A single-molecule study on polymer fluid dynamics in porous media.

Understanding the dynamic behavior of polymeric fluids in porous media is essential for vast geoscience applications, particularly enhanced oil recovery and polymer-enhanced soil washing, to clean up soil contamination. During the past decades, the behavior of polymeric fluids in microscopic space has only been investigated using ensemble-averaged experimental methods in which a bulk phase behavior of the fluids characterizes flow mechanisms. Multiple flow mechanisms have been proposed based on ensemble-averaged data; however, microscale characterization of the interactions between polymers and solid surfaces and the mechanisms governing polymer retention and permeability reduction as well as the reversibility of polymer retention are lacking, resulting in a limited understanding of the flow mechanisms. Here we report direct visualization and multi-scale characterization of the dynamic behavior of polymer molecules in a representative porous medium by integrating microfluidics with single-molecule imaging. We demonstrate that the polymers' adsorption, entrapment and hydrodynamic retention contribute to their overall retention in porous media. Our study illustrates how microfluidics can help in understanding the dynamic behavior of polymers, their interactions with the solid/fluid interface and their effects on flow properties. Additionally, it demonstrates the role of microfluidic platforms in providing a more representative and accurate model for polymer retention and permeability reduction in porous media. The obtained insights encourage the development of improved models that better capture the behavior of complex fluids in confined environments and have significant implications for a wide range of applications in geoscience, materials science, and rheology.

Experimental and numerical investigation of polymer pore-clogging in micromodels.

Polymers have been used effectively in the Oil & Gas Industry for a variety of field applications, such as enhanced oil recovery (EOR), well conformance, mobility control, and others. Polymer intermolecular interactions with the porous rock, in particular, formation clogging and the associated alterations to permeability, is a common problem in the industry. In this work, fluorescent polymers and single-molecule imaging are presented for the first time to assess the dynamic interaction and transport behavior of polymer molecules utilizing a microfluidic device. Pore-scale simulations are performed to replicate the experimental observations. The microfluidic chip, also known as a "Reservoir-on-a-Chip" functions as a 2D surrogate to evaluate the flow processes that take place at the pore-scale. The pore-throat sizes of an oil-bearing reservoir rock, which range from 2 to 10 nm, are taken into consideration while designing the microfluidic chip. Using soft lithography, we created the micromodel from polydimethylsiloxane (PDMS). The conventional use of tracers to monitor polymers has a restriction due to the tendency of polymer and tracer molecules to segregate. For the first time, we develop a novel microscopy method to observe the dynamic behavior of polymer pore-clogging and unclogging processes. We provide direct dynamic observations of polymer molecules during their transport within the aqueous phase and their clustering and accumulations. Pore-scale simulations were carried out to simulate the phenomena using a finite-element simulation tool. The simulations revealed a decline in flow conductivity over time within the flow channels that experienced polymer accumulation and retention, which is consistent with the experimental observation of polymer retention. The performed single-phase flow simulations allowed us to assess the flow behavior of the tagged polymer molecules within the aqueous phase. Additionally, both experimental observation and numerical simulations are used to evaluate the retention mechanisms that emerge during flow and how they affect apparent permeability. This work provides new insights to assessing the mechanisms of polymer retention in porous media.

Single-molecule imaging and microfluidic platform reveal molecular mechanisms of leukemic cell rolling.

Hematopoietic stem/progenitor cell (HSPC) and leukemic cell homing is an important biological phenomenon that occurs through key interactions between adhesion molecules. Tethering and rolling of the cells on endothelium, the crucial initial step of the adhesion cascade, is mediated by interactions between selectins expressed on endothelium to their ligands expressed on HSPCs/leukemic cells in flow. Although multiple factors that affect the rolling behavior of the cells have been identified, molecular mechanisms that enable the essential slow and stable cell rolling remain elusive. Here, using a microfluidics-based single-molecule live cell fluorescence imaging, we reveal that unique spatiotemporal dynamics of selectin ligands on the membrane tethers and slings, which are distinct from that on the cell body, play an essential role in the rolling of the cell. Our results suggest that the spatial confinement of the selectin ligands to the tethers and slings together with the rapid scanning of a large area by the selectin ligands, increases the efficiency of selectin-ligand interactions during cell rolling, resulting in slow and stable rolling of the cell on the selectins. Our findings provide novel insights and contribute significantly to the molecular-level understanding of the initial and essential step of the homing process.

Geometry-Based Self-Assembly of Histone-DNA Nanostructures at Single-Nucleotide Resolution.

Histones are basic protein monomers capable of interacting with DNA, providing the mechanism of DNA compaction inside the cell nucleus. The well-ordered assembly process of histone and DNA is a potential candidate as the approach for building DNA-protein nanostructures. Here, utilizing the sequence-independent histone-DNA interaction, we present an approach to self-assemble histones and single-stranded DNA (ssDNA) to form well-defined histone-DNA (sHD) nanoparticles and their multidimensional cross-linked complexes (cHD). By using various molecular biology and microscopy techniques, we elucidate the structure of these complexes, and we show that they are formed at carefully controlled conditions of temperature, ionic strength, concentration, and incubation time. We also demonstrate using a set of ssDNA molecular rulers and a geometric accommodation model that the assembly of sHD and cHD particles proceeds with precise geometry so that the number of ssDNA in these particles can be programmed by the length of ssDNA. We further show that the formation of cHD amplifies the effect of the length of ssDNA on the self-assembly, allowing for distinguishing ssDNA of different lengths at single nucleotide resolution. We envision that our geometry-directed approach of self-assembling histone-DNA nanostructures and the fundamental insights can serve as a structural platform to advance building precisely ordered DNA-protein nanostructures.

Entangled polymer dynamics beyond reptation.

Macroscopic properties of polymers arise from microscopic entanglement of polymer chains. Entangled polymer dynamics have been described theoretically by time- and space-averaged relaxation modes of single chains occurring at different time and length scales. However, theoretical and experimental studies along this framework provide oversimplified picture of spatiotemporally heterogeneous polymer dynamics. Characterization of entangled polymer dynamics beyond this paradigm requires a method that enables to capture motion and relaxation occurring in real space at different length and time scales. Here we develop new single-molecule characterization platform by combining super-resolution fluorescence imaging and recently developed single-molecule tracking method, cumulative-area tracking, which enables to quantify the chain motion in the length and time scale of nanometres to micrometres and milliseconds to minutes. Using linear and cyclic dsDNA molecules as model systems, our new method reveals chain-position-dependent motion of the entangled linear chains, which is beyond the scope of current theoretical framework.

Conserved linear dynamics of single-molecule Brownian motion.

Macromolecular diffusion in homogeneous fluid at length scales greater than the size of the molecule is regarded as a random process. The mean-squared displacement (MSD) of molecules in this regime increases linearly with time. Here we show that non-random motion of DNA molecules in this regime that is undetectable by the MSD analysis can be quantified by characterizing the molecular motion relative to a latticed frame of reference. Our lattice occupancy analysis reveals unexpected sub-modes of motion of DNA that deviate from expected random motion in the linear, diffusive regime. We demonstrate that a subtle interplay between these sub-modes causes the overall diffusive motion of DNA to appear to conform to the linear regime. Our results show that apparently random motion of macromolecules could be governed by non-random dynamics that are detectable only by their relative motion. Our analytical approach should advance broad understanding of diffusion processes of fundamental relevance.

Single-molecule diffusion and conformational dynamics by spatial integration of temporal fluctuations.

Single-molecule localization and tracking has been used to translate spatiotemporal information of individual molecules to map their diffusion behaviours. However, accurate analysis of diffusion behaviours and including other parameters, such as the conformation and size of molecules, remain as limitations to the method. Here, we report a method that addresses the limitations of existing single-molecular localization methods. The method is based on temporal tracking of the cumulative area occupied by molecules. These temporal fluctuations are tied to molecular size, rates of diffusion and conformational changes. By analysing fluorescent nanospheres and double-stranded DNA molecules of different lengths and topological forms, we demonstrate that our cumulative-area method surpasses the conventional single-molecule localization method in terms of the accuracy of determined diffusion coefficients. Furthermore, the cumulative-area method provides conformational relaxation times of structurally flexible chains along with diffusion coefficients, which together are relevant to work in a wide spectrum of scientific fields.

Spatiotemporal visualization of subcellular dynamics of carbon nanotubes.

To date, there is no consensus on the relationship between the physicochemical characteristics of carbon nanotubes (CNTs) and their biological behavior; however, there is growing evidence that the versatile characteristics make their biological fate largely unpredictable and remain an issue of limited knowledge. Here we introduce an experimental methodology for tracking and visualization of postuptake behavior and the intracellular fate of CNTs based on the spatial distribution of diffusion values throughout the plant cell. By using raster scan image correlation spectroscopy (RICS), we were able to generate highly quantitative spatial maps of CNTs diffusion in different cell compartments. The spatial map of diffusion values revealed that the uptake of CNTs is associated with important subcellular events such as carrier-mediated vacuolar transport and autophagy. These results show that RICS is a useful methodology to elucidate the intracellular behavior mechanisms of carbon nanotubes and potentially other fluorescently labeled nanoparticles, which is of relevance for the important issues related to the environmental impact and health hazards.

The plant cell uses carbon nanotubes to build tracheary elements.

Since their discovery, carbon nanotubes (CNTs) have been eminent members of the nanomaterial family. Because of their unique physical, chemical and mechanical properties, they are regarded as new potential materials to bring enormous benefits in cell biology studies. Undoubtedly, the first step to prove the advantages of CNTs is to understand the basic behavior of CNTs inside the cells. In a number of studies, CNTs have been demonstrated as new carrier systems for the delivery of DNA, proteins and therapeutic molecules into living cells. However, post-uptake behavior of CNTs inside the cells has not received much consideration. Utilizing the plant cell model, we have shown in this study that the plant cells, differentiating into tracheary elements, incorporate cup-stacked carbon nanotubes (CSCNTs) into cell structure via oxidative cross-linking of monolignols to the nanotubes surface during lignin biosynthesis. This finding highlights the fate of CNTs inside plant cells and provides an example on how the plant cell can handle internalized carbon nanomaterials.

Functional platform for controlled subcellular distribution of carbon nanotubes.

As nanoparticles can cross different cellular barriers and access different tissues, control of their uptake and cellular fate presents a functional approach that will be broadly applicable to nanoscale technologies in cell biology. Here we show that the trafficking of single-walled carbon nanotubes (SWCNTs) through various subcellular membranes of the plant cell is facilitated or inhibited by attaching a suitable functional tag and controlling medium components. This enables a unique control over the uptake and the subcellular distribution of SWCNTs and provides a key strategy to promote their cellular elimination to minimize toxicity. Our results also demonstrate that SWCNTs are involved in a carrier-mediated transport (CMT) inside cells; this is a phenomenon that scientists could use to obtain novel molecular insights into CMT, with the potential translation to advances in subcellular nanobiology.

Trafficking and subcellular localization of multiwalled carbon nanotubes in plant cells.

Major barriers to delivery of biomolecules are crossing the cellular membranes and achieving a high cytoplasmic concentration by circumventing entrapment into endosomes and other lytic organelles. Motivated by such aim, we have investigated the capability of multiwalled carbon nanotubes (MWCNTs) to penetrate the cell membrane of plant protoplasts (plant cells made devoid of their cell walls via enzymatic treatment) and studied their internalization mechanism via confocal imaging and TEM techniques. Our results indentified an endosome-escaping uptake mode of MWCNTs by plant protoplasts. Moreover, short MWCNTs (<100 nm) were observed to target specific cellular substructures including the nucleus, plastids, and vacuoles. These findings are expected to have a significant impact on plant cell biology and transformation technologies.