Aseem Salhotra

In nature, colors can originate from pigments or structural effects, with the latter producing brilliant hues through the interference of light with nanoscale structures. This study describes a feasible strategy to achieve structurally colored films based on acetylated lignin nanoparticles. Lignin nanoparticles were prepared by using membrane emulsification and subsequently self-assembled into multilayered films on silicon substrates through an evaporative process. These films exhibit vivid structural colors resulting from thin-film interference, with hues that vary with film thickness. Spectroscopic reflectance measurements and structural analysis reveal a wide range of colors spanning across the visible spectrum. The observed colors are ascribed to interference effects and could be modeled using the transfer matrix method. Furthermore, we demonstrate that increasing relative humidity causes clear color shifts associated with reflectance peak position changes.

Plant cells represent smart cargo carriers with great socioeconomic potential in oral drug delivery applications. The two exterior barriers, featuring a rigid cell wall and a dense plasma membrane, are unique with complementary structural, mechanical, and chemical properties. Current strategies for producing therapeutic drugs within plant cells for oral delivery are efficient, but largely limited to recombinant pharmaceutical proteins, and involve complex genetic modification of plants. To address this, we engineer plant cell–inspired delivery systems with cellulose nanofiber–based shells and lipid layers through a bottom-up assembly strategy, which offers greater flexibility to encapsulate nonprotein compounds and nanoparticles. Notably, the layered shell structure resists degradation in acidic environments, and two barriers respond differently to external stimuli in simulated gastrointestinal medium, resulting in size-dependent dual-triggered release mechanisms. The cytocompatibility was shown by incubation with Caco-2 cells. Our results open avenues for developing next generation of bioinspired oral delivery systems for multisite-specific gastrointestinal release in a low-cost and sustainable manner.

In the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21–26 °C, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21–26 °C, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.

For certain nanotechnological applications of the contractile proteins actin and myosin, e.g., in biosensing and network-based biocomputation, it would be desirable to temporarily switch on/off motile function in parts of nanostructured devices, e.g., for sorting or programming. Myosin XI motor constructs, engineered with a light-switchable domain for switching actin motility between high and low velocities (light-sensitive motors (LSMs) below), are promising in this regard. However, they were not designed for use in nanotechnology, where longevity of operation, long shelf life, and selectivity of function in specific regions of a nanofabricated network are important. Here, we tested if these criteria can be fulfilled using existing LSM constructs or if additional developments will be required. We demonstrated extended shelf life as well as longevity of the actin-propelling function compared to those in previous studies. We also evaluated several approaches for selective immobilization with a maintained actin propelling function in dedicated nanochannels only. Whereas selectivity was feasible using certain nanopatterning combinations, the reproducibility was not satisfactory. In summary, the study demonstrates the feasibility of using engineered light-controlled myosin XI motors for myosin-driven actin transport in nanotechnological applications. Before use for, e.g., sorting or programming, additional work is however needed to achieve reproducibility of the nanofabrication and, further, optimize the motor properties.

Molecular motor-driven filament systems have been extensively explored for biomedical and nanotechnological applications such as lab-on-chip molecular detection or network-based biocomputation. In these applications, filament transport conventionally occurs in two dimensions (2D), often guided along open, topographically and/or chemically structured channels which are coated by molecular motors. However, at crossing points of different channels the filament direction is less well determined and, though crucial to many applications, reliable guiding across the junction can often not be guaranteed. We here present a three-dimensional (3D) approach that eliminates the possibility for filaments to take wrong turns at junctions by spatially separating the channels crossing each other. Specifically, 3D junctions with tunnels and overpasses were manufactured on glass substrates by two-photon polymerization, a 3D fabrication technology where a tightly focused, femtosecond-pulsed laser is scanned in a layer-to-layer fashion across a photo-polymerizable inorganic–organic hybrid polymer (ORMOCER^®) with µm resolution. Solidification of the polymer was confined to the focal volume, enabling the manufacturing of arbitrary 3D microstructures according to computer-aided design data. Successful realization of the 3D junction design was verified by optical and electron microscopy. Most importantly, we demonstrated the reliable transport of filaments, namely microtubules propelled by kinesin-1 motors, across these 3D junctions without junction errors. Our results open up new possibilities for 3D functional elements in biomolecular transport systems, in particular their implementation in biocomputational networks.

Significant advancements have been made towards exploitation of naturally available molecular motors and their associated cytoskeletal filaments in nanotechnological applications. For instance, myosin motors and actin filaments from muscle have been used with the aims to establish new approaches in biosensing and network-based biocomputation. The basis for these developments is a version of the in vitro motility assay (IVMA) where surface-adsorbed myosin motors propel the actin filaments along suitably derivatized nano-scale channels on nanostructured chips. These chips are generally assembled into custom-made microfluidic flow cells. For effective applications, particularly in biocomputation, it is important to appreciably prolong function of the biological system. Here, we systematically investigated potentially critical factors necessary to achieve this, such as biocompatibility of different components of the flow cell, the degree of air exposure, assay solution composition and nanofabrication methods. After optimizing these factors we prolonged the function of actin and myosin in nanodevices for biocomputation from 60 min. In addition, we demonstrated that further optimizations could increase motility run times to >20 h. Of great importance for the latter development was a switch of glucose oxidase in the chemical oxygen scavenger system (glucose oxidase–glucose–catalase) to pyranose oxidase, combined with the use of blocking actin (non-fluorescent filaments that block dead motors). To allow effective testing of these approaches we adapted commercially available microfluidic channel slides, for the first time demonstrating their usefulness in the IVMA. As part of our study, we also demonstrate that myosin motor fragments can be stored at −80 °C for more than 10 years before use for nanotechnological purposes. This extended shelf-life is important for the sustainability of network-based biocomputation.

Over the last 25 years, extensive progress has been made in developing a range of nanotechnological applications where cytoskeletal filaments and molecular motors are key elements. This includes novel, highly miniaturized lab on a chip systems for biosensing, nanoseparation etc but also new materials and parallel computation devices for solving otherwise intractable mathematical problems. For such approaches, both actin-based and microtubule-based cytoskeletal systems have been used. However, in accordance with their different cellular functions, actin filaments and microtubules have different properties and interaction kinetics with molecular motors. Therefore, the two systems obviously exhibit different advantages and encounter different challenges when exploited for applications. Specifically, the achievable filament velocities, the capability to guide filaments along nanopatterned tracks and the capability to attach and transport cargo differ between actin- and microtubule-based systems. Our aim here is to systematically elucidate these differences to facilitate design of new devices and optimize future developments. We first review the cellular functions and the fundamental physical and biochemical properties of actin filaments and microtubules. In this context we also consider their interaction with molecular motors and other regulatory proteins that are of relevance for applications. We then relate these properties to the advantages and challenges associated with the use of each of the motor-filament systems for different tasks. Finally, fundamental properties are considered in relation to some of the most interesting future development paths e.g. in biosensing and biocomputation.

Benefits of single molecule studies of biomolecules include the need for minimal amounts of material and the potential to reveal phenomena hidden in ensembles. However, results from recent single molecule studies of fluorescent ATP turnover by myosin are difficult to reconcile with ensemble studies. We found that key reasons are complexities due to dye photophysics and fluorescent contaminants. After eliminating these, through surface cleaning and use of triple state quenchers and redox agents, the distributions of ATP binding dwell times on myosin are best described by 2 to 3 exponential processes, with and without actin, and with and without the inhibitor para-aminoblebbistatin. Two processes are attributable to ATP turnover by myosin and actomyosin respectively, whereas the remaining process (rate constant 0.2–0.5 s⁻¹) is consistent with non-specific ATP binding to myosin, possibly accelerating ATP transport to the active site. Finally, our study of actin-activated myosin ATP turnover without sliding between actin and myosin reveals heterogeneity in the ATP turnover kinetics consistent with models of isometric contraction.

Single molecule enzymology using fluorescent substrate requires truly minimal amounts of proteins. This is highly beneficial when the protein source is either advanced expression systems or samples from humans/animals with ethical and economic implications. Further benefits of single molecule analysis is the potential to reveal phenomena hidden in ensemble studies. However, dye photophysics and fluorescent contaminants complicate interpretation of the single molecule data. We here corroborate the importance of such complexities using fluorescent Alexa647 ATP to study ATP turnover by myosin and actomyosin. We further show that the complexities are largely eliminated by aggressive surface cleaning and use of a range of triple state quenchers and redox agents with minor effects on actin-myosin function. Using optimized assay conditions, we then show that the distributions of ATP binding dwell times on myosin are best described by the sum of 2 to 3 exponential processes. This applies in the presence and absence of actin and in the presence and absence of the drug para-aminoblebbistatin. Two of the processes are attributable to ATP turnover by myosin and actomyosin, respectively. A remaining process with rate constant in the range 0.2-0.5 s ^-1 is consistent with non-specific ATP binding to myosin and bioinformatics modelling suggests that such binding may be important for accelerated ATP transport to the active site. Finally, we report studies of the actin-activated myosin ATP turnover under conditions with no sliding between actin and myosin, as in isometrically contracting muscle, revealing heterogeneity in the ATP turnover kinetics between different molecules.

The guided gliding of cytoskeletal filaments, driven by biomolecular motors on nano/micro-structured chips, enables novel applications in biosensing and biocomputation. However, expensive and time-consuming chip production hampers the developments. It is therefore important to establish protocols to regenerate the chips, preferably without the need to dismantle the assembled microfluidic devices which contain the structured chips. We here describe a novel method toward this end. Specifically, we use the small, nonselective proteolytic enzyme, proteinase K to cleave all surface-adsorbed proteins, including myosin and kinesin motors. Subsequently, we apply a detergent (5 % SDS or 0.05 % Triton X100) to remove the protein remnants. After this procedure, fresh motor proteins and filaments can be added for new experiments. Both, silanized glass surfaces for actin-myosin motility and pure glass surfaces for microtubule-kinesin motility were repeatedly regenerated using this approach. Moreover, we demonstrate the applicability of the method for the regeneration of nano/micro-structured silicon-based chips with selectively functionalized areas for supporting or suppressing gliding motility for both motor systems. The results demonstrate the versatility and a promising broad use of the method for regenerating a wide range of protein-based nano/microdevices.