

Side-view imaging of the contact angle between an extended planar solid surface and a liquid is p... more Side-view imaging of the contact angle between an extended planar solid surface and a liquid is problematic. Even when aligning the view perfectly parallel to the contact line, focusing one point of the contact line is not possible. We describe a new measurement technique for determining contact angles with the reflection of a widened laser sheet on a moving contact line. We verified this new technique measuring the contact angle on a cylinder, rotating partially immersed in a liquid. A laser sheet is inclined under an angle ϕ to the unperturbed liquid surface and is reflected off the meniscus. Collected on a screen, the reflection image contains information to determine the contact angle. When dividing the laser sheet into an array of laser rays by placing a mesh into the beam path, the shape of the meniscus can be reconstructed from the reflection image. We verified the method by measuring the receding contact angle versus speed for aqueous cetyltrimethyl ammonium bromide solutions on a smooth hydrophobized as well as on a rough polystyrene surface.

$Figure 1. Schematic of the experimental setup. The cylinder was rotating in a bath filled with a liquid. A widened laser sheet with a wavelength of \ = 635 nm was inclined under an angle y to the liquid surface and was reflected on the cylinder (green dashed), the meniscus (red) and the unperturbed liquid surface (blue dotted). The reflection image was collected on a screen where the angle @ref; can be detected.$

$with the scaling parameter \ and the direction vector that depends on the array index 7. Here the last approx- imation is only valid for small opening angles of the laser sheet. € is the angle between a single ray and the x-axis. In further calculations we focus on one ray and omit the index ¢ for clarity. Details of the calculation can be found in the Appendix [A]$

Phototunable Response in Caged Polymer Brushes

A light-responsive brush was obtained by surface-initiated ATRP of a methacrylate monomer contain... more A light-responsive brush was obtained by surface-initiated ATRP of a methacrylate monomer containing ionizable −COOH side groups caged with the photoremovable group 4,5-dimethoxy-2-nitrobenzyl (NVOC). In the caged form, the polymer brush (PNVOCMA) is neutral and hydrophobic due to the presence of the aromatic chromophore. Upon irradiation the NVOC group is removed and a polyanion (polymethacrylic acid, PMAA) chain is generated. The charged brush can swell and collapse depending on the pH and the exposure dose (i.e., uncaging degree). The behavior and properties of the brush layer for different photoconversion degrees were studied. On the basis of quartz crystal microbalance measurements, a threshold of 50% uncaging was identified in order to achieve significant swelling and pH response of the brush. Between 50 and 80% the photoconversion the response of the brush could be light-modulated. For photoconversions >80% only small changes in the response were detectable. X-ray reflectivity (XRR) and scanning force microscopy allowed us to measure thickness, roughness and swelling of the brushes at intermediate photoconversions. Combined XRR and grazing-incidence small-angle scattering experiments evidenced a change in the internal structure of the brush upon exposure and indicated the occurrence of domain segregation as a consequence of the coexistence of hydrophobic and charged groups in the brush structure.

ARTICLE IN PRESS G Model Insights into the interaction of CD4 with anti-CD4 antibodies

Knowledge about the mechanism by which some antibodies can block HIV-1 entry is critical to our u... more Knowledge about the mechanism by which some antibodies can block HIV-1 entry is critical to our understanding of their function and may offer new avenues for controlling the adhesion of HIV-1 to the host cells. While much progress has been made, this mechanism remains unclear. Here, atomic force microscopy, isothermal titration calorimetry (ITC), and circular dichroism spectroscopy were used to measure some biophysical characteristics of the interaction of four-domains (D1–D4) membrane protein CD4 with anti-D3 antibody OKT4 and with HIV-1 entry blocking anti-D1 antibody Leu3a. The results showed that at 37 • C they bind with similar binding strength, thermodynamics, and kinetics but with different assembly states. Further analyzing the interactions at different temperatures by ITC showed that binding of CD4 with Leu3a is characteristic for specific hydrophobic binding as well as for protein folding while with OKT4 comes from an extensive additional hydration upon binding and charge-related interactions within the binding site. Comparing these characteristics with those of HIV-1 gp120-CD4 interaction revealed that Leu3a binds to CD4 faster than HIV-1 followed by changing local structure of D1 to which HIV-1 binds leading to a prevention of viral entry.

An improved measurement of dsDNA elasticity using AFM An improved measurement of dsDNA elasticity using AFM

The mechanical properties of a small fragment (30 bp) of an individual double-stranded deoxyribon... more The mechanical properties of a small fragment (30 bp) of an individual double-stranded deoxyribonucleic acid (dsDNA) in water have been investigated by atomic force microscopy (AFM). We have stretched three systems including ssDNA, double-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3-end and thiolated at the 5-end, this was reversed for the other complementary strand) and single-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3-end and thiolated at the 5-end, whereas the other complementary strand was biotinylated at only the 5-end). The achieved thiolation and biotinylation were to bind ds-or ssDNA to the gold surface and streptavidin-coated AFM tip, respectively. Analysis of the force versus displacement (F–D) curves from tip–DNA–substrate systems shows that the pull-off length (L o) and stretch length (δ) from the double-fixed system were shorter than those observed in the ssDNA and the single-fixed system. The obtained stretch force (F st) from the single-fixed dsDNA was much greater than that from the ssDNA even though it was about 10 pN greater than the one obtained in the double-fixed system. As a result, the Young's modulus of the double-fixed dsDNA was greater than that of the single-fixed dsDNA and the ssDNA. A more reliable stiffness of the dsDNA was observed via the double-fixed system, since there is no effect of the unpaired molecules during stretching, which always occurred in the single-fixed system. The unpaired molecules were also observed by comparing the stiffness of ssDNA and single-fixed dsDNA in which the end of one strand was left free.

Influence of surfactants in forced dynamic dewetting

In this work we show that the forced dynamic dewetting of surfactant solutions depends sensitivel... more In this work we show that the forced dynamic dewetting of surfactant solutions depends sensitively on the surfactant concentration. To measure this effect, a hydrophobic rotating cylinder was horizontally half immersed in aqueous surfactant solutions. Dynamic contact angles were measured optically by extrapolating the contour of the meniscus to the contact line. Anionic (sodium 1-decanesulfonate, S-1DeS), cationic (cetyl trimethylammonium bromide, CTAB) and nonionic surfactants (C 4 E 1 , C 8 E 3 and C 12 E 5) with critical micelle concentrations (CMCs) spanning four orders of magnitude were used. The receding contact angle in water decreased with increasing velocity. This decrease was strongly enhanced when adding surfactant, even at surfactant concentrations of 10% of the critical micelle concentration. Plots of the receding contact angle-versus-velocity almost superimpose when being plotted at the same relative concentration (concentration/ CMC). Thus the rescaled concentration is the dominating property for dynamic dewetting. The charge of the surfactants did not play a role, thus excluding electrostatic effects. The change in contact angle can be interpreted by local surface tension gradients, i.e. Marangoni stresses, close to the three-phase contact line. The decrease of dynamic contact angles with velocity follows two regimes. Despite the existence of Marangoni stresses close to the contact line, for a dewetting velocity above 1–10 mm s À1 the hydrodynamic theory is able to describe the experimental results for all surfactant concentrations. At slower velocities an additional steep decrease of the contact angle with velocity was observed. Particle tracking velocimetry showed that the flow profiles do not differ with and without surfactant on a scales 4100 mm.

Study on the Binding Force Between λ-DNA and Various Types of Substrates

Binding forces connecting bio-molecules to solid substrates have a strong influence on both atomi... more Binding forces connecting bio-molecules to solid substrates have a strong influence on both atomic force microcopy (AFM) imaging and force spectroscopy. We have investigated the binding forces between λ-DNA and several kinds of substrates, such as glass, mica, and Si, and observed some detailed features representing various binding states between them. The strongest binding was observed on the glass substrate silanized by 0.5 % APTES due to 'APTES bridges'. The interaction forces are in the range of ∼pN for DNA on mica and glass, and ∼nN for DNA on Si and APTES-treated glass substrates. In addition, the force-distance curve shows different features depending on the position of the cantilever tip on the DNA. The variation of the binding forces with the substrate materials and the measurement conditions will be discussed in detail.

$Fig. 2. Typical DNA images obtained before and after F-D curve measurement. F-D curve measurement was performec on both the tail and the train of \A-DNA. (a) This image was obtained from a A-DNA on a glass substrate. The insert was obtained after F-D measurement, and the change of the tai shape was observed. (b) Image of A-DNA on APTES/glas: substrate.$

Measuring Single Small Molecule Binding via Rupture Forces of a Split Aptamer

The rupture force of a split (bipartite) aptamer that forms binding pockets for adenosine monopho... more The rupture force of a split (bipartite) aptamer that forms binding pockets for adenosine monophosphate (AMP) was measured by atomic force spectroscopy. Changes in the rupture force were observed in the presence of AMP, while this effect was absent when mutant aptamers or inosine were used. Thus, changes in the rupture force were a direct consequence of specific binding of AMP to the split aptamer. The split aptamer concept allowed the detection of nonlabeled AMP and enabled us to determine the dissociation constant on a single-molecule level.

Nanomechanical measurement of astrocyte stiffness correlated with cytoskeletal maturation

by Huong Nguyen, Thi Nguyen, and Kyo Hwang

Astrocytes are known to serve as scaffolding cells that shape the brain. The physical properties ... more Astrocytes are known to serve as scaffolding cells that shape the brain. The physical properties of astrocytes, such as stiffness, are important for their scaffolding function. These properties may be altered in certain pathological conditions , such as in brain cancer. However, actual stiffness of astrocytes is not yet well understood. Here, we report that the astrocyte stiffness is positively correlated with the density of cytoskeletal proteins, such as actin filaments, microtu-bules, and intermediate filaments. The value of the stiffness of astrocytes as measured by atomic force microscopy (AFM) increases 38-fold in five-week-old rats compared to postnatal-day zero pups. Using multicolor confocal microscopy, we found that the complexity of cytoskeletal proteins, such as actin filaments, microtubules, and intermediate filaments, increase as the animal gets older. Our findings indicate that the change of stiffness positively correlates with the matura-tion of cytoskeletal proteins, and suggest that AFM can be useful as an analytical and diagnostic tool for neuroscience.

The role of CD4 on mechanical properties of live cell membrane

Although much progress has been made in the characterization and identification of CD4 functions,... more Although much progress has been made in the characterization and identification of CD4 functions, its role in mechanical properties of cell membrane remains largely unknown. Here an atomic force microscopy (AFM) was used to investigate the roles of CD4 in the elasticity of the leukemic human Jurkat (clone E6-1) cell membranes. Analysis of the approach force curves with Hertz model for a completely elastic soft sample measured on the selected CD41 and CD4-cells showed that CD41 cell membrane was softer than CD4-one. To confirm that CD4 plays a role in altering cell elasticity, human embryonic kidney 293T cells were transiently trans-fected with wild type (wt) CD4 plasmid before being used in AFM nanoindentation experiments. The results also demonstrated CD4-membrane was stiffer than CD41 one suggesting that CD4 integrated into plasma membrane and altered its mechanical properties. The study gives insights into the role of CD4 on cell membrane mechanical characteristics and might be helpful for development of cell biology and medicine. V C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 239–244, 2016.

Single‐molecule force spectroscopy applied to heparin‐induced thrombocytopenia

Heparin‐induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiv... more Heparin‐induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiving the antithrombotic drug heparins, has a complex pathogenesis involving multiple partners ranging from small molecules to cells/platelets. Recently, insights into the mechanism of HIT have been achieved by using single‐molecule force spectroscopy (SMFS), a methodology that allows direct measurements of interactions among HIT partners. Here, the potential of SMFS in unraveling the mechanism of the initial steps in the pathogenesis of HIT at single‐molecule resolution is highlighted. The new findings ranging from the molecular binding strengths and kinetics to the determination of the boundary between risk and non‐risk heparin drugs or platelet‐surface and platelet‐platelet interactions will be reviewed. These novel results together have contributed to elucidate the mechanisms underlying HIT and demonstrate how SMFS can be applied to develop safer drugs with a reduced risk profile.

Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds

Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetram... more Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly understood. In this study, single molecule force spectroscopy (SMFS) is utilized to characterize the interaction of PF4 with heparins of defined length (5-, 6-, 8-, 12-, and 16-mers). Analysis of the force–distance curves shows that PF4/heparin binding strength rises with increasing heparin length. In addition, two binding pathways in the PF4/short heparins (≤8-mers) and three binding pathways in the PF4/long heparins (≥8-mers) are identified. We provide a model for the PF4/heparin complexes in which short heparins bind to one PF4 tetramer, while long heparins bind to two PF4 tetramers. We propose that the interaction between long heparins and PF4s is not only due to charge differences as generally assumed, but also due to hydrophobic interaction between two PF4s which are brought close to each other by long heparin. This complicated interaction induces PF4/heparin complexes more stable than other ligand–receptor interactions. Our results also reveal that the boundary between antigenic and non-antigenic heparins is between 8-and 12-mers. These observations are particularly important to understand processes in which PF4–heparin interactions are involved and to develop new heparin-derived drugs.

Rupture Forces among Human Blood Platelets at different Degrees of Activation

by Huong Nguyen and Thi-Huong Nguyen

Little is known about mechanics underlying the interaction among platelets during activation and ... more Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects. Blood platelets are discoidal, anuclear cell fragments, 1–2 μ m in size and are produced from the cytoplasm of bone marrow megakaryocytes 1. They prevent blood loss upon vascular injury 2 , can induce arterial thrombosis 3 , and have additional roles in immune defense 4 , wound healing 5 , and cancer metastasis 6,7. In vivo, platelets do not normally interact with the inner surfaces of blood vessels, but they adhere promptly when the vessel wall is altered. Several glycoproteins (GPs) on the platelet surface interact with extracellular matrix proteins on the vessel wall (e.g. GPIbIX with von Willebrand factor [vWF], GPIaIIa and GPVI with collagen) leading to platelet activation 1. Activated platelets expose GPIIbIIIa (α IIbβ 3) in an activated form, which allows binding of fibrinogen 8,9 and triggers platelet activation and aggregation 10. In parallel, activated platelets release prothrombotic substances from their granules 11. Some of these substances recruit and activate neighboring platelets, while others enhance thrombin generation together with plasma clotting factors. Finally, this leads to formation of a hemostatic plug at the site of endothelial damage which eventually results in blood vessel closure 12,13. An increasingly important issue for biotechnological applications in medicine is modulation of platelet activation on various surfaces 14,15. However, these processes are not well understood as platelets are difficult to handle because they activate immediately after short contact with non-physiological surfaces 16–18 , at elevated shear stress 19 and upon air contact 20. Among these, platelet activation on surfaces is not only a major drawback for microfluidic devices 21 , micro-and nano-particulate drug delivery systems 22,23 , and intravascular devices 24 , but it is also essential for developing tools for direct measuring of platelet mechanics 25,26. Platelet-surface interactions have been quantified in real-time using calcium mobilization assays, showing that the lag time until platelets activate depends on the physico-chemical properties of the surface 27. It has been shown that platelets are activated nearly instantaneously upon coming in contact with immobilized fibrinogen and completing their contraction after 15 min 28. Others have provided information regarding morphology 17,18,29 , and elastic moduli 29–31 of activated platelets under physiological conditions. Although these studies have successfully identified significant characteristics of platelets on a single molecular level, none of these previous studies describes quantitatively single platelet-platelet interactions, neither in activated, nor in non-/weakly activated states.

SARS-CoV-2 spike protein-induced platelet activation: Mechanism for virus and vaccine-induced thrombotic thrombocytopenia

Covid-19 pandemic stimulated an extremely fast development of effective vaccines. Recent studies ... more Covid-19 pandemic stimulated an extremely fast development of effective vaccines. Recent studies found platelet-activating antibodies against platelet factor 4 (PF4) in both clinically ill Covid-19 patients and vaccine-induced thrombotic thrombocytopenia (VITT) patients. Here, we use various tools to identify the binding reaction of the SARS-CoV-2 spike glycoprotein (SP) with PF4 that results in immunogenic platelet-activating PF4/SP complexes. This binding is evidenced by an increase in mass, optical intensity, and stable binding force observed by quartz crystal microbalance, enzyme immune assay, and force spectroscopy, respectively. The SP induced an increase in the size of PF4 and switched the surface zeta potential of the PF4 from positive to negative values as evaluated by dynamic light scattering. The SP-induced platelet aggregation was identified by functional assay and flow cytometry but in a concentration-dependent manner. Our results indicated that the formed PF4/SP comple...

Investigation of Structural Transition of dsDNA on Various Substrates Studied by Atomic Force Microscopy

Structural transition of single dsDNA molecule which is immobilized on 3-aminopropyltriethoxysila... more Structural transition of single dsDNA molecule which is immobilized on 3-aminopropyltriethoxysilane (APTES) treated substrate (APTES/substrate) or alkylthiol treated substrate (alkylthiol/substrate) has been investigated by atomic force microscopy (AFM). The obtained force versus distance (F –D) curves are used to dissect the transition from B-form to S-form, the melting from double stranded (ds) to single stranded (ss) DNA, and its Young's modulus as well as persistence length. The melt from dsDNA to ssDNA is evidenced by fitting with freely jointed chain (FJC) model. FJC fit and Young's modulus or persistence length values when the molecules are fixed on alkylthiol/substrate are more agreeable with other studies than those on APTES. We have clarified the different results of those experiments by analyzing the binding force between DNA molecules and APTES or alkylth-iol linkers on the substrate. The DNA binding to APTES linker is much stronger than that on alkylthiol/substrate.

Tip Functionalization by Biotin Linkers: A Technique for DNA Elasticity Measurement Studied by Atomic Force Microscopy

We have investigated the mechanical properties of a double strands deoxyribonucleic acid (dsDNA) ... more We have investigated the mechanical properties of a double strands deoxyribonucleic acid (dsDNA) in deionized water and in 10 mM NaCl solution by using atomic force microscopy (AFM). In order to do this, a number of the force vs. displacement (F-D) curves between the AFM tip functionalized by biotin linkers and the dsDNA immobilized on 3-aminopropyltriethoxysilane treated glass substrate has been measured. These force curves will be compared to those measured by untreated tip to compare the difference of the adhesion forces between the two tip types. The retraction parts of the force curves reveal that the binding force between the DNA and the biotin treated tip is increased up to four times compared to those measured by the untreated one. Analysis of the force curves measured by biotin treated tip shows that the dissipated force for the molecular stretching in salt solution (702 ± 19 pN) is greater than that in DI water (558 ± 21 pN) due to the effect of electrostatic attraction generated by the Na + ions around the DNA segments. It also shows that the Young's modulus and the persistence length of the dsDNA measured in DI water are smaller than those measured in NaCl medium which implies that the salt solution increased the rigidity of the biological system.

Nanoscience and Nanotechnology CONFOCAL MICROSCOPY AND ATOMIC FORCE MICROSCOPY OF SACCHAROMYCES CEREVISIAE

We examined the physical properties of the surrounding yeast cell walls by using atomic force mic... more We examined the physical properties of the surrounding yeast cell walls by using atomic force microscopy (AFM). The yeast cells were prepared on a cleaned glass substrate for CM observation and were mechanically trapped into a porous membrane for AFM measurement. The confocal image of the yeast cells was measured in air meanwhile the AFM topography images of the cells were measured in both de-ionized (DI) water (pH = 6.9) and phosphate-buffered saline (PBS) solution (pH = 7.4). No significant differences between the AFM topography images of the yeast cells measured in DI water and in phosphate buffered saline (PBS) solution could be inferred. In order to get the quantitative information on the sample elasticity, the force curves between an AFM tip and the yeast cell have been measured. These curves were measured in both DI water and in PBS solution on the same yeast cell using the same AFM cantilever to get the reliable result. The contact region of these force curves in approach mode was then converted into force versus indentation curves. These curves would be fitted with Hertz-Sneddon model for the calculation of the elasticity. Analysis of the curves indicates that there is a difference of the Young's modulus values of the yeast cell in various environments. These data show that the salt buffer solution increases the rigidity of the biological system.

Measuring contact angle and meniscus shape with a reflected laser beam

Side-view imaging of the contact angle between an extended planar solid surface and a liquid is p... more Side-view imaging of the contact angle between an extended planar solid surface and a liquid is problematic. Even when aligning the view perfectly parallel to the contact line, focusing one point of the contact line is not possible. We describe a new measurement technique for determining contact angles with the reflection of a widened laser sheet on a moving contact line. We verified this new technique measuring the contact angle on a cylinder, rotating partially immersed in a liquid. A laser sheet is inclined under an angle ϕ to the unperturbed liquid surface and is reflected off the meniscus. Collected on a screen, the reflection image contains information to determine the contact angle. When dividing the laser sheet into an array of laser rays by placing a mesh into the beam path, the shape of the meniscus can be reconstructed from the reflection image. We verified the method by measuring the receding contact angle versus speed for aqueous cetyltrimethyl ammonium bromide solutions on a smooth hydrophobized as well as on a rough polystyrene surface.

$Figure 1. Schematic of the experimental setup. The cylinder was rotating in a bath filled with a liquid. A widened laser sheet with a wavelength of \ = 635 nm was inclined under an angle y to the liquid surface and was reflected on the cylinder (green dashed), the meniscus (red) and the unperturbed liquid surface (blue dotted). The reflection image was collected on a screen where the angle @ref; can be detected.$

$with the scaling parameter \ and the direction vector that depends on the array index 7. Here the last approx- imation is only valid for small opening angles of the laser sheet. € is the angle between a single ray and the x-axis. In further calculations we focus on one ray and omit the index ¢ for clarity. Details of the calculation can be found in the Appendix [A]$

Phototunable Response in Caged Polymer Brushes

A light-responsive brush was obtained by surface-initiated ATRP of a methacrylate monomer contain... more A light-responsive brush was obtained by surface-initiated ATRP of a methacrylate monomer containing ionizable −COOH side groups caged with the photoremovable group 4,5-dimethoxy-2-nitrobenzyl (NVOC). In the caged form, the polymer brush (PNVOCMA) is neutral and hydrophobic due to the presence of the aromatic chromophore. Upon irradiation the NVOC group is removed and a polyanion (polymethacrylic acid, PMAA) chain is generated. The charged brush can swell and collapse depending on the pH and the exposure dose (i.e., uncaging degree). The behavior and properties of the brush layer for different photoconversion degrees were studied. On the basis of quartz crystal microbalance measurements, a threshold of 50% uncaging was identified in order to achieve significant swelling and pH response of the brush. Between 50 and 80% the photoconversion the response of the brush could be light-modulated. For photoconversions >80% only small changes in the response were detectable. X-ray reflectivity (XRR) and scanning force microscopy allowed us to measure thickness, roughness and swelling of the brushes at intermediate photoconversions. Combined XRR and grazing-incidence small-angle scattering experiments evidenced a change in the internal structure of the brush upon exposure and indicated the occurrence of domain segregation as a consequence of the coexistence of hydrophobic and charged groups in the brush structure.

ARTICLE IN PRESS G Model Insights into the interaction of CD4 with anti-CD4 antibodies

Knowledge about the mechanism by which some antibodies can block HIV-1 entry is critical to our u... more Knowledge about the mechanism by which some antibodies can block HIV-1 entry is critical to our understanding of their function and may offer new avenues for controlling the adhesion of HIV-1 to the host cells. While much progress has been made, this mechanism remains unclear. Here, atomic force microscopy, isothermal titration calorimetry (ITC), and circular dichroism spectroscopy were used to measure some biophysical characteristics of the interaction of four-domains (D1–D4) membrane protein CD4 with anti-D3 antibody OKT4 and with HIV-1 entry blocking anti-D1 antibody Leu3a. The results showed that at 37 • C they bind with similar binding strength, thermodynamics, and kinetics but with different assembly states. Further analyzing the interactions at different temperatures by ITC showed that binding of CD4 with Leu3a is characteristic for specific hydrophobic binding as well as for protein folding while with OKT4 comes from an extensive additional hydration upon binding and charge-related interactions within the binding site. Comparing these characteristics with those of HIV-1 gp120-CD4 interaction revealed that Leu3a binds to CD4 faster than HIV-1 followed by changing local structure of D1 to which HIV-1 binds leading to a prevention of viral entry.

An improved measurement of dsDNA elasticity using AFM An improved measurement of dsDNA elasticity using AFM

The mechanical properties of a small fragment (30 bp) of an individual double-stranded deoxyribon... more The mechanical properties of a small fragment (30 bp) of an individual double-stranded deoxyribonucleic acid (dsDNA) in water have been investigated by atomic force microscopy (AFM). We have stretched three systems including ssDNA, double-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3-end and thiolated at the 5-end, this was reversed for the other complementary strand) and single-fixed dsDNA (one strand of the dsDNA molecules was biotinylated at the 3-end and thiolated at the 5-end, whereas the other complementary strand was biotinylated at only the 5-end). The achieved thiolation and biotinylation were to bind ds-or ssDNA to the gold surface and streptavidin-coated AFM tip, respectively. Analysis of the force versus displacement (F–D) curves from tip–DNA–substrate systems shows that the pull-off length (L o) and stretch length (δ) from the double-fixed system were shorter than those observed in the ssDNA and the single-fixed system. The obtained stretch force (F st) from the single-fixed dsDNA was much greater than that from the ssDNA even though it was about 10 pN greater than the one obtained in the double-fixed system. As a result, the Young's modulus of the double-fixed dsDNA was greater than that of the single-fixed dsDNA and the ssDNA. A more reliable stiffness of the dsDNA was observed via the double-fixed system, since there is no effect of the unpaired molecules during stretching, which always occurred in the single-fixed system. The unpaired molecules were also observed by comparing the stiffness of ssDNA and single-fixed dsDNA in which the end of one strand was left free.

Influence of surfactants in forced dynamic dewetting

In this work we show that the forced dynamic dewetting of surfactant solutions depends sensitivel... more In this work we show that the forced dynamic dewetting of surfactant solutions depends sensitively on the surfactant concentration. To measure this effect, a hydrophobic rotating cylinder was horizontally half immersed in aqueous surfactant solutions. Dynamic contact angles were measured optically by extrapolating the contour of the meniscus to the contact line. Anionic (sodium 1-decanesulfonate, S-1DeS), cationic (cetyl trimethylammonium bromide, CTAB) and nonionic surfactants (C 4 E 1 , C 8 E 3 and C 12 E 5) with critical micelle concentrations (CMCs) spanning four orders of magnitude were used. The receding contact angle in water decreased with increasing velocity. This decrease was strongly enhanced when adding surfactant, even at surfactant concentrations of 10% of the critical micelle concentration. Plots of the receding contact angle-versus-velocity almost superimpose when being plotted at the same relative concentration (concentration/ CMC). Thus the rescaled concentration is the dominating property for dynamic dewetting. The charge of the surfactants did not play a role, thus excluding electrostatic effects. The change in contact angle can be interpreted by local surface tension gradients, i.e. Marangoni stresses, close to the three-phase contact line. The decrease of dynamic contact angles with velocity follows two regimes. Despite the existence of Marangoni stresses close to the contact line, for a dewetting velocity above 1–10 mm s À1 the hydrodynamic theory is able to describe the experimental results for all surfactant concentrations. At slower velocities an additional steep decrease of the contact angle with velocity was observed. Particle tracking velocimetry showed that the flow profiles do not differ with and without surfactant on a scales 4100 mm.

Study on the Binding Force Between λ-DNA and Various Types of Substrates

Binding forces connecting bio-molecules to solid substrates have a strong influence on both atomi... more Binding forces connecting bio-molecules to solid substrates have a strong influence on both atomic force microcopy (AFM) imaging and force spectroscopy. We have investigated the binding forces between λ-DNA and several kinds of substrates, such as glass, mica, and Si, and observed some detailed features representing various binding states between them. The strongest binding was observed on the glass substrate silanized by 0.5 % APTES due to 'APTES bridges'. The interaction forces are in the range of ∼pN for DNA on mica and glass, and ∼nN for DNA on Si and APTES-treated glass substrates. In addition, the force-distance curve shows different features depending on the position of the cantilever tip on the DNA. The variation of the binding forces with the substrate materials and the measurement conditions will be discussed in detail.

$Fig. 2. Typical DNA images obtained before and after F-D curve measurement. F-D curve measurement was performec on both the tail and the train of \A-DNA. (a) This image was obtained from a A-DNA on a glass substrate. The insert was obtained after F-D measurement, and the change of the tai shape was observed. (b) Image of A-DNA on APTES/glas: substrate.$

Measuring Single Small Molecule Binding via Rupture Forces of a Split Aptamer

The rupture force of a split (bipartite) aptamer that forms binding pockets for adenosine monopho... more The rupture force of a split (bipartite) aptamer that forms binding pockets for adenosine monophosphate (AMP) was measured by atomic force spectroscopy. Changes in the rupture force were observed in the presence of AMP, while this effect was absent when mutant aptamers or inosine were used. Thus, changes in the rupture force were a direct consequence of specific binding of AMP to the split aptamer. The split aptamer concept allowed the detection of nonlabeled AMP and enabled us to determine the dissociation constant on a single-molecule level.

Nanomechanical measurement of astrocyte stiffness correlated with cytoskeletal maturation

by Huong Nguyen, Thi Nguyen, and Kyo Hwang

Astrocytes are known to serve as scaffolding cells that shape the brain. The physical properties ... more Astrocytes are known to serve as scaffolding cells that shape the brain. The physical properties of astrocytes, such as stiffness, are important for their scaffolding function. These properties may be altered in certain pathological conditions , such as in brain cancer. However, actual stiffness of astrocytes is not yet well understood. Here, we report that the astrocyte stiffness is positively correlated with the density of cytoskeletal proteins, such as actin filaments, microtu-bules, and intermediate filaments. The value of the stiffness of astrocytes as measured by atomic force microscopy (AFM) increases 38-fold in five-week-old rats compared to postnatal-day zero pups. Using multicolor confocal microscopy, we found that the complexity of cytoskeletal proteins, such as actin filaments, microtubules, and intermediate filaments, increase as the animal gets older. Our findings indicate that the change of stiffness positively correlates with the matura-tion of cytoskeletal proteins, and suggest that AFM can be useful as an analytical and diagnostic tool for neuroscience.

The role of CD4 on mechanical properties of live cell membrane

Although much progress has been made in the characterization and identification of CD4 functions,... more Although much progress has been made in the characterization and identification of CD4 functions, its role in mechanical properties of cell membrane remains largely unknown. Here an atomic force microscopy (AFM) was used to investigate the roles of CD4 in the elasticity of the leukemic human Jurkat (clone E6-1) cell membranes. Analysis of the approach force curves with Hertz model for a completely elastic soft sample measured on the selected CD41 and CD4-cells showed that CD41 cell membrane was softer than CD4-one. To confirm that CD4 plays a role in altering cell elasticity, human embryonic kidney 293T cells were transiently trans-fected with wild type (wt) CD4 plasmid before being used in AFM nanoindentation experiments. The results also demonstrated CD4-membrane was stiffer than CD41 one suggesting that CD4 integrated into plasma membrane and altered its mechanical properties. The study gives insights into the role of CD4 on cell membrane mechanical characteristics and might be helpful for development of cell biology and medicine. V C 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 239–244, 2016.

Single‐molecule force spectroscopy applied to heparin‐induced thrombocytopenia

Heparin‐induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiv... more Heparin‐induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiving the antithrombotic drug heparins, has a complex pathogenesis involving multiple partners ranging from small molecules to cells/platelets. Recently, insights into the mechanism of HIT have been achieved by using single‐molecule force spectroscopy (SMFS), a methodology that allows direct measurements of interactions among HIT partners. Here, the potential of SMFS in unraveling the mechanism of the initial steps in the pathogenesis of HIT at single‐molecule resolution is highlighted. The new findings ranging from the molecular binding strengths and kinetics to the determination of the boundary between risk and non‐risk heparin drugs or platelet‐surface and platelet‐platelet interactions will be reviewed. These novel results together have contributed to elucidate the mechanisms underlying HIT and demonstrate how SMFS can be applied to develop safer drugs with a reduced risk profile.

Quantitative description of thermodynamic and kinetic properties of the platelet factor 4/heparin bonds

Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetram... more Heparin is the most important antithrombotic drug in hospitals. It binds to the endogenous tetrameric protein platelet factor 4 (PF4) forming PF4/heparin complexes which may cause a severe immune-mediated adverse drug reaction, so-called heparin-induced thrombocytopenia (HIT). Although new heparin drugs have been synthesized to reduce such a risk, detailed bond dynamics of the PF4/heparin complexes have not been clearly understood. In this study, single molecule force spectroscopy (SMFS) is utilized to characterize the interaction of PF4 with heparins of defined length (5-, 6-, 8-, 12-, and 16-mers). Analysis of the force–distance curves shows that PF4/heparin binding strength rises with increasing heparin length. In addition, two binding pathways in the PF4/short heparins (≤8-mers) and three binding pathways in the PF4/long heparins (≥8-mers) are identified. We provide a model for the PF4/heparin complexes in which short heparins bind to one PF4 tetramer, while long heparins bind to two PF4 tetramers. We propose that the interaction between long heparins and PF4s is not only due to charge differences as generally assumed, but also due to hydrophobic interaction between two PF4s which are brought close to each other by long heparin. This complicated interaction induces PF4/heparin complexes more stable than other ligand–receptor interactions. Our results also reveal that the boundary between antigenic and non-antigenic heparins is between 8-and 12-mers. These observations are particularly important to understand processes in which PF4–heparin interactions are involved and to develop new heparin-derived drugs.

Rupture Forces among Human Blood Platelets at different Degrees of Activation

by Huong Nguyen and Thi-Huong Nguyen

Little is known about mechanics underlying the interaction among platelets during activation and ... more Little is known about mechanics underlying the interaction among platelets during activation and aggregation. Although the strength of a blood thrombus has likely major biological importance, no previous study has measured directly the adhesion forces of single platelet-platelet interaction at different activation states. Here, we filled this void first, by minimizing surface mediated platelet-activation and second, by generating a strong adhesion force between a single platelet and an AFM cantilever, preventing early platelet detachment. We applied our setup to measure rupture forces between two platelets using different platelet activation states, and blockade of platelet receptors. The rupture force was found to increase proportionally to the degree of platelet activation, but reduced with blockade of specific platelet receptors. Quantification of single platelet-platelet interaction provides major perspectives for testing and improving biocompatibility of new materials; quantifying the effect of drugs on platelet function; and assessing the mechanical characteristics of acquired/inherited platelet defects. Blood platelets are discoidal, anuclear cell fragments, 1–2 μ m in size and are produced from the cytoplasm of bone marrow megakaryocytes 1. They prevent blood loss upon vascular injury 2 , can induce arterial thrombosis 3 , and have additional roles in immune defense 4 , wound healing 5 , and cancer metastasis 6,7. In vivo, platelets do not normally interact with the inner surfaces of blood vessels, but they adhere promptly when the vessel wall is altered. Several glycoproteins (GPs) on the platelet surface interact with extracellular matrix proteins on the vessel wall (e.g. GPIbIX with von Willebrand factor [vWF], GPIaIIa and GPVI with collagen) leading to platelet activation 1. Activated platelets expose GPIIbIIIa (α IIbβ 3) in an activated form, which allows binding of fibrinogen 8,9 and triggers platelet activation and aggregation 10. In parallel, activated platelets release prothrombotic substances from their granules 11. Some of these substances recruit and activate neighboring platelets, while others enhance thrombin generation together with plasma clotting factors. Finally, this leads to formation of a hemostatic plug at the site of endothelial damage which eventually results in blood vessel closure 12,13. An increasingly important issue for biotechnological applications in medicine is modulation of platelet activation on various surfaces 14,15. However, these processes are not well understood as platelets are difficult to handle because they activate immediately after short contact with non-physiological surfaces 16–18 , at elevated shear stress 19 and upon air contact 20. Among these, platelet activation on surfaces is not only a major drawback for microfluidic devices 21 , micro-and nano-particulate drug delivery systems 22,23 , and intravascular devices 24 , but it is also essential for developing tools for direct measuring of platelet mechanics 25,26. Platelet-surface interactions have been quantified in real-time using calcium mobilization assays, showing that the lag time until platelets activate depends on the physico-chemical properties of the surface 27. It has been shown that platelets are activated nearly instantaneously upon coming in contact with immobilized fibrinogen and completing their contraction after 15 min 28. Others have provided information regarding morphology 17,18,29 , and elastic moduli 29–31 of activated platelets under physiological conditions. Although these studies have successfully identified significant characteristics of platelets on a single molecular level, none of these previous studies describes quantitatively single platelet-platelet interactions, neither in activated, nor in non-/weakly activated states.