Building a community to engineer synthetic cells and organelles from the bottom-up.

Employing concepts from physics, chemistry and bioengineering, 'learning-by-building' approaches are becoming increasingly popular in the life sciences, especially with researchers who are attempting to engineer cellular life from scratch. The SynCell2020/21 conference brought together researchers from different disciplines to highlight progress in this field, including areas where synthetic cells are having socioeconomic and technological impact. Conference participants also identified the challenges involved in designing, manipulating and creating synthetic cells with hierarchical organization and function.

Centrifugal Generation of Droplet-Based 3D Cell Cultures.

Quickly and easily producing uniform populations of microsphere-based 3D cell cultures using droplet-based templating methods has the potential to enable widespread use of such platforms in drug discovery or cancer research. Here, we advance the design of centrifuge-based droplet generation devices, describe the use of this platform for droplet generation with controlled cell occupancy, and demonstrate weeklong culture duration. Using simple-to-construct devices and easily implemented protocols, the initial concentration of encapsulated cells is adjustable up to hundreds of cells per microsphere.

Single-Cell Response to the Rigidity of Semiconductor Nanomembranes on Compliant Substrates.

Single-crystalline semiconductor nanomembranes (NMs) bonded to compliant substrates are increasingly used for biomedical research and in health care. Nevertheless, there is a limited understanding of how individual cells sense the unique mechanical properties of these substrates and adjust their behavior in response to them. In this work, we performed proliferation assays, cytoskeleton analysis, and focal adhesion (FA) studies for NIH-3T3 fibroblasts on 220 and 20 nm single-crystalline Si on polydimethylsiloxane (PDMS) substrates with an elastic modulus of ∼31 kPa.

Oil-Free Acoustofluidic Droplet Generation for Multicellular Tumor Spheroid Culture.

We present an easy-to-assemble microfluidic system for synthesizing cell-loaded dextran/alginate (DEX/ALG) hydrogel spheres using an aqueous two-phase system (ATPS) for templated fabrication of multicellular tumor spheroids (MTSs). An audio speaker driven by an amplified output of a waveform generator or smartphone provides acoustic modulation to drive the breakup of an ATPS into MTS template droplets within microcapillary fluidic devices. We apply extensions of Plateau-Rayleigh theory to help define the flow and frequency parameter space necessary for acoustofluidic ATPS droplet formation in these devices.

Line-Focused Optical Excitation of Parallel Acoustic Focused Sample Streams for High Volumetric and Analytical Rate Flow Cytometry.

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 μL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.

A Microsphere-Supported Lipid Bilayer Platform for DNA Reactions on a Fluid Surface.

We report a versatile microsphere-supported lipid bilayer system that can serve as a general-purpose platform for implementing DNA nanotechnologies on a fluid surface. To demonstrate our platform, we implemented both toehold-mediated strand displacement (TMSD) and DNAzyme reactions, which are typically performed in solution and which are the cornerstone of DNA-based molecular logic and dynamic DNA nanotechnology, on the surface. We functionalized microspheres bearing supported lipid bilayers (μSLBs) with membrane-bound nucleic acid components.

Method for measuring the unbinding energy of strongly-bound membrane-associated proteins.

We describe a new method to measure the activation energy for unbinding (enthalpy ΔH* and free energy ΔG*) of a strongly-bound membrane-associated protein from a lipid membrane. It is based on measuring the rate of release of a liposome-bound protein during centrifugation on a sucrose gradient as a function of time and temperature. The method is used to determine ΔH* and ΔG* for the soluble dengue virus envelope protein (sE) strongly bound to 80:20 POPC:POPG liposomes at pH5.

DNA-assisted photoinduced charge transfer between a cationic poly(phenylene vinylene) and a cationic fullerene.

Water-soluble cationic conjugated poly(phenylene vinylene) (PPV) and cationic fullerene were complexed with negatively charged single stranded DNA and double stranded DNA via electrostatic interactions to achieve photoinduced charge transfer with efficiencies as high as those observed from oppositely charged, cationic PPV and anionic fullerene but with distinctly different quenching mechanisms.

Diblock copolymer micelles and supported films with noncovalently incorporated chromophores: a modular platform for efficient energy transfer.

We report generation of modular, artificial light-harvesting assemblies where an amphiphilic diblock copolymer, poly(ethylene oxide)-block-poly(butadiene), serves as the framework for noncovalent organization of BODIPY-based energy donor and bacteriochlorin-based energy acceptor chromophores. The assemblies are adaptive and form well-defined micelles in aqueous solution and high-quality monolayer and bilayer films on solid supports, with the latter showing greater than 90% energy transfer efficiency. This study lays the groundwork for further development of modular, polymer-based materials for light harvesting and other photonic applications.

Relaxation mechanism of the hydrated electron.

The relaxation dynamics of the photoexcited hydrated electron have been subject to conflicting interpretations. Here, we report time-resolved photoelectron spectra of hydrated electrons in a liquid microjet with the aim of clarifying ambiguities from previous experiments. A sequence of three ultrashort laser pulses (~100 femtosecond duration) successively created hydrated electrons by charge-transfer-to-solvent excitation of dissolved anions, electronically excited these electrons via the s→p transition, and then ejected them into vacuum.

Lipid membrane domains for the selective adsorption and surface patterning of conjugated polyelectrolytes.

Conjugated polyelectrolytes (CPEs) are promising materials for generating optoelectronics devices under environmentally friendly processing conditions, but challenges remain to develop methods to define lateral features for improved junction interfaces and direct optoelectronic pathways. We describe here the potential to use a bottom-up approach that employs self-assembly in lipid membranes to form structures to template the selective adsorption of CPEs. Phase separation of gel phase anionic lipids and fluid phase phosphocholine lipids allowed the formation of negatively charged domain assemblies that selectively adsorb a cationic conjugated polyelectrolyte (P2).

Effects of solvent properties on the spectroscopy and dynamics of alkoxy-substituted PPV oligomer aggregates.

Conjugated systems are frequently studied in their nanoaggregate form to probe the effects of solvent and of film formation on their spectral and dynamical properties. This article focuses on the emission spectra and dynamics of nanoaggregates of alkoxy-substituted PPV oligomers with the goal of interpreting the vibronic emission envelopes observed in these systems (J. Phys.

Bright two-photon emission and ultra-fast relaxation dynamics in a DNA-templated nanocluster investigated by ultra-fast spectroscopy.

Metal nanoclusters have interesting steady state fluorescence emission, two-photon excited emission and ultrafast dynamics. A new subclass of fluorescent silver nanoclusters (Ag NCs) are NanoCluster Beacons. NanoCluster Beacons consist of a weakly emissive Ag NC templated on a single stranded DNA ("Ag NC on ssDNA") that becomes highly fluorescent when a DNA enhancer sequence is brought in proximity to the Ag NC by DNA base pairing ("Ag NC on dsDNA").

A DNA-templated fluorescent silver nanocluster with enhanced stability.

We report the discovery of a DNA sequence that templates a highly stable fluorescent silver nanocluster. In contrast to other DNA templated silver nanoclusters that have a relatively short shelf-life, the fluorescent species templated in this new DNA sequence retains significant fluorescence for at least a year. Moreover, this new silver nanocluster possesses low cellular toxicity and enhanced thermal, oxidative, and chemical stability.

Quantum interference between the third and fourth exciton states in semiconducting carbon nanotubes using resonance Raman spectroscopy.

We exploit an energy level crossover effect [Haroz et al., Phys. Rev.

Ion specific influences on the stability and unfolding transitions of a naturally aggregating protein; RecA.

The Escherichia coli RecA protein is a naturally aggregated protein complex that is affected by the presence of salts. In order to gain further insight into the nature of the ion-interactions on a naturally aggregating protein we used circular dichroism (CD), fluorescence and dynamic light scattering (DLS) to study the effects of different concentrations of MgCl2, CaCl2, NaCl, Na2SO4, and MgSO4 on RecA structure and thermal unfolding. The results show unique ion influences on RecA structure, aggregation, unfolding transitions and stability and the anion effects correlate with the reverse Hofmeister series.

Ag K-edge EXAFS analysis of DNA-templated fluorescent silver nanoclusters: insight into the structural origins of emission tuning by DNA sequence variations.

DNA-templated silver nanoclusters are promising biological fluorescence probes due to their useful fluorescence properties, including tunability of emission wavelength through DNA template sequence variations. Ag K-edge EXAFS analysis of DNA-templated silver nanoclusters has been used to obtain insight into silver nanocluster bonding, size, and structural correlations to fluorescence. The results indicate the presence of small silver nanoclusters (<30 silver atoms) containing Ag-Ag bonds and Ag-N/O ligations to DNA.

Violation of the condon approximation in semiconducting carbon nanotubes.

The Condon approximation is widely applied in molecular and condensed matter spectroscopy and states that electronic transition dipoles are independent of nuclear positions. This approximation is related to the Franck-Condon principle, which in its simplest form holds that electronic transitions are instantaneous on the time scale of nuclear motion. The Condon approximation leads to a long-held assumption in Raman spectroscopy of carbon nanotubes: intensities arising from resonance with incident and scattered photons are equal.

Implementation of time-resolved step-scan fourier transform infrared (FT-IR) spectroscopy using a kHz repetition rate pump laser.

Time-resolved step-scan Fourier transform infrared (FT-IR) spectroscopy has been shown to be invaluable for studying excited-state structures and dynamics in both biological and inorganic systems. Despite the established utility of this method, technical challenges continue to limit the data quality and more wide ranging applications. A critical problem has been the low laser repetition rate and interferometer stepping rate (both are typically 10 Hz) used for data acquisition.

Fullerene derivatives induce premature senescence: a new toxicity paradigm or novel biomedical applications.

Engineered fullerenes (C(60)) are extensively used for commercial and clinical applications based on their unique physicochemical properties. Such materials have also been recognized as byproducts of many industrial activities. Functionalization of C(60) may significantly influence the nature of its interactions with biological systems, impacting its applications and raising uncertainties about its health effects.

Anion photoelectron spectroscopy of C3N- and C5N-.

Anion photoelectron spectroscopy of C(3)N(-) and C(5)N(-) is performed using slow electron velocity-map imaging (SEVI) and field-free time-of-flight (TOF), respectively. The SEVI spectrum exhibits well-resolved vibrational transitions from the linear C(3)N(-) ground state to the corresponding C(3)N ground state. The TOF spectrum comprises transitions arising from the linear C(5)N(-) ground state to the corresponding neutral ground and excited states.