Immobilization of proteins on carboxylic acid functionalized nanopatterns

The immobilization of proteins on nanopatterned surfaces was investigated using in situ atomic force microscopy (AFM) and ex situ infrared reflectance–absorption spectroscopy (IRAS). The AFM-based lithography technique of nanografting provided control of the size, geometry, and spatial placement of nanopatterns within self-assembled monolayers (SAMs). Square nanopatterns of carboxylate-terminated SAMs were inscribed within methyl-terminated octadecanethiolate SAMs and activated using carbodiimide/succinimide coupling chemistry. Staphylococcal protein A was immobilized on the activated nanopatterns before exposure to rabbit immunoglobulin G. In situ AFM was used to monitor changes in the topography and friction of the nanopatterns in solution upon protein immobilization. Complementary studies with ex situ IRAS confirmed the surface chemistry that occurred during the steps of SAM activation and subsequent protein immobilization on unpatterned samples. Since carbodiimide/succinimide coupling chemistry can be used for surface attachment of different biomolecules, this protocol shows promise for development of other aqueous-based studies for nanopatterned protein immobilization.     

Hybridization with nanostructures of single-stranded DNA

Nanostructures of single-stranded DNA (ssDNA) were produced within alkanethiol self-assembled monolayers using nanografting, an atomic force microscopy (AFM) based lithography technique. Next, variations of the fabrication parameters, such as the concentration of ssDNA or lines per frame, allowed for the regulation of the density of ssDNA molecules within the nanostructures. The label-free hybridization of nanostructures, monitored using high-resolution AFM imaging, has proven to be highly selective and sensitive; as few as 50 molecules can be detected. The efficiency of the hybridization reaction at the nanometer scale highly depends on the ssDNA packing density within the nanostructures. This investigation provides a fundamental step toward sensitive DNA detection and construction of complex DNA architectures on surfaces.     

Nanografting versus solution self-assembly of alpha,omega-alkanedithiols on Au(111) investigated by AFM

The solution self-assembly of alpha,omega-alkanedithiols onto Au(111) was investigated using atomic force microscopy (AFM). A heterogeneous surface morphology is apparent for 1,8-octanedithiol and for 1,9-nonanedithiol self-assembled monolayers (SAMs) prepared by solution immersion as compared to methyl-terminated n-alkanethiols. Local views from AFM images reveal a layer of mixed molecular orientations for alpha,omega-alkanedithiols, which evidence surface structures with heights corresponding to both lying-down and standing-up orientations. For dithiol SAMs prepared by solution self-assembly, the majority of alpha,omega-alkanedithiol molecules chemisorb with both thiol end groups bound to the Au(111) surface with the backbone of the alkane chain aligned parallel to the surface. However, AFM images disclose that there are also islands of standing molecules scattered throughout the surface. To measure the thickness of alpha,omega-alkanedithiol SAMs with angstrom sensitivity, methyl-terminated n-alkanethiols with known dimensions were used as molecular rulers. Under conditions of spatially constrained self-assembly, nanopatterns of alpha,omega-alkanedithiols written by nanografting formed monolayers with heights corresponding to an upright configuration.     

Nanografting of alkanethiols by tapping mode atomic force microscopy

Nanografting, an atomic force microscopy (AFM) based nanolithography technique, is becoming a popular method for patterning self-assembled monolayers (SAMs). In this technique, a nanoscale patch of a thiol-on-gold SAM is exchanged with a different thiol by the action of an AFM tip operated in contact mode at high load. The results are then imaged in topographic or lateral force microscopy again at low values of the load. One of the problems of contact mode nanografting is that monolayers of large molecules such as proteins are likely to be deformed, damaged, or even removed from the surface by contact mode imaging even when small loads are used. Furthermore, we need to note that the stiffness of the cantilevers used in contact mode is different than that of the cantilevers used in tapping mode and that tip changing in the course of an experiment can be quite inconvenient. Here, we show that a monolayer on a gold substrate can be nanografted using tapping mode AFM (also referred to as amplitude modulation AFM) rather than the commonly used contact mode. While the grafting parameters are somewhat trickier to choose, the results demonstrate that nanografting in tapping mode can make patches of the same quality as those made by contact mode, therefore allowing for gentle imaging of the grafted molecules and the whole SAM without changing the microscope tip.     

Atomic force microscopy (AFM)-based nanografting for the study of self-assembled monolayer formation of organophosphonic acids on Al2O3 single-crystal surfaces

Adsorption, stability, and organization kinetics of organophosphonic acids on single-crystalline alumina surfaces were investigated by means of atomic force microscopy (AFM)-based imaging, nanoshaving, and nanografting. AFM friction and phase imaging have shown that chemical etching and subsequent annealing led to heterogeneities on single-crystalline surfaces with (0001) orientation. Self-assembly and stability of octadecylphosphonic acid (ODPA) were shown to be strictly dependent upon the observed heterogeneities of the surface termination, where it was locally shown that ODPA can loosely or strongly bind on different terminations of the crystal surface. Organization kinetics of ODPA was monitored with nanografting on (0001) surfaces. Supported by measurements of surface wettability and diffuse reflectance infrared Fourier transform spectroscopy (DRIFTS), it was demonstrated that the lack of organization within the protective adsorbed hexylphosphonic acid (HPA) monolayer on alumina surfaces facilitated the reduced confinement effect during nanografting, such that kinetics information on the organization process of ODPA could be obtained.     

Viscoelastic modeling of highly hydrated laminin layers at homogeneous and nanostructured surfaces: quantification of protein layer properties using QCM-D and SPR

The adsorption of proteins at material surfaces is important in applications such as biomaterials, drug delivery, and diagnostics. The interaction of cells with artificial surfaces is mediated through adsorbed proteins, where the type of protein, amount, orientation, and conformation are of consequence for the cell response. Laminin, an important cell adhesive protein that is central in developmental biology, is studied by a combination of quartz crystal microbalance with dissipation (QCM-D) and surface plasmon resonance (SPR) to characterize the adsorption of laminin on surfaces of different surface chemistries. The combination of these two techniques allows for the determination of the thickness and effective density of the protein layer as well as the adsorbed mass and viscoelastic properties. We also evaluate the capacity of QCM-D to be used as a quantitative technique on a nanostructured surface, where protein is adsorbed specifically in a nanopattern exploiting PLL-g-PEG as a protein-resistant background. We show that laminin forms a highly hydrated protein layer with different characteristics depending on the underlying substrate. Using a combination of QCM-D and atomic force microscopy (AFM) data from nanostructured surfaces, we model laminin and antibody binding to nanometer-scale patches. A higher amount of laminin was found to adsorb in a thicker layer of a lower effective density in nanopatches compared to equivalent homogeneous surfaces. These results suggest that modeling of QCM-D data of soft viscoelastic layers arranged in nanopatterns may be applied where an independent measure of the "dry" mass is known.     

Friction and adhesion between C60 single crystal surfaces and AFM tips: effects of the orientational phase transition

We have investigated the nanotribological properties of C60 single crystal (111) and (100) surfaces around its orientational order-disorder phase transition temperature, approximately 260 K, by atomic force microscopy and frictional force microscopy (AFM/FFM) in high vacuum. Results show that for both surfaces across the phase transition temperature, the friction force and the adhesive force between a C60 coated AFM tip and the C60 crystal surfaces exhibit discontinuous behavior. The friction force within the applied external load range in the low temperature phase is significantly larger than that in the high temperature phase, with no obvious change in the slope of the friction force curves (the friction coefficient) in the low and high temperature phases. The abrupt change in friction was found to be caused mainly by the abrupt change in adhesion, which, in turn, can be qualitatively understood through changes in the van der Waals interaction and the short-range Coulomb interaction associated with the structural changes across the phase transition. Compared to most other degrees of freedom, the rotation of C60 molecules was found to have little effect on friction and is an ineffective energy dissipation channel.     

Comparative study of the adhesion, friction, and mechanical properties of CF3- and CH3-terminated alkanethiol monolayers

We report the results of a direct comparison of the adhesion, friction, and mechanical properties between alkanethiol self-assembled monolayer films terminated by either CH(3) or CF(3) end groups using both interfacial force (IFM) and atomic force (AFM) microscopies. The purpose of this work is to gain insight into the detailed origins of the differing frictional behavior previously observed with AFM. The IFM results reveal an increased adhesive interaction for the CF(3)-terminated film due to the highly polar nature of the end groups. In agreement with earlier studies, the AFM results show two linear regions with differing frictional slopes for the CH(3)-terminated film but only a single slope for the CF(3)-terminated film. We contrast the differences between these techniques, approximately 100 times smaller tips for the AFM, and discuss the role of the mechanical properties, the increased adhesive interaction, and the amount of disorder present in the film in creating differences in frictional behavior between the two systems. We conclude that increased adhesion for the CF(3)-terminated film plays an important role in the observed differences in frictional behavior, while the differences between the two techniques can be traced to the different tip sizes and the consequent responses to the presence of disorder in the films.     

Investigating and characterizing the binding activity of the immobilized calmodulin to calmodulin-dependent protein kinase I binding domain with atomic force microscopy

Protein–protein interactions are responsible for many biological processes, and the study of how proteins undergo a conformational change induced by other proteins in the immobilized state can help us to understand a protein’s function and behavior, empower the current knowledge on molecular etiology of disease, as well as the discovery of putative protein targets of therapeutic interest. In this study, a bottom-up approach was utilized to fabricate micro/nanometer-scale protein patterns. One cysteine mutated calmodulin (CaM), as a model protein, was immobilized on thiol-terminated pattern surfaces. Atomic Force Microscopy (AFM) was then employed as a tool to investigate the interactions between CaM and CaM kinase I binding domain, and show that the immobilized CaM retains its activity to interact with its target protein. Our work demonstrate the potential of employing AFM to the research and assay works evolving surface-based protein–protein interactions biosensors, bioelectronics or drug screening.     

DNA as invisible ink for AFM nanolithography

We have used nanografting, an atomic force microscopy (AFM)-based nanolithography technique, to fabricate thiolated DNA nanostructures on gold surfaces. The tip-guided assembly offers opportunities for locally controlling the packing order, density, and thus the thickness of the DNA patterns. By selecting proper nanografting parameters, we can embed single-stranded DNA (ssDNA) patches into a background composed of the same DNA molecule prepared by self-assembly, in which the patches remain topographically (and chemically) invisible but have much improved packing order. When the complementary DNA (cDNA) is added, the thickness of the nanografted layer increases much more dramatically than that of the self-assembled layer during the hybridization process, and as a result, the pattern emerges. Interestingly, the pattern can be reversibly hidden and shown with high fidelity simply by dehybridizing and appending the cDNA repeatedly.     

A modular nanoparticle-based system for reagentless small molecule biosensing

Metalloprotein tethered CdSe nanoparticles have been generated to provide selective and reagentless maltose biosensing. As opposed to cell or protein detection by semiconducting nanoparticle bioconjugates, a modular method for small-molecule detection using semiconducting nanoparticle bioconjugates has been difficult. Here we report a method for reagentless protein-based semiconducting nanoparticle biosensors. This method uses Ru(II) complex-CdSe nanoparticle interactions and the maltose-induced conformation changes of maltose binding protein to alter the CdSe nanoparticle fluorescence emission intensity. In this proof-of-principle system, the maltose-induced protein conformation changes alter the Ru(II) complex-CdSe nanoparticle interaction, which increases the CdSe emission intensity. Altered CdSe emission intensity effects are best described as electron transfer from the Ru(II) complex to the CdSe excited state forming the nonfluorescent CdSe anion. Four surface-cysteine, Ru(II) complex-attached maltose-binding proteins have been studied for maltose dependent alteration of CdSe emission intensities. With 3.0-3.5 nm diameter CdSe nanoparticles, all ruthenated maltose-binding proteins display similar maltose-dependent increases (1.4-fold) in CdSe emission intensity and maltose binding affinities (KA = 3 x 106 M-1). For these four systems, the only difference was the sample-to-sample variation in maltose-dependent responses. Thus, very few surface cysteine mutations need to be examined to find a successful biosensor, as opposed to analogous systems using organic fluorophores. This strategy generates a unimolecular, or reagentless, semiconducting nanoparticle biosensor for maltose, which could be applied to other proteins with ligand-dependent conformation changes.     

Effect of protein solution components in the adsorption of Herbaspirillum seropedicae GlnB protein on mica

The adsorption of proteins and its buffer solution on mica surfaces was investigated by atomic force microscopy (AFM). Different salt concentration of the Herbaspirillum seropedicae GlnB protein (GlnB-Hs) solution deposited on mica was investigated. This protein is a globular, soluble homotrimer (36 kDa), member of PII-like proteins family involved in signal transducing in prokaryote. Supramolecular structures were formed when this protein was deposited onto bare mica surface. The topographic AFM images of the GlnB-Hs films showed that at high salt concentration the supramolecular structures are spherical-like, instead of the typical doughnut-like shape for low salt concentration. AFM images of NaCl and Tris from the buffer solution showed structures with the same pattern as those observed for high salt protein solution, misleading the image interpretation. XPS experiments showed that GlnB protein film covers the mica surface without chemical reaction.     

Dependence of the Nanoscale Composite Morphology of Fe3O4 Nanoparticle-Infused Lysozyme Amyloid Fibrils on Timing of Infusion: A Combined SAXS and AFM Study

Understanding the formation process and the spatial distribution of nanoparticle (NP) clusters on amyloid fibrils is an essential step for the development of NP-based methods to inhibit aggregation of amyloidal proteins or reverse the assembling trend of the proto-fibrillary complexes that prompts pathogenesis of neuro degeneration. For this, a detailed structural determination of the diverse hybrid assemblies that are forming is needed, which can be achieved by advanced X-ray scattering techniques. Using a combined solution small angle X-ray scattering (SAXS) and atomic force microscopy (AFM) approach, this study investigates the intrinsic trends of the interaction between lysozyme amyloid fibrils (LAFs) and Fe₃O₄ NPs before and after fibrillization at nanometer resolution. AFM images reveal that the number of NP clusters interacting with the lysozyme fibers does not increase significantly with NP volume concentration, suggesting a saturation in NP aggregation on the fibrillary surface. The data indicate that the number of non-adsorbed Fe₃O₄ NPs is highly dependent on the timing of NP infusion within the synthesis process. SAXS data yield access to the spatial distribution, aggregation manner and density of NP clusters on the fibrillary surfaces. Employing modern data analysis approaches, the shape and internal structural morphology of the so formed nanocomposites are revealed. The combined experimental approach suggests that while Fe₃O₄ NPs infusion does not prevent the fibril-formation, the variation of NP concentration and size at different stages of the fibrillization process can impose a pronounced impact on the superficial and internal structural morphologies of these nanocomposites. These findings may be applicable in devising advanced therapeutic treatments for neurodegenerative diseases and designing novel bio-inorganic magnetic devices. Our results further demonstrate that modern X-ray methods give access to the structure of—and insight into the formation process of—biological–inorganic hybrid structures in solution.     

Frictional drag and electrical manipulation of recombinant proteins in polymer-supported membranes

We establish a lipid monolayer supported by a polymer interface that offers advantages over conventional solid-supported membranes for determining the frictional drag at the membrane-protein interface as well as for electric field manipulation of membrane-anchored proteins. Polymer-supported monolayers with functional lipid anchors allow for the specific docking of His-tagged green fluorescent protein variants (His-EGFP and His-DsRed tetramer) onto the membrane surface at a defined surface density. In the first part, we measure the lateral diffusion coefficients of lipids and proteins and calculate the frictional drag at the protein-membrane interface. The second part deals with the electric field-induced accumulation of recombinant proteins on a patterned surface. The mean drift velocity of proteins, which can be obtained analytically from the shape of the steady-state concentration gradient, can be controlled by tuning the interplay of electrophoresis and electroosmosis. The results demonstrate the potential of such molecular constructs for the local functionalization of solid substrates with membrane-associated proteins.     

Systematic manipulation of surface chemical reaction on the nanoscale: a novel approach for constructing three-dimensional nanostructures

Nanoscale patches, created by nanografting a maleimide-terminated thiol into a self-assembled monolayer, were elaborated by sequential chemical reactions. Each stage in the nanofabrication was followed by atomic force microscopy (AFM), providing a controlled approach to the fabrication of novel three-dimensional (3D) surface nanostructures.     

Interaction forces measured using AFM between colloids and surfaces coated with both dextran and protein

Both proteins and polysaccharides are biopolymers present on a bacterial surface that can simultaneously affect bacterial adhesion. To better understand how the combined presence of proteins and polysaccharides might influence bacterial attachment, adhesion forces were examined using atomic force microscopy (AFM) between colloids (COOH- or protein-coated) and polymer-coated surfaces (BSA, lysozyme, dextran, BSA+dextran and lysozyme+dextran) as a function of residence time and ionic strength. Protein and dextran were competitively covalently bonded onto glass surfaces, forming a coating that was 22-33% protein and 68-77% dextran. Topographic and phase images of polymer-coated surfaces obtained with tapping mode AFM indicated that proteins at short residence times (<1 s) were shielded by dextran. Adhesion forces measured between colloid and polymer-coated surfaces at short residence times increased in the order protein+dextran < or = protein < dextran. However, the adhesion forces for protein+dextran-coated surface substantially increased with longer residence times, producing the largest adhesion forces between polymer coated surfaces and the colloid over the longest residence times (50-100 s). It was speculated that with longer interaction times the proteins extended out from beneath the dextran and interacted with the colloid, leading to a molecular rearrangement that increased the overall adhesion force. These results show the importance of examining the effect of the combined adhesion force with two different types of biopolymers present and how the time of interaction affects the magnitude of the force obtained with two-polymer-coated surfaces.     

原子力显微镜在蛋白单分子结构与功能研究中的应用

原子力显微镜(AFM)以其超常的信噪比、空间分辨率和灵活的探测环境使得单个蛋白分子能在生理条件下成像,在蛋白单分子结构与功能研究中得到广泛地应用。论文介绍了AFM在分子马达、光合蛋白、分子伴侣等蛋白表面结构表征中的应用;AFM在蛋白单分子表面的粘弹性、电荷分布、分子间相互作用等物理属性研究中的进展;总结了AFM在蛋白分子功能研究和单分子操纵中的应用。     

Nanografting: modeling and simulation

We present a simple phenomenological model of the nanografting process with an emphasis on the formation of binary self-assembled monolayers. This model includes dynamical processes that are involved in natural growth experiments, including molecular deposition, surface diffusion, and the phase transition from physisorption to chemisorption, and we show that it predicts domain formation in ungrafted deposition that matches experiment. The one-order-of-magnitude faster kinetics that is found in the nanografting experiments compared to natural self-assembly (or unconstrained self-assembly) is described with a key assumption that the deposition rate is greatly enhanced in the small region confined between the back side of the AFM tip and the edge of the previously deposited self-assembled monolayer. Monte Carlo simulations based on this model reproduce experimental observations concerning the variation of SAM heterogeneity with AFM tip speed. Our simulations demonstrate that the faster the AFM tip displaces adsorbed molecules in a monolayer, the more heterogeneous are the monolayers formed behind the tip, as this allows space and time for the formation of phase-segregated domains.     

A nanoengineering approach to regulate the lateral heterogeneity of self-assembled monolayers

Using a scanning probe lithography method known as nanografting in conjunction with knowledge of self-assembly chemistry, regulation of the heterogeneity of self-assembled monolayers (SAMs) is demonstrated. While nanografting in single-component thiols produces areas of SAMs with designed geometry and size, nanofabrication in mixed thiol solution yields segregated domains. The reaction mechanism in nanografting differs significantly from self-assembly in mix-and-grow methods, as proven in systematic studies reported in this article and a companion paper of theoretical calculations of the nanografting process. Knowledge of the reaction pathways enables development of methods for shifting the interplay between the kinetics and thermodynamics in SAM formation, and thus the heterogeneity of mixed SAMs. By varying fabrication parameters, such as shaving speed, and reaction conditions, such as concentration and ratio of the components, the lateral heterogeneity can be adjusted ranging from near molecular mixing to segregated domains of several to tens of nanometers.     

AFM studies of inhibition effect in binding of antimicrobial peptide and immune proteins

By using atomic force microscopy (AFM), we clearly show that the antimicrobial peptide affects the molecular interaction between lipopolysaccharide (LPS) and immune proteins (lipopolysaccharide binding protein [LBP] and CD14). To reconstruct an in vivo interaction, LBP and LPS (the Ra, Rc, and Re forms from Salmonella minnesota, with varying lengths of the saccharide region) were immobilized onto the AFM tip using a chemical spacer linker. We examined the interaction between the proteins on the tip and model lipid bilayer biomembranes including CD14, in both the presence and absence of the antimicrobial peptide, polymyxin B (PMB). When LPS was present, the binding force between the LBP-LPS complex and CD14 increased dramatically, compared to that seen between LBP and CD14 alone. Longer LPS saccharide regions resulted in higher binding forces. The data suggest that LPS may have an important influence on the binding of LBP to CD14 and that the saccharide region of LPS is influential in this regard. It was also found that the antimicrobial peptide PMB, at or above a particular concentration, specifically inhibited the binding between LBP-LPS and CD14.