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Abhijit H Phakatkar,1 Emre Firlar,1– 3 Laura Alzate1,†, Boao Song,2 Surya Narayanan,1 Ramin Rojaee,2 Tara Foroozan,2 Ramasubramonian Deivanayagam,2 David James Banner,1 Reza Shahbazian-Yassar,2 Tolou Shokuhfar1 1Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60607, USA; 2Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, Chicago, IL 60607, USA; 3Institute for Quantitative Biomedicine, Rutgers University, Piscataway, NJ 08854, USA†Laura Alzate passed away on October 20, 2019Correspondence: Tolou ShokuhfarDepartment of Bioengineering, University of Illinois at Chicago, Chicago, IL 60607, USATel +1 312 413 9872Email tolou@uic.eduReza Shahbazian-YassarDepartment of Mechanical and Industrial Engineering, University of Illinois at Chicago, IL 60607, USATel +1 312 996 3440Email rsyassar@uic.eduPurpose: Recently, two-dimensional (2D) nanomaterials are gaining tremendous attention as novel antibacterial platforms to combat against continuously evolving antimicrobial resistance levels. Among the family of 2D nanomaterials, black phosphorus (BP) nanosheets have demonstrated promising potential for biomedical applications. However, there is a need to gain nanoscale insights of the antibacterial activity of BP nanosheets which lies at the center of technical challenges.Methods: Ultra-large BP nanosheets were synthesized by liquid-exfoliation method in the eco-friendly deoxygenated water. Synthesized BP nanosheets were characterized by TEM, AFM, and Raman spectroscopy techniques and their chemical stability was evaluated by EDS and EELS elemental analysis. The antibacterial activity of BP nanosheets was evaluated at nanoscale by the ultramicrotome TEM technique. Further, HAADF-STEM image and EDS elemental line map of the damaged bacterium were utilized to analyze the presence of diagnostic ions. Supportive SEM and ATR-FTIR studies were carried out to confirm the bacterial cell wall damage. In vitro colony counting method was utilized to evaluate the antibacterial performance of ultra-large BP nanosheets.Results: Elemental EELS and EDS analysis of BP nanosheets stored in deoxygenated water confirmed the absence of oxygen peak. TEM studies indicate the various events of bacterial cell damage with the lost cellular metabolism and structural integrity. Colony counting test results show that as-synthesized BP nanosheets (100 μg/mL) can kill ∼ 95% bacteria within 12 hours.Conclusion: TEM studies demonstrate the various events of E. coli membrane damage and the loss of structural integrity. These events include the BP nanosheets interaction with the bacterial cell wall, cytoplasmic leakage, detachment of cytoplasm from the cell membrane, reduced density of lipid bilayer and agglomerated DNA structure. The EDS elemental line mapping of the damaged bacterium confirms the disrupted cell membrane permeability and the lost cellular metabolism. SEM micrographs and ATR-FTIR supportive results confirm the bacterial cell wall damage.Keywords: phosphorene, transmission electron microscopy, black phosphorus nanosheet, two-dimensional materials, antibacterial

David J Banner,1 Emre Firlar,1,2 Justas Jakubonis,1 Yusuf Baggia,1 Jodi K Osborn,1 Reza Shahbazian-Yassar,2 Constantine M Megaridis,2 Tolou Shokuhfar1,2 1Department of Bioengineering, University of Illinois at Chicago, Chicago, IL 60607, USA; 2Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, Chicago, IL 60607, USACorrespondence: Tolou ShokuhfarDepartment of Bioengineering (MC 063), University of Illinois at Chicago, 851 S. Morgan St, SEO 218, Chicago, IL 60607, USATel +1 312 996-2335Fax +1 312 996-5291Email tolou@uic.eduBackground: Nanoscale surface roughness has been suggested to have antibacterial and antifouling properties. Several existing models have attempted to explain the antibacterial mechanism of nanoscale rough surfaces without direct observation. Here, conventional and liquid-cell TEM are implemented to observe nanoscale bacteria/surface roughness interaction. The visualization of such interactions enables the inference of possible antibacterial mechanisms.Methods and Results: Nanotextures are synthesized on biocompatible polymer microparticles (MPs) via plasma etching. Both conventional and liquid-phase transmission electron microscopy observations suggest that these MPs may cause cell lysis via bacterial binding to a single protrusion of the nanotexture. The bacterium/protrusion interaction locally compromises the cell wall, thus causing bacterial death. This study suggests that local mechanical damage and leakage of the cytosol kill the bacteria first, with subsequent degradation of the cell envelope.Conclusion: Nanoscale surface roughness may act via a penetrative bactericidal mechanism. This insight suggests that future research may focus on optimizing bacterial binding to individual nanoscale projections in addition to stretching bacteria between nanopillars. Further, antibacterial nanotextures may find use in novel applications employing particles in addition to nanotextures on fibers or films.Keywords: liquid TEM, graphene liquid cell, antibacterial nanopatterns, antibacterial surface topology, antibacterial microparticles

Emre Firlar,1,2 Meagan Ouy,1 Leigha Covnot,1 Yuan Xing,3 Daniel Lee,1,4 Alessandro Chan,1,4 Yi He,3 Boao Song,2 Solomon Afelik,4 Yong Wang,3 Reza Shahbazian-Yassar,2 Jose Oberholzer,1,3 Tolou Shokuhfar1 1Department of Bioengineering, University of Illinois at Chicago, Chicago, IL, USA; 2University of Illinois at Chicago, Department of Mechanical and Industrial Engineering, Chicago, IL, USA; 3University of Virginia, Department of Surgery, Charlottesville, VA, USA; 4University of Illinois at Chicago, Department of Surgery, Chicago, IL, USA Background: Islet cell transplantation is one of the key treatments for type 1 diabetes. Understanding the mechanisms of insulin fusion and exocytosis are of utmost importance for the improvement of the current islet cell transplantation and treatment of diabetes. These phenomena have not been fully evaluated due either to the lack of proper dynamic imaging, or the lack of proper cell preservation during imaging at nanoscales. Methods: By maintaining the native environment of pancreatic β-cells between two graphene monolayer sheets, we were able to monitor the subcellular events using in situ graphene liquid cell (GLC)-transmission electron microscopy (TEM) with both high temporal and high spatial resolution. Results: For the first time, the nucleation and growth of insulin particles until the later stages of fusion were imaged at nanometer scales. The release of insulin from plasma membrane involves the degradation of plasma membrane and drastic reductions in the shorter axis of the insulin particles. Sequential exocytosis results indicated the nucleation, growth and attachment of the new insulin particles to the already anchored ones, which is thermodynamically favorable due to the reduction in total surface, further reducing the Gibbs free energy. The retraction of the already anchored insulin toward the cell is also monitored for the first time live at nanoscale resolution. Conclusion: Investigation of insulin granule dynamics in β-cells can be investigated via GLC-TEM. Our findings with this technology open new realms for the development of novel drugs on pathological pancreatic β-cells, because this approach facilitates observing the effects of the stimuli on the live cells and insulin granules. Keywords: transmission electron microscopy, graphene liquid cell, insulin secretion, exocytosis

Yu Zhao,1 Qi Xing,2 Jagadeesh Janjanam,3 Kun He,1,4 Fei Long,1 Ke-Bin Low,5 Ashutosh Tiwari,3 Feng Zhao,2 Reza Shahbazian-Yassar,1 Craig Friedrich,1,8 Tolou Shokuhfar1,6–81Department of Mechanical Engineering-Engineering Mechanics, Michigan Technological University, Houghton, MI, USA; 2Department of Biomedical Engineering, Michigan Technological University, Houghton, MI, USA; 3Department of Chemistry, Michigan Technological University, Houghton, MI, USA; 4School of Materials Science and Engineering, Shandong University, Jinan, People’s Republic of China; 5Research Resources Center, University of Illinois at Chicago, Chicago, IL, USA; 6Department of Physics, University of Illinois at Chicago, Chicago, IL, USA; 7Mechanical and Industrial Engineering Department, University of Illinois at Chicago, Chicago, IL, USA; 8Multi-Scale Technologies Institute, Michigan Technological University, Houghton, MI, USAAbstract: Infection-related complications have been a critical issue for the application of titanium orthopedic implants. The use of Ag nanoparticles offers a potential approach to incorporate antimicrobial properties into the titanium implants. In this work, a novel and simple method was developed for synthesis of Ag (II) oxide deposited TiO2 nanotubes (TiNTs) using electrochemical anodization followed by Ag electroplating processes in the same electrolyte. The quantities of AgO nanoparticles deposited in TiNT were controlled by selecting different electroplating times and voltages. It was shown that AgO nanoparticles were crystalline and distributed throughout the length of the nanotubes. Inductively coupled plasma mass spectrometry tests showed that the quantities of released Ag were less than 7 mg/L after 30 days at 37°C. Antimicrobial assay results show that the AgO-deposited TiNTs can effectively kill the Escherichia coli bacteria. Although the AgO-deposited TiNTs showed some cytotoxicity, it should be controllable by optimization of the electroplating parameters and incorporation of cell growth factor. The results of this study indicated that antimicrobial properties could be added to nanotextured medical implants through a simple and cost effective method. Keywords: TiO2 nanotube arrays, anodization, AgO nanoparticles, antimicrobial, cytotoxicity

Tolou Shokuhfar,1–3 Azhang Hamlekhan,1 Jen-Yung Chang,1 Chang Kyoung Choi,1 Cortino Sukotjo,4 Craig Friedrich11Department of Mechanical Engineering-Engineering Mechanics, Multi Scale Technologies Institute, Michigan Technological University, Houghton, MI, 2Department of Physics, University of Illinois at Chicago, Chicago, IL, 3Department of Mechanical and Industrial Engineering, University of Illinois at Chicago, IL, 4Department of Restorative Dentistry, Comprehensive Dental Implant Center, University of Illinois at Chicago, College of Dentistry, Chicago, IL, USAAbstract: After the implantation of a biomaterial in the body, the first interaction occurs between the cells in contact with the biomaterial surface. Therefore, evaluating the cell–substrate interface is crucial for designing a successful implant. In this study, the interaction of MC3T3 osteoblasts was studied on commercially pure and alloy (Ti6Al4V) Ti surfaces treated with amorphous and crystalline titanium dioxide nanotubes. The results indicated that the presence of nanotubes increased the density of osteoblast cells in comparison to bare surfaces (no nanotubes). More importantly, our finding shows that the chemistry of the substrate affects the cell density rather than the morphology of the cells. A novel approach based on the focused ion beam technique was used to investigate the biophysical cell–substrate interaction. The analysis revealed that portions of the cells migrated inside the crystalline nanotubes. This observation was correlated with the super hydrophilic properties of the crystalline nanotubes. Keywords: nanotubes, osteoblasts, titanium dioxide

Vellaisamy Selvaraj,1 Sravanthi Bodapati,1 Elizabeth Murray,2 Kevin M Rice,1 Nicole Winston,1,3 Tolou Shokuhfar,4 Yu Zhao,4 Eric Blough1,3,5 1Center for Diagnostic Nanosystems, 2Department of Integrated Science and Technology, 3Department of Pharmaceutical Sciences and Research, School of Pharmacy, Marshall University, Huntington, WV, USA; 4Department of Mechanical Engineering and Engineering Mechanics, Michigan Technological University, Houghton, MI, USA; 5Department of Pharmacology, Physiology and Toxicology, School of Medicine, Marshall University, Huntington, WV, USA Background: The increased use of engineered nanoparticles (NPs) has caused new concerns about the potential exposure to biological systems and the potential risk that these materials may pose on human health. Here, we examined the effects of exposure to different concentrations (0–50 µg/mL) and incubation times (10 hours, 24 hours, or 48 hours) of yttrium oxide (Y2O3) NPs on human embryonic kidney (HEK293) cells. Changes in cellular morphology, cell viability, cell membrane integrity, reactive oxygen species levels, mitochondrial membrane potential, cell death (apoptosis and necrosis), and the DNA damage after NP exposure were compared to the effects seen following incubation with paraquat, a known toxicant. Results: The 24-hour inhibitory concentration 50 (IC50) of Y2O3 NPs (41±5 nm in size) in the HEK293 cells was found to be 108 µg/mL. Incubation with Y2O3 NPs (12.25–50 µg/mL) increased the ratio of Bax/Bcl-2, caspase-3 expression and promoted apoptotic- and necrotic-mediated cell death in both a concentration and a time-dependent manner. Decreases in cell survivability were associated with elevations in cellular reactive oxygen species levels, increased mitochondrial membrane permeability, and evidence of DNA damage, which were consistent with the possibility that mitochondria impairment may play an important role in the cytotoxic response. Conclusion: These data demonstrate that the Y2O3 NP exposure is associated with increased cellular apoptosis and necrosis in cultured HEK293 cells. Keywords: yttrium oxide nanoparticles, cytotoxicity, mitochondria damage, DNA damage, ROS production, apoptosis, necrosis

We report the structural instability of TiO2 nanotubes subjected to treatment with ammonium hydroxide (NH4OH) solution prior to calcination at elevated temperatures. The nanotubes were disintegrated into nanoparticles and the tubular morphology was vanished after 2 h of calcination at 500 °C. High-resolution transmission electron microscopy, Raman spectroscopy, x-ray diffraction, and atomic force microscopy were used to understand the nature of structural collapse in the NH4OH treated TiO2 nanotubes. It was concluded that the volumetric changes during amorphous to anatase phase transformation and surface cracking was the key role during the collapse of NH4OH-treated TiO2 nanotubes. [ABSTRACT FROM AUTHOR]

The structural instabilities exhibited by the Ti[O.sub.2] nanotubes are examined by the study. The different mechanisms associated with the collapse taking place in the [NH.sub.4]OH treated Ti[O.sub.2] nanotubes are also analyzed.

Despite extensive research on the nucleation and growth of calcium oxalate (CaOx) crystals, there are still several challenges and unknowns that remain. In particular, the role of trace metal elements in the promotion or inhibition of CaOx crystals is not well understood. In the present study, in situ graphene liquid cell transmission electron microscopy ( in situ GLC TEM) was used to observe real-time, nanoscale transformations of CaOx crystals in the presence of nickel ions (Ni 2+ ). The results showed that Ni 2+ form Ni-water complexes, acting as a shape-directing species, generating a unique morphology and altering growth kinetics. Transient adsorption of Ni-water complexes resulted in a metastable phase formation of calcium oxalate trihydrate. Atomistic molecular dynamics simulations confirmed that Ni 2+ acts as a weak inhibitor which slows down the CaOx crystallization, elucidating that Ni 2+ impacts small-sized CaOx clusters by bringing more water into the clusters. This work highlighted the intricacies behind the effect of Ni 2+ on CaOx biomineralization that were made possible to discern using in situ GLC TEM.

Artificial bone grafting materials such as collagen are gaining interest due to the ease of production and implantation. However, collagen must be supplemented with additional coating materials for improved osteointegration. Here, we report room-temperature atomic layer deposition (ALD) of MgO, a novel method to coat collagen membranes with MgO. Characterization techniques such as X-ray photoelectron spectroscopy, Raman spectroscopy, and electron beam dispersion mapping confirm the chemical nature of the film. Scanning electron and atomic force microscopies show the surface topography and morphology of the collagen fibers were not altered during the ALD of MgO. Slow release of magnesium ions promotes bone growth, and we show the deposited MgO film leaches trace amounts of Mg when incubated in phosphate-buffered saline at 37 °C. The coated collagen membrane had a superhydrophilic surface immediately after the deposition of MgO. The film was not toxic to human cells and demonstrated antibacterial properties against bacterial biofilms. Furthermore, in vivo studies performed on calvaria rats showed MgO-coated membranes (200 and 500 ALD) elicit a higher inflammatory response, leading to an increase in angiogenesis and a greater bone formation, mainly for Col-MgO500, compared to uncoated collagen. Based on the characterization of the MgO film and in vitro and in vivo data, the MgO-coated collagen membranes are excellent candidates for guided bone regeneration., Competing Interests: The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript., (© 2023 The Authors.)

While polyelemental alloys are shown to be promising for healthcare applications, their effectiveness in promoting bacterial growth remains unexplored. In the present work, we evaluated the interaction of polyelemental glycerolate particles (PGPs) with Escherichia coli ( E. coli ) bacteria. PGPs were synthesized using the solvothermal route, and nanoscale random distribution of metal cations in the glycerol matrix of PGPs was confirmed. We observed 7-fold growth of E. coli bacteria upon 4 h of interaction with quinary glycerolate (NiZnMnMgSr-Gly) particles in comparison to control E. coli bacteria. Nanoscale microscopic studies on bacteria interactions with PGPs showed the release of metal cations in the bacterium cytoplasm from PGPs. The electron microscopy imaging and chemical mapping indicated bacterial biofilm formation on PGPs without causing significant cell membrane damage. The data showed that the presence of glycerol in PGPs is effective in controlling the release of metal cations, thus preventing bacterial toxicity. The presence of multiple metal cations is expected to provide synergistic effects of nutrients needed for bacterial growth. The present work provides key microscopic insights of mechanisms by which PGPs enhance biofilm growth. This study opens the door for future applications of PGPs in areas where bacterial growth is essential including healthcare, clean energy, and the food industry.

The study of ice nucleation and growth at the nanoscale is of utmost importance in geological and atmospheric sciences. However, existing transmission electron microscopy (TEM) approaches have been unsuccessful in imaging ice formation directly. Herein, we demonstrate how radical scavengers - such as TiO 2 - encased with water in graphene liquid cells (GLCs) facilitate the observation of ice nucleation phenomena at low temperatures. Atomic-resolution imaging reveals the nucleation and growth of cubic ice-phase crystals at close proximity to TiO 2 -water nanointerfaces at low temperatures. Interestingly, both heterogeneously and homogeneously nucleated ice crystals exhibited this cubic phase. Ice crystal nuclei were observed to be more stable at the TiO 2 -water nanointerface, as compared with crystals in the bulk liquid (homogeneous nucleation), suggesting the radical scavenging efficacy of TiO 2 nanoparticles mitigating the electron beam by-products. The present work demonstrates that the use of radical scavengers in GLC TEM shows great promise towards unveiling the nanoscale pathways for ice nucleation and growth dynamic events.