Robust acoustic trapping and perturbation of single-cell microswimmers illuminate three-dimensional swimming and ciliary coordination

We report a label-free acoustic microfluidic method to confine single, cilia-driven swimming cells in space without limiting their rotational degrees of freedom. Our platform integrates a surface acoustic wave (SAW) actuator and bulk acoustic wave (BAW) trapping array to enable multiplexed analysis with high spatial resolution and trapping forces that are strong enough to hold individual microswimmers. The hybrid BAW/SAW acoustic tweezers employ high-efficiency mode conversion to achieve submicron image resolution while compensating for parasitic system losses to immersion oil in contact with the microfluidic chip. We use the platform to quantify cilia and cell body motion for wildtype biciliate cells, investigating effects of environmental variables like temperature and viscosity on ciliary beating, synchronization, and three-dimensional helical swimming. We confirm and expand upon the existing understanding of these phenomena, for example determining that increasing viscosity promotes asynchronous beating. Motile cilia are subcellular organelles that propel microorganisms or direct fluid and particulate flow. Thus, cilia are critical to cell survival and human health. The unicellular alg

Raman spectroscopy: techniques and applications in the life sciences

Raman spectroscopy is an increasingly popular technique in many areas including biology and medicine. It is based on Raman scattering, a phenomenon in which incident photons lose or gain energy via interactions with vibrating molecules in a sample. These energy shifts can be used to obtain information regarding molecular composition of the sample with very high accuracy. Applications of Raman spectroscopy in the life sciences have included quantification of biomolecules, hyperspectral molecular imaging of cells and tissue, medical diagnosis, and others. This review briefly presents the physical origin of Raman scattering explaining the key classical and quantum mechanical concepts. Variations of the Raman effect will also be considered, including resonance, coherent, and enhanced Raman scattering. We discuss the molecular origins of prominent bands often found in the Raman spectra of biological samples. Finally, we examine several variations of Raman spectroscopy techniques in practice, looking at their applications, strengths, and challenges. This review is intended to be a starting resource for scientists new to Raman spectroscopy, providing theoretical background and practical examples as the foundation for further study and exploration

High-performance artificial micro/nanomachines and their bioapplications

Artificial micro/nanomachines are micrometer or nanometer scale mechanical devices that convert diverse energy sources into controlled mechanical motions. The development and applications of these micro/nanomachines are among the most pressing challenges in the research field of nanoscience and nanotechnology. In this dissertation, we report innovative designs and operations of artificial micro/nanomachines for bioapplications in biochemical sensing, biomolecule capture, drug delivery and release. Based on the electric tweezers, innovative rotary nanomotors are bottom-up assembled with high efficiency from nanoscale building blocks, which are massively fabricated and less than 1 μm in all dimensions. After assembling, the rotary nanomotors achieve an ultrafast speed up to 18,000 rpm, an ultradurable operation lifetime of 80 hours, and over 1.1 million rotation cycles. To explore diverse alternative energy inputs for nanomotors, we also applied electric tweezers in the guided manipulation of chemical nanomotors: the motions of chemical nanomotors are aligned along the direction of AC electric fields and their speeds are modulated by the DC electric fields. The prowess of the manipulation of chemical nanomotors by the electric tweezers is demonstrated for applications in cargo delivery to designated microdocks and assembling of chemical nanomotors for powering rotary nanoelectromechanical system (NEMS) devices. To integrate the function of Raman sensing on the micro/nanomachines, plasmonic nanomotors and bio-photonic-plasmonic micromotors with silver (Ag) nanoparticle coating are designed and fabricated, which provide ultrasensitive detection of biochemicals by Surface-enhanced Raman spectroscopy (SERS). The plasmonic nanomotors are designed with nanoporous superstructures, providing high capacities of drug loading and large numbers of hotspots. The plasmonic nanomotors also actively manipulate molecules and tune the release rate in electric fields due to the induced electrokinetic effect. The bio-photonic-plasmonic micromotors based on biosilica diatom frustules are applied in the capture and detection of DNA molecules, which are significantly accelerated during the rotation of the micromotors. The fundamental mechanism is investigated and attributed to the reduction of Nernst diffusion layer caused by the rotation. The innovations of artificial micro/nanomachines including concept, design, fabrication, manipulation, and bioapplications in this dissertation, are expected to inspire various research areas including NEMS, nanorobotics, microfluidics, biochemical delivery, and diagnostic sensingMaterials Science and Engineerin

Directing Matter and Energy: Five Challenges for Science and the Imagination

The twin aspects of energy and control (or direction) are the underlying concepts. Matter and energy are closely linked, and their understanding and control will have overwhelming importance for our civilization, our planet, our science, and our technology. This importance ranges even beyond the large portfolio of BES, both because these truly significant Grand Challenges confront many other realms of science and because even partial solutions to these challenges will enrich scientists’ collective imagination and ability to solve problems with new ideas and new methods

Novel Electrochemical Biosensors for Clinical Assays

Biosensors, i.e., devices where biological molecules or bio(mimetic)structures are intimately coupled to a chemo/physical transducer for converting a biorecognition event into a measurable signal, have recently gained a wide (if not huge) academic and practical interest for the multitude of their applications in analysis, especially in the field of bioanalysis, medical diagnostics, and clinical assays. Indeed, thanks to their very simple use (permitting sometimes their application at home), the minimal sample pretreatment requirement, the higher selectivity, and sensitivity, biosensors are an essential tool in the detection and monitoring of a wide range of medical conditions from glycemia to Alzheimer’s disease as well as in the monitoring of drug responses. Soon, we expect that their importance and use in clinical diagnostics will expand rapidly so as to be of critical importance to public health in the coming years. This Special Issue would like to focus on recent research and development in the field of biosensors as analytical tools for clinical assays and medical diagnostics

The interplay of lipids and respiratory enzymes in synthetic ATP producing systems

All organisms require energy to maintain vital macroscopic and microscopic processes. Primary energy supplied in form of light or as nutrients has to be converted to the universal energy currency ATP before it can be used by the cells. The majority of ATP is produced at energy-converting membranes where a series of respiratory enzyme complexes couple electron transport reactions to the generation of an electrochemical proton gradient across the membrane. Similar to power generation at a hydroelectric power turbine through water pressure, the electrochemical proton gradient energizes the production of ATP by driving rotation of the molecular turbine of the ATP synthase. While the structures and mech-anisms of most respiratory enzymes and the ATP synthase are well understood, relatively little is known how these respiratory complexes interact with each other, including the influence of the environment on the complexes and their interactions. Here, we used a bottom-up approach, in which the individual enzymes are purified and reinserted into a lipid bilayer to investigate the functional interplay of these enzymes. In the simplest scenario, the terminal respiratory bo3 oxidase and the ATP synthase from E. coli are coreconstituted into liposomes to form a minimal respiratory chain system, where proton pumping by bo3 oxidase is initiated by adding its reduced substrate ubiquinol Q1, and ATP synthesis is monitored in real-time under steady-state conditions. Nilsson et al. recently found a strong influence of the lipid composition on the ATP synthesis rate in this system 1 where the stepwise insertion of neg-atively charged lipids into zwitterionic liposomes dramatically reduced ATP synthesis. The data indicate lipid-dependent changes in the lateral distance between the two enzymes. In this PhD thesis, we aimed to test this hypothesis by reversibly coupling bo3 oxidase and ATP synthase, reconstituting the complex subsequently into liposomes of varying lipid composition and comparing coupled ATP synthesis rates of the coupled complex with rates of freely floating enzymes. We describe three different approaches for coupling of these two large complexes and the different challenges. While perfect stoichiometric coupling of the two enzymes has not yet been achieved here, the results are promising and are the basis for current experiments. Instead of using the artificial DTT/Q1 electron donor system, we have expanded the minimal respiratory chain by the monotopic complex I analogue NDH-2 to supply the system with reduced quinol Q8, thereby generating a more natural alternative. If this alternative system was subjected to a lipid screen-ing, we observed an inverted lipid dependency than with the Q1 system, with a strong requirement of negatively charged lipids for coupled ATP synthesis. We were able to pinpoint this effect to an increased NADH:ubiquinone oxidoreductase activity in presence of anionic liposomes in experiments with solu-bilized NDH-2. We identified negatively charged liposomes to be essential for proper NDH-2 activity, indicating a charge-mediated binding of NDH-2 to the membrane. The inverted lipid dependency of the two systems created doubt that a lipid-dependent lateral differ-ence of the proteins is the only reason for the observed lipid effect. We thus wanted to analyze the effect of the lipid composition on the orientation of the bo3 oxidase. To this end, we established a novel method to determine the relative orientation of membrane proteins in liposomes that is independent of protein function. Instead, the membrane protein is site-specifically labeled with a fluorophore that is quenched stepwise after reconstitution into liposomes with membrane-impermeable quencher. A strongly lipid-dependent orientation of bo3 oxidase in liposomes with lower fraction of desired inside-out orientation in liposomes carrying a net negative charge compared to uncharged liposomes was in-deed observed, in good agreement with the reduced ATP synthesis activity in negatively charged lipo-somes described above. Furthermore, we show that the fraction of inside-out orientation could be in-creased when reconstitution was carried out in presence of salt, suggesting an electrostatic-mediated insertion of bo3 oxidase into liposomes. To suppress this unwanted effect on orientation, we aimed to achieve unidirectional inside-out orien-tation of bo3 oxidase. Based on the observation that the large head group of ATP synthase is unable to cross the membrane during reconstitution, a large soluble protein (~100 kDa) was coupled to bo3 oxi-dase using the SpyTag/SpyCatcher methodology to guide its insertion in the favored inside-out orien-tation. We present the successful procedure to produce the desired product keeping its native func-tionality and preliminary experiments with ATP synthase look very promising and are the basis for on-going trials of unidirectional bo3 oxidase reconstitution

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

This work was supported by the National Institute of General Medical Sciences [GM131919].In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.PostprintPeer reviewe