Atomic Force Microscopy Substrate Research Articles

Predicting the placement of biomolecular structures on AFM substrates based on electrostatic interactions

Atomic force microscopy (AFM) and high-speed AFM allow direct observation of biomolecular structures and their functional dynamics. Based on scanning the molecular surface of a sample deposited on a supporting substrate by a probing tip, topographic images of its dynamic shape are obtained. Critical to successful AFM observations is a balance between immobilization of the sample while avoiding too strong perturbations of its functional conformational dynamics. Since the sample placement on the supporting substrate cannot be directly controlled in experiments, the relative orientation is a priori unknown, and, due to limitations in the spatial resolution of images, difficult to infer from a posteriori analysis, thus hampering the interpretation of measurements. We present a method to predict the macromolecular placement of samples based on electrostatic interactions with the AFM substrate and demonstrate applications to HS-AFM observations of the Cas9 endonuclease, an aptamer-protein complex, the Monalysin protein, and the ClpB molecular chaperone. The model also allows predictions of imaging stability taking into account buffer conditions. We implemented the developed method within the freely available BioAFMviewer software package. Predictions based on available structural data can therefore be made even prior to an actual experiment, and the method can be applied for post-experimental analysis of AFM imaging data.

Atomic Force Microscopy Study of the Effect of an Electric Field, Applied to a Pyramidal Structure, on Enzyme Biomolecules.

The influence of an external constant strong electric field, formed using a pyramidal structure under a high electric potential, on an enzyme located near its apex, is studied. Horseradish peroxidase (HRP) is used as a model. In our experiments, a 27 kV direct current (DC) voltage was applied to two electrodes with a conducting pyramidal structure attached to one of them. The enzyme particles were visualized by atomic force microscopy (AFM) after the adsorption of the enzyme from its 0.1 µM solution onto mica AFM substrates. It is demonstrated that after the 40 min exposure to the electric field, the enzyme forms extended structures on mica, while in control experiments compact HRP particles are observed. After the exposure to the electric field, the majority of mica-adsorbed HRP particles had a height of 1.2 nm (as opposed to 1.0 nm in the case of control experiments), and the contribution of higher (>2.0 nm) particles was also considerable. This indicates the formation of high-order HRP aggregates under the influence of an applied electric field. At that, the enzymatic activity of HRP against its substrate 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) remains unaffected. These results are important for studying macroscopic effects of strong electromagnetic fields on enzymes, as well as for the development of cellular structure models.

An Improved Substrate for Superior Imaging of Individual Biomacromolecules with Atomic Force Microscopy

High-resolution atomic force microscopy (AFM) of biomacromolecules is a valuable method for structural studies in biology. Traditionally, the surfaces used for AFM imaging of individual molecules are limited to mica, graphite, and glass. Because these substrates have certain shortcomings, new or modified surfaces that improve the quality of AFM imaging are highly desirable. Here, we describe an improved substrate for imaging of individual biomacromolecules with high-resolution AFM based on graphite surfaces coated by physical adsorption. We provide a detailed methodology, including the chemical structure, synthesis, characterization and the use of a substance that modifies the surface of freshly cleaved graphite, making it suitable for adsorption and AFM visualization of various biomacromolecules while minimizing spatial distortions. We illustrate the advantages of the modified graphite over regular surfaces with examples of high-resolution single-molecule imaging of proteins, polysaccharides, DNA and DNA-protein complexes. The proposed methodology is easy to use and helps to improve substantially AFM imaging of biomacromolecules of various natures, including flexible and/or unstructured sub-molecular regions that are not seen on other AFM substrates. The proposed technique has the potential to improve the use of AFM in structural biology for visualization and morphometric characterization of macromolecular objects.

Preparation of a Si(111) Atomically Flat Substrate via Wet Etching and Evaluation as an AFM Substrate for Observations of Isolated Chains, Crystals, and Crystallization of Isotactic Poly(methyl methacrylate) at the Molecular Level.

To observe a polymer chain deposited on a substrate by atomic force microscopy (AFM) at the molecular level, the substrate should be atomically flat and stable under laboratory conditions and adsorb polymer chains firmly. Therefore, substrates used under laboratory conditions are practically limited to mica, highly ordered pyrolytic graphite, and atomically stepped sapphire, and polymers observed by AFM at the molecular level are also limited. A silicon wafer is frequently used as a substrate for AFM observation for somewhat macroscopic observations, but the surface of the silicon wafer is too rough to observe polymer chains deposited on it at the molecular level. In this study, we prepared an atomically stepped Si(111) substrate via wet etching in NH4F and evaluated it as an AFM substrate. The Si(111) substrate was stable as an AFM substrate, and isolated poly(methyl methacrylate) (it-PMMA) chains and a crystalline monolayer deposited on the substrate were observed by AFM at the molecular level. An it-PMMA amorphous monolayer deposited on mica crystallized under high humidity, but that on the Si(111) substrate did not because of the difference in the surface nature and the crystal structure of the substrates. The Si(111) substrate was hydrophobic, and the it-PMMA monolayers could be deposited as a multilayer, which could not be formed on hydrophilic mica. The crystallization behavior of an it-PMMA amorphous multilayer and an amorphous/crystalline mixed multilayer on the Si(111) substrate was also evaluated.

AFM Imaging of Protein Aggregation in Studying the Impact of Knotted Electromagnetic Field on A Peroxidase

The phenomenon of knotted electromagnetic field (KEMF) is now actively studied, as such fields are characterized by a nontrivial topology. The research in this field is mainly aimed at technical applications – for instance, the development of efficient communication systems. Until present, however, the influence of KEMF on biological objects (including enzyme systems) was not considered. Herein, we have studied the influence of KEMF on the aggregation and enzymatic activity of a protein with the example of horseradish peroxidase (HRP). The test HRP solution was irradiated in KEMF (the radiation power density was 10−12 W/cm2 at 2.3 GHz frequency) for 40 min. After the irradiation, the aggregation of HRP was examined by atomic force microscopy (AFM) at the single-molecule level. The enzymatic activity was monitored by conventional spectrophotometry. It has been demonstrated that an increased aggregation of HRP, adsorbed on the AFM substrate surface, was observed after irradiation of the protein sample in KEMF with low (10−12 W/cm2) radiation power density; at the same time, the enzymatic activity remained unchanged. The results obtained herein can be used in the development of models describing the interaction of enzymes with electromagnetic field. The obtained data can also be of importance considering possible pathological factors that can take place upon the influence of KEMF on biological objects— for instance, changes in hemodynamics due to increased protein aggregation are possible; the functionality of protein complexes can also be affected by aggregation of their protein subunits. These effects should also be taken into account in the development of novel highly sensitive systems for human serological diagnostics of breast cancer, prostate cancer, brain cancer and other oncological pathologies, and for diagnostics of diseases in animals, and crops.

Probing the surface ultrastructure of Brevibacillus laterosporus using atomic force microscopy

One of the main obstacles to studying the surface ultrastructure of microbial cells by atomic force microscopy (AFM) is determining how to immobilize live cells on the AFM substrates. Each method has its own advantages and disadvantages. The aim of this study was to characterize a new simple and inexpensive method using two types of polyethersulfone (PES) membrane filters that differ in pore size (micropore and nanopore) to immobilize live and dead Brevibacillus laterosporus for AFM imaging. B. laterosporus was easily trapped by the microporous PES membrane, facilitating the successful AFM scanning of the bacterial surface ultrastructure. In addition, B. laterosporus strongly attached to the nanoporous membranes and withstood the pulling forces exerted by the AFM tip during scanning. These methods of immobilization did not affect the cell viability. The nanostructure and roughness of the bacterial surface were also observed for live, fixed, and air-dried cells. Live and dead bacteria displayed similar morphologies at low resolution, while at high resolution, live bacteria displayed a more convoluted surface ("brain-like structure").

DNA Sequence Mediates Apparent Length Distribution in Single-Walled Carbon Nanotubes.

Single-walled carbon nanotubes (SWCNTs) functionalized with short single-stranded DNA have been extensively studied within the last decade for biomedical applications due to the high dispersion efficiency and intrinsic biocompatibility of DNA as well as the photostable and tunable fluorescence of SWCNTs. Characterization of their physical properties, particularly their length distribution, is of great importance regarding their application as a bioengineered research tool and clinical diagnostic agent. Conventionally, atomic force microscopy (AFM) has been used to quantify the length of DNA-SWCNTs by depositing the hybrids onto an electrostatically charged flat surface. Here, we demonstrate that hybrids of DNA-SWCNTs with different oligomeric DNA sequences ((GT)6 and (GT)30) differentially deposit on the AFM substrate, resulting in significant inaccuracies in the reported length distributions of the parent solutions. Using a solution-based surfactant exchange technique, we placed both samples into a common surfactant wrapping and found identical SWCNT length distributions upon surface deposition. Additionally, by spin-coating the surfactant-wrapped SWCNTs onto a substrate, thus mitigating effects of electrostatic interactions, we found length distributions that did not depend on DNA sequence but were significantly longer than electrostatic deposition methods, illuminating the inherent bias of the surface deposition method. Quantifying the coverage of DNA molecules on each SWCNT through both absorbance spectroscopy and direct observation, we found that the density of DNA per SWCNT was significantly higher in short (GT)6-SWCNTs (length < 100 nm) compared to long (GT)6-SWCNTs (length > 100 nm). In contrast, we found no dependence of the DNA density on SWCNT length in (GT)30-SWCNT hybrids. Thus, we attribute differences in the observed length distributions of DNA-SWCNTs to variations in electrostatic repulsion induced by sequence-dependent DNA density.

The Influence of Anions on the Release of Ag(I) Ions from Silver Nanoparticles (AgNPs)

Silver nanoparticles (AgNPs) are widely being incorporated into products such as socks [1, 2], sports wears and wound dressings [3, 4] to serve as antibacterials and to inhibit unwanted odours. As these products are washed, the silver nanoparticles leach out, dispersing in water where they may release silver ions. Although the only contraindication of high levels of silver in humans is argyria [5], it is said to be very toxic to aquatic organisms that make up the base of a series of food chains. In this study, silver nanoparticles were synthesised by the citrate reduction method following the procedure of Lee and Meisel [6]. The nanoparticles were prepared in the presence of various anions and in pure water in order to examine the effect of the medium on the release of Ag(I). AgNPs were characterised by UV-Vis spectrometry, dynamic light scattering (DLS) and atomic force microscopy (AFM). The decrease in surface plasmon resonance (SPR) peak of the nanoparticles after dialysis indicates that quite a number of conduction electrons have gone into reaction, hence lowering of resonant frequency. The diameters of the nanoparticles as measured by DLS ranged from 102 nm in the 10 mM chloride AgNPs before dialysis to 892 nm in the 50 mM sample after dialysis, indicating aggregation over the dialysis period. On the other hand, particle sizes in terms of both height and diameter as measured by AFM decreased after dialysis. This could be explained that in spite of aggregation in solution, the AgNPs deposit as islands about 1 particle in height on the AFM substrate (Si wafer). Release of Ag(I) from the NPs was monitored by anodic stripping voltammetry at glassy carbon electrodes. Two interesting observations were made: (i) there is substantial (90 mM) Ag(I) present in the initial NP preparation and (ii) the concentration of Ag(I) after 73 h of dialysis remained easily detectable at about 4 µM irrespective of the concentration of chloride present. References 1 T.M. Benn, P.Westerhoff. Environ. Sci. Technol. 2008, 42, 4133-4139. 2 D.E Meyer, M.A. Curran, M.A. Gonzalez. J Nanopart Res. 2011, 13, 147-156. 3 A. Hebeish, M.H. El-Rafie, M.A. El-Sheikh, A.A. Saleem, M.E. El-Naggar. Intnal. Journ. of Macromolecules. 2014, 65, 509-515. 4 T. Maneerung, S. Tokura, R. Rujiavanit. Carbohydrate Polymers. 2008, 72, 43-51. 5 WHO Guidelines for Drinking-water quality. 2003. World Health Organisation, Geneva. 6 P.C. Lee, D. Meisel. J. Phys. Chem. 1982, 86, 3391-3395. Figure 1

A Flexible Nanoarray Approach for the Assembly and Probing of Molecular Complexes

Immobilization is a key step involved in probing molecular interactions using single-molecule force spectroscopy methods, including atomic force microscopy (AFM). To our knowledge, we describe a novel approach termed flexible nanoarray (FNA) in which the interaction between the two internally immobilized amyloid β peptides is measured by pulling of the tether. The FNA tether was synthesized with nonnucleotide phosphoramidite monomers using the DNA synthesis chemistry. The two anchoring points for immobilization of the peptides inside the tether were incorporated at defined distances between them and from the ends of the polymer. Decamers of amyloid β peptide capable of dimer formation were selected as a test system. The formation of the peptide dimers was verified by AFM force spectroscopy by pulling the tether at the ends. In these experiments, the thiolated end of the FNA tether was covalently immobilized on the AFM substrate functionalized with maleimide. The other end of the FNA tether was functionalized with biotin to form a noncovalent link with the streptavidin functionalized AFM tip during the approach stage. The dimers’ rupture fingerprint was unambiguously identified on the force curves by its position and the force value. The FNA design allowed reversible experiments in which the monomers were allowed to associate after the rupture of the dimers by performing the approach stage before the rupture of the biotin-streptavidin link. This suggests that the FNA technique is capable of analyzing multiple intermolecular interactions in the same molecular complex. The computational analysis showed that the tethered peptides assemble into the same dimer structure as that formed by nontethered peptides, suggesting that the FNA tether has the necessary flexibility to enable assembly of the dimer even during the course of the force spectroscopy experiment.

Mapping single molecular binding kinetics of carbohydrate-binding module with crystalline cellulose by atomic force microscopy recognition imaging.

We studied the binding kinetics of family 3 carbohydrate-binding module (CBM3a) molecules to crystalline cellulose fibrils extracted from the poplar cell wall by atomic force microscopy (AFM) recognition imaging. The free CBM3a molecules of different concentrations were added to the buffer solution to bind to the crystalline cellulose sample immobilized on the AFM substrate. During in-situ AFM imaging, the CBM molecules were observed to bind to cellulose efficiently and regularly, especially in the first 60-120 min. A 1:1 single-molecule binding model was used to study the kinetics of the CBM3a-cellulose interaction. The saturation time when the concentration of occupied binding sites is 99% of the maximum bound CBM3a concentration at the end of reaction, t(0.99), was determined by fitting different concentrations of CBM3a against reaction time using the high resolution AFM images and the single-molecule kinetics equations. Based on the experimental data and kinetics calculations, the minimal effective initial CBM3a concentration was estimated to be 5.1 × 10(-7) M at 287 min reaction time. This study provides an in-depth understanding of the binding mechanism of CBM with crystalline cellulose at single molecule level.

Strand-specific Recognition of DNA Damages by XPD Provides Insights into Nucleotide Excision Repair Substrate Versatility

Recognition and removal of DNA damages is essential for cellular and organismal viability. Nucleotide excision repair (NER) is the sole mechanism in humans for the repair of carcinogenic UV irradiation-induced photoproducts in the DNA, such as cyclobutane pyrimidine dimers. The broad substrate versatility of NER further includes, among others, various bulky DNA adducts. It has been proposed that the 5'-3' helicase XPD (xeroderma pigmentosum group D) protein plays a decisive role in damage verification. However, despite recent advances such as the identification of a DNA-binding channel and central pore in the protein, through which the DNA is threaded, as well as a dedicated lesion recognition pocket near the pore, the exact process of target site recognition and verification in eukaryotic NER still remained elusive. Our single molecule analysis by atomic force microscopy reveals for the first time that XPD utilizes different recognition strategies to verify structurally diverse lesions. Bulky fluorescein damage is preferentially detected on the translocated strand, whereas the opposite strand preference is observed for a cyclobutane pyrimidine dimer lesion. Both states, however, lead to similar conformational changes in the resulting specific complexes, indicating a merge to a "final" verification state, which may then trigger the recruitment of further NER proteins.

Interaction of cement model systems with superplasticizers investigated by atomic force microscopy, zeta potential, and adsorption measurements

Polyelectrolyte-based dispersants are commonly used in a wide range of industrial applications to provide specific workability to colloidal suspensions. Their working mechanism is based on adsorption onto the surfaces of the suspended particles. The adsorbed polymer layer can exercise an electrostatic and/or a steric effect which is responsible for achieving dispersion. This study is focused on the dispersion forces induced by polycarboxylate ether-based superplasticizers (PCEs) commonly used in concrete. They are investigated by atomic force microscopy (AFM) applying standard silicon nitride tips exposed to solutions with different ionic compositions in a wet cell. Adsorption isotherms and zeta potential analysis were performed to characterize polymer displacement in the AFM system on nonreactive model substrates (quartz, mica, calcite, and magnesium oxide) in order to avoid the complexity of cement hydration products. The results show that PCE is strongly adsorbed by positively charged materials. This fact reveals that, being silicon nitride naturally positively charged, in most cases the superplasticizer adsorbs preferably on the silicon nitride tip than on the AFM substrate. However, the force–distance curves displayed repulsive interactions between tip and substrates even when polymer was poorly adsorbed on both. These observations allow us to conclude that the dispersion due to PCE strongly depends on the particle charge. It differs between colloids adsorbing and not adsorbing PCE, and leads to different forces acting between the particles.

Synthesis of carbon nanotubes by CO 2-laser-assisted chemical vapour deposition

The field of carbon nanotube research is remarkable not only because of the unique properties of this new material but also because of the various possible schemes of their synthesis and their applications. In the present study, we have explored the suitability of laser-assisted chemical vapour deposition for the formation and growth of carbon nanotubes. A medium-power continuous-wave CO 2 laser was employed to irradiate a sensitized mixture of Fe(CO) 5 vapour and acetylene and to simultaneously heat a silicon substrate on which the carbon nanotubes were grown. Scanning and transmission electron microscopy (TEM and HRTEM) as well as atomic force microscopy (AFM) were used to analyze the as-grown films and samples specially prepared on TEM grids and AFM substrates. Carbon nanotubes with different structures (straight, curved and even branched), including single- and multi-walled nanotubes were observed. Some nanotubes were found to be partially filled with a solid material (probably metallic iron) that seems to catalyze the nanotube growth. Some regions of the deposit also revealed the presence of nanoparticles. The present experimental conditions should be suitable to produce locally structured deposits of carbon nanotubes for various applications.

Use of Self-Assembled Monolayers as Substrates for Atomic Force Imaging of Hydroxyapatite Crystals from Mammalian Skeletal Tissues

Atomic force microscopy (AFM) has recently been successfully used to describe the surface topography of hydroxyapatite crystals from mammalian skeletal tissues. To further characterize the growth mechanisms of skeletal hydroxyapatite crystals and the role of adsorbed proteins in these processes, imaging under biological fluids is essential. However, under aqueous solutions, these crystals do not bind to the usual AFM substrates such as mica and graphite and therefore alternative substrates are necessary. The aim of the present study was to evaluate the use of self-assembled monolayer technology with controllable chemical functionality to provide “designer surfaces” for crystal binding in fluid environments which simulate the normal physiological milieu. We have found that hydroxyapatite crystals from developing enamel are bound most effectively by negatively charged self-assembled monolayer (COO- and SO3-) surfaces, demonstrating an important role for such substrates in AFM imaging of biological samples under aqueous fluids and suggesting that the prevalent charge on enamel crystal surfaces is positive.