Egor Ukraintsev, Ph.D.

Research investigating the interface between biological organisms and nanomaterials nowadays requires multi-faceted microscopic methods to elucidate the interaction mechanisms and effects. Here we describe a novel approach and methodology correlating data from an atomic force microscope inside a scanning electron microscope (AFM-in-SEM). This approach is demonstrated on bacteria-diamond-metal nanocomposite samples relevant in current life science research. We describe a procedure for preparing such multi-component test samples containing E. coli bacteria and chitosan-coated hydrogenated nanodiamonds decorated with silver nanoparticles on a carbon-coated gold grid. Microscopic topography information (AFM) is combined with chemical, material, and morphological information (SEM using SE and BSE at varied acceleration voltages) from the same region of interest and processed to create 3D correlative probe-electron microscopy (CPEM) images. We also establish a novel 3D RGB color image algorithm for merging multiple SE/BSE data from SEM with the AFM surface topography data which provides additional information about microscopic interaction of the diamond-metal nanocomposite with bacteria, not achievable by individual analyses. The methodology of CPEM data interpretation is independently corroborated by further in-situ (EDS) and ex-situ (micro-Raman) chemical characterization as well as by force volume AFM analysis. We also discuss the broader applicability and benefits of the methodology for life science research.

Prostate cancer (PCa) is the second most common cancer. In this paper, the isolation and properties of exosomes as potential novel liquid biopsy markers for early PCa liquid biopsy diagnosis are investigated using two prostate human cell lines, i.e., benign (control) cell line RWPE1 and carcinoma cell line 22Rv1. Exosomes produced by both cell lines are characterised by various methods including nanoparticle-tracking analysis, dynamic light scattering, scanning electron microscopy and atomic force microscopy. In addition, surface plasmon resonance (SPR) is used to study three different receptors on the exosomal surface (CD63, CD81 and prostate-specific membrane antigen-PMSA), implementing monoclonal antibodies and identifying the type of glycans present on the surface of exosomes using lectins (glycan-recognising proteins). Electrochemical analysis is used to understand the interfacial properties of exosomes. The results indicate that cancerous exosomes are smaller, are produced at higher concentrations, and exhibit more negative zeta potential than the control exosomes. The SPR experiments confirm that negatively charged α-2,3- and α-2,6-sialic acid-containing glycans are found in greater abundance on carcinoma exosomes, whereas bisecting and branched glycans are more abundant in the control exosomes. The SPR results also show that a sandwich antibody/exosomes/lectins configuration could be constructed for effective glycoprofiling of exosomes as a novel liquid biopsy marker.

Plasma provides specific adjustment of solid-state surface properties offering an alternative to high temperature treatment. Herein, hydrogen plasma treatment of monocrystalline (0001) ZnO surface is studied in an inductively coupled plasma reactor with reduced capacitively coupled plasma mode. The crucial role of electrical grounding of the sample holder for plasma etching and related changes in the morphology, optical, and electrical properties of surfaces exposed to electron and ion bombardment are explained. The effects on the chemical composition of the surface are analyzed by X-ray photoelectron spectroscopy (XPS), optical properties by photoluminescence spectroscopy, topography, roughness, and surface measurements by atomic force microscopy (AFM) and Kelvin probe force microscopy (KPFM). All methods show altered ZnO surface properties before and after plasma treatment strongly depending on the electrical potential of the holder.

The present study investigates the effect of an oxidized nanocrystalline diamond (O-NCD) coating functionalized with bone morphogenetic protein 7 (BMP-7) on human osteoblast maturation and extracellular matrix mineralization in vitro and on new bone formation in vivo. The chemical structure and the morphology of the NCD coating and the adhesion, thickness and morphology of the superimposed BMP-7 layer have also been assessed. The material analysis proved synthesis of a conformal diamond coating with a fine nanostructured morphology on the Ti6Al4V samples. The homogeneous nanostructured layer of BMP-7 on the NCD coating created by a physisorption method was confirmed by AFM. The osteogenic maturation of hFOB 1.19 cells in vitro was only slightly enhanced by the O-NCD coating alone without any increase in the mineralization of the matrix. Functionalization of the coating with BMP-7 resulted in more pronounced cell osteogenic maturation and increased extracellular matrix mineralization. Similar results were obtained in vivo from micro-CT and histological analyses of rabbit distal femurs with screws implanted for 4 or 12 weeks. While the O-NCD-coated implants alone promoted greater thickness of newly-formed bone in direct contact with the implant surface than the bare material, a further increase was induced by BMP-7. It can be therefore concluded that O-NCD coating functionalized with BMP-7 is a promising surface modification of metallic bone implants in order to improve their osseointegration.

This study presents a sensor based on quartz crystal microbalance (QCM) coated with nanocrystalline diamond (NCD) thin film, functionalization method and novel application of such sensor. Diamond-coated QCMs (DQCMs) were superficially terminated by hydrogen and oxygen (H-NCD and O-NCD) to control its surface dipole/potential. Two protein solutions were tested: bovine serum albumin (BSA) and fibronectin (FN). We performed reference measurements of serial resonant frequency (SRF) of clean QCMs loaded with protein and compared them with SRF shifts DQCMs loaded with proteins. In order to investigate the influence of the deposited NCD thin film on QCM measuring capabilities, additional FEM analysis was performed. The simulation results showed that QCM sensors maintain the sensing capabilities with a rigid thin film of NCD on its surface. The shift of SRF was demonstrably caused by the weight of protein adhered to the diamond film's surface. We compared masses estimated from the Sauerbrey equation to characterize the adhesive properties of the studied proteins. Comparing bare QCM and DQCM, we discovered diamonds enhance the sensing performance for proteins. At the same time, it saturates quickly with phosphate buffer saline used as a diluent solution for proteins. Results showed a significant increase in protein adhesion confirmed by the increase of the mass for both oxygen and hydrogen-terminated DQCMs. Moreover, a different time-dependent behaviour (i.e. different adsorption rate, degrees of physisorption and/or preference of the diamond surface functionalization) of the O-NCD and H-NCD QCMs was observed for BSA and FN proteins. In this meaning, we propose a schematic model which describes the detection principle of BSA and FN proteins on H- and O-terminated DQCM sensors. Finally, a simple proof of concept for using the functionalized diamond-coated sensors with current stimulation and EQCM (Electrochemical Quartz Crystal Microbalance) is also proposed.

Synthesis of inherently chiral materials and investigation of their self-assembly into molecular wires or 2D crystals belongs to hot topics in nanotechnology. Here we report an inherent splitting of orientational symmetry of 10e500 nm long molecular wires assembled from 3 nm chiral helicene-based macrocycles on atomically flat HOPG surface. The symmetry splitting of molecular wire orientation by a small angle of (6.5 ± 0.5) is attributed to interaction of individual helicene-based macrocycle with HOPG. There is a good correlation between experimental AFM data and theoretical simulations using ReaxFF force field and Lennard-Jones (L-J) potential. In particular the new 1000 times faster computational method based on L-J potential and sequential addition of molecules is able to simulate formation of molecular wires and emergence of their symmetry splitting, which was not possible to do by conventional molecular dynamics. The method is also sensitive to chirality of the studied molecules; enantiomers and racemates placed on HOPG form different arrangements. This opens new possibilities for simulations of large molecular systems.

ZnO biointerfaces with serum albumin have attracted noticeable attention due to the increasing interest in developing ZnO-based materials for biomedical applications. ZnO surface morphology and chemistry are expected to play a critical role on the structural, optical, and electronic properties of albumin-ZnO complexes. Yet there are still large gaps in the understanding of these biological interfaces. Herein we comprehensively elucidate the interactions at such interfaces by using atomic force microscopy and nanoshaving experiments to determine roughness, thickness, and adhesion properties of BSA layers adsorbed on the most typical polar and non-polar ZnO single-crystal facets. These experiments are corroborated by force field (FF) and density-functional tight-binding (DFTB) calculations on ZnO-BSA interfaces. We show that BSA adsorbs on all the studied ZnO surfaces while interactions of BSA with ZnO are found to be considerably affected by the atomic surface structure of ZnO. BSA layers on the (0001?) surface have the highest roughness and thickness, hinting at a specific upright BSA arrangement. BSA layers on (101?0) surface have the strongest binding, which is well correlated with DFTB simulations showing atomic rearrangement and bonding between specific amino acids (AAs) and ZnO. Besides the structural properties, the ZnO interaction with these AAs also controls the charge transfer and HOMO-LUMO energy positions in the BSA-ZnO complexes. This ZnO facet-specific protein binding and related structural and electronic effects can be useful for improving the design and functionality of ZnO-based materials and devices.

Electron emission plays an important role in diverse applications, from cold cathodes to chemical processes (solvated electrons, water purification), energy generation (thermionic or dye-sensitized solar cells), and even cancer treatment. Here we show that by surface treatment using electrochemically grown polypyrrole the secondary-electron emission and photoelectron emission from boron-doped diamond is enhanced even above the intensity of electron emission from the hydrogen-terminated surface with negative electron affinity. This enhancement is stable in air for at least one month and it persists also in vacuum after thermal annealing. Scanning electron microscopy, Kelvin probe force microscopy, total photoelectron yield spectroscopy as well as surface mapping by Auger and secondary ion mass spectroscopies are used to characterize and correlate the surface electronic and chemical properties. A model of the electron emission enhancement is provided.

Properties and functions of various ZnO materials are intensively investigated in biological systems for diagnostics, therapy, health risks assessment as well as bactericidal and decontamination purposes. Herein, the interface between ZnO and biological environment is studied by characterizing adsorption of bovine serum albumin (BSA) and fetal bovine serum (FBS) using atomic force microscopy with CF4-treated tips. Similar molecular morphologies (thickness around 2 nm) yet different binding forces to ZnO (10–25 nN) are observed. These observations are corroborated by atomic scale simulations of BSA on (0001) ZnO surface using force-field method and showing rearrangements of Zn surface atoms. Such binding may have an impact also on other properties of ZnO–BSA complex.

We introduce a straightforward mathematical method for improving the AFM image resolution, applied to image analysis of helicene-based macrocycles adsorbed on HOPG. The method reveals structural details from insufficiently resolved AFM images and attributes them to internal structure and ordering of the macrocycles. Our findings are also corroborated by molecular mechanics simulations, validating that the structure provided by the method has lower potential energy compared to other tested macrocycle arrangements.

Study of friction and energy dissipation always relied on direct observations. Actual theories provide limited prediction on the frictional and dissipative properties if only the material chemistry and geometry are known. We here develop a framework to study intrinsic friction and energy dissipation based on the only knowledge of the normal modes of the system at equilibrium. We derive an approximated expression for the first anharmonic term in the potential energy expansion which does not require the computation of the third-order force constants. Moreover, we show how to characterize the frequency content of observed physical quantities and individuate the dissipative processes active during experimental measurements. As a case study, we consider the relative sliding motion of atomic layers in molybdenum disulfide dry lubricant, and we discuss how to extract information on the energetics of sliding from atomic force microscopy signals. The presented framework switches the investigation paradigm on friction and energy dissipation from dynamic to static studies, paving avenues to explore for the design of alternative anisotropic tribological and thermal materials.

Nanocrystalline diamond (NCD) layers functionalized with amine-containing functional groups have generated considerable interest as biocompatible substrates for attachment of biomolecules and cells with a view to biosensor and tissue engineering applications. Here we prepare nanoporous diamond layers with the surfaces modified by hydrogen plasma, oxygen plasma, and conformal 7 nm amine-containing plasma polymer (PP). Immobilization of bovine serum albumin (BSA) molecules is characterized on such surfaces. Grazing angle reflectance infrared spectroscopy as well as X-ray photoelectron spectroscopy show that concentration of amine-containing bonds after BSA exposure depends on the type of NCD surface modification. AFM measurements reveal that BSA proteins are physisorbed on H- and O-terminated diamond surfaces in different thicknesses and morphology. When the diamond layers are coated with the amine-containing PP, BSA molecules assume similar thickness and morphology, and their adhesion is significantly increased on both types of the diamond surfaces.

The easy and fast detection of drug content and concentration levels is demanded in biological research as well as in clinical practice. Here we study on microscopic level how nanodiamonds and gold nanoparticles interact with a multifunctional drug molecule directly on a biosensor surface. The sensors are made of interdigitated Au electrodes coated by 5 nm hydrogenated or oxidized nanodiamonds and further combined with Au colloidal nanoparticles (size 20 nm) providing nanoscale composite (spacing 100 nm). Atomic force microscopy is employed to measure local tip-surface adhesion forces and surface topography. AFM adhesion maps show that the drug binds to all types of nanoparticles and the adhesion is also significantly influenced by the substrates on which the nanoparticles are deposited. Role of local AFM tip interaction with nanostructured surface is also discussed.

The present work aims to understand the sliding of ordered/disordered molybdenum disulfide against itself by combination of nanoscale sliding experiments and atomistic simulations. Tribological experiments were performed using lateral force microscopy with tips covered by a thin sputtered MoS2film. Nanoscale contact area between the MoS2-coated tips and MoS2 samples opened up the possibility for close comparison with classical molecular dynamics simulations. Our simulations replicated well the coefficient of friction obtained by experiments for various contact conditions and shed light on nanoscale sliding of both crystalline and amorphous MoS2. Experimental sliding at humid environment demonstrated detrimental effect of water molecules on friction. However, such effect was much less pronounced when compared to that observed in macroscopic sliding experiments.

Polytetrafluoroethylene (PTFE) is widely used for constructing tissue replacements, particularly clinically used vascular prostheses, and is also applied in dental and orthopedic surgery, thanks to its non-toxicity, high chemical resistance, low surface energy and excellent thermal stability. We report here on a comparative study in which PTFE is modified with the use of DC argon plasma (8W, 240s) and is coated with hydrogenated or oxidized nanodiamonds (mean size 5nm), with a view to achieving improved body acceptance of the bio-inert pristine material. The surface morphology characterized by scanning electron microscopy reveals a microscale and nanoscale structuring of the PTFE foils with comparable roughness among all samples (analyzed by atomic force microscopy). The water contact angle remains in the highly hydrophobic range (above 100 degrees). However, the proliferation and metabolic activity of primary human hFOB 1.19 osteoblasts (studied for up to 7 days) are significantly enhanced by the plasma and/or by hydrogenated nanodiamond treatment (rather than by oxidized nanodiamond treatment) of the PTFE foil.

Cell fate modulation by adapting the surface of a biocompatible material is nowadays a challenge in implantology, tissue engineering as well as in construction of biosensors. Nanocrystalline diamond (NCD) thin films are considered promising in these fields due to their extraordinary physical and chemical properties and diverse ways in which they can be modified structurally and chemically. The initial cell distribution, the rate of cell adhesion, distance of cell migration and also the cell proliferation are influenced by the NCD surface termination. Here, we use real-time live-cell imaging to investigate the above-mentioned processes on oxidized NCD (NCD-O) and hydrogenated NCD (NCD-H) to elucidate cell preference to the NCD-O especially on surfaces with microscopic surface termination patterns. Cells adhere more slowly and migrate farther on NCD-H than on NCD-O. Cells seeded with a fetal bovine serum (FBS) supplement in the medium move across the surface prior to adhesion. In the absence of FBS, the cells adhere immediately, but still exhibit different migration and proliferation on NCD-O/H regions. We discuss the impact of these effects on the formation of cell arrays on micropatterned NCD. (C) 2017 Wiley Periodicals, Inc.

Diamond is considered as a promising tissue equivalent material in radiation therapies as well as for bio-electronic sensors due to its unique set of properties. These features are combined in this work where effects of gamma irradiation (Co-60, up to 300 Gy) on function and stability of microscopic (60 x 20 mu m(2)) hydrogen-terminated diamond (H-diamond) solution-gated field effect transistors (SG-FET5) are studied. The H-diamond SG-FETs were prepared using 300 nm thin diamond films deposited on glass from methane and hydrogen gas mixture by microwave plasma. Prior to gamma irradiation they were interfaced to proteins (fetal bovine serum) and cells (human sarcoma osteogenic cell line - SAOS2) in cell growth medium. Blank H-diamond SG-FETs did not degrade after the irradiation. With adsorbed proteins and cells they showed specific changes in gate current characteristics (about 100% increase) after the irradiation. These current changes are attributed to modified protein layer and cell morphology on the diamond surface. The presented results establish a first step towards real-time electronic monitoring of cell growth during the irradiation by therapeutically relevant doses. (C) 2015 Elsevier B.V. All rights reserved.

Two profoundly different carbon allotropes - nanocrystalline diamond and graphene - are of considerable interest from the viewpoint of a wide range of biomedical applications including implant coating, drug and gene delivery, cancer therapy, and biosensing. Osteoblast adhesion and proliferation on nanocrystalline diamond and graphene are compared under various conditions such as differences in wettability, topography, and the presence or absence of protein interlayers between cells and the substrate. The materials are characterized in detail by means of scanning electron microscopy, atomic force microscopy, photoelectron spectroscopy, Raman spectroscopy, and contact angle measurements. In vitro experiments have revealed a significantly higher degree of cell proliferation on graphene than on nanocrystalline diamond and a tissue culture polystyrene control material. Proliferation is promoted, in particular, by hydrophobic graphene with a large number of nanoscale wrinkles independent of the presence of a protein interlayer, i.e., substrate fouling is not a problematic issue in this respect. Nanowrinkled hydrophobic graphene, thus, exhibits superior characteristics for those biomedical applications where high cell proliferation is required under differing conditions.

Cell migration plays an important role in many biological systems. A relatively simple stochastic model is developed and used to describe cell behavior on chemically patterned substrates. The model is based on three parameters: the speed of cell movement (own and external), the probability of cell adhesion, and the probability of cell division on the substrate. The model is calibrated and validated by experimental data obtained on hydrogen- and oxygen-terminated patterns on diamond. Thereby, the simulations reveal that: (1) the difference in the cell movement speed on these surfaces (about 1.5×) is the key factor behind the formation of cell arrays on the patterns, (2) this difference is provided by the presence of fetal bovine serum (validated by experiments), and (3) the directional cell flow promotes the array formation. The model also predicts that the array formation requires mean distance of cell travel at least 10% of intended stripe width. The model is generally applicable for biosensors using diverse cells, materials, and structures.