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. A key conclusion is the need to build an international and interdisciplinary research community through enhanced communication, resource-sharing, and educational initiatives.
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.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.
The formation of condensed phase nucleoprotein assemblies, such as membraneless organelles (MLOs), that contribute to gene regulation and signaling within the cell is garnering widespread attention. ...A critical technical challenge is understanding how interactions between intrinsically disordered protein (IDP) and nucleic acid molecular components affect liquid–liquid phase separation (LLPS) into nucleoprotein condensates. To better understand the physics of LLPS that drive the formation of biomolecular condensates (known as coacervates), we investigate a model IDP system using a cationic elastin-like polypeptide (ELP), “E3”, that is engineered to phase separate and bind DNA upon coacervate formation. Using mean field Flory–Huggins (FH) theory, we create ternary phase diagrams to quantify DNA component partitioning within discrete protein- and solvent-rich phases across a range of salt and E3 compositions. We suggest a modified FH theory that combines canonical FH interaction parameters with an approximation of the Debye–Hückel theory to predict the strength of E3–DNA interactions and partitioning with a variable salt concentration. Finally, we establish a simple two-step DNA solution separation/purification assay to highlight the potential utility of our system. This model LLPS biopolymer platform represents an important chemical engineering-based contribution to synthetic biology and DNA technologies, with possible implications for origin of life discussions.
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. We also characterized cell response on bulk Si as a reference. Our in vitro studies show that varying the thickness of the NM between 20 and 220 nm affects the proliferation rate of the cells, their cytoskeleton, fiber organization, spread area, and degree of FA. For example, cultured cells on 220 nm Si/PMDS exhibit the same response as on bulk Si, that is, they are well-spread with a pentagonal (or dendritic) shape and show a good organization of stress fibers and FAs. On the other hand, the cells on 20 nm Si/PDMS are spherical, with fiber organization and FAs in undetectable levels. We explained the results of our in vitro studies through a shear-lag mechanical model. The calculated FA–substrate contact stiffnesses for fibroblasts on bulk Si and 220 nm Si/PDMS closely match, and they are significantly higher than the stiffness of the integrin clutches and the plaque. Conversely, focal contacts with 20 nm Si/PDMS have comparable lateral compliance to adhesion-mediating intracellular organisms. In conclusion, our work relies on recent advances in NM technology to fill a critical knowledge gap about how individual cells sense and react to the mechanical properties of NM-based substrates. Our findings will have a major impact on the design of flexible electronic materials for applications in biomedical science and health care.
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.
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"). Steady state fluorescence was observed at 540 nm for both Ag NC on ssDNA and dsDNA; emission at 650 nm is observed for Ag NC on dsDNA. The emission at 550 nm is eight times weaker than that at 650 nm. Fluorescence up-conversion was used to study the dynamics of the emission. Bi-exponential fluorescence decay was recorded at 550 nm with lifetimes of 1 ps and 17 ps. The emission at 650 nm was not observed at the time scale investigated but has been reported to have a lifetime of 3.48 ns. Two-photon excited fluorescence was detected for Ag NC on dsDNA at 630 nm when excited at 800 nm. The two-photon absorption cross-section was calculated to be ∼3000 GM. Femtosecond transient absorption experiments were performed to investigate the excited state dynamics of DNA-Ag NC. An excited state unique to Ag NC on dsDNA was identified at ∼580 nm as an excited state bleach that related directly to the emission at 650 nm based on the excitation spectrum. Based on the optical results, a simple four level system is used to describe the emission mechanism for Ag NC on dsDNA.
The emission mechanism and two-photon excited emission for silver nanoclusters on a DNA scaffold found a new excited state when an enhancer sequence is introduced.
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•Computationally efficient design of random assemblies of multiple chromophores for panchromatic light-harvesting.•Use of approximate modeling scheme validated against both experiment ...and numerical computational methods.•Inputs to model can be tabulated as function of pairwise Förster radius values and chromophore concentrations.•Results useful for design of light-harvesting systems, sensors or optical materials using mesoscale self-assembly.
Developing efficient panchromatic light harvesting systems that exploit the energy available from the entire solar spectrum in an economically feasible and scalable fashion is of great importance. Light harvesting by incorporating multiple chromophores into molecular assemblies such as micelles and vesicles is one method for accomplishing this result. In this paper, we describe panchromatic light harvesting in lipid-based vesicle bilayers that contain a random distribution of lipid-bound chromophores. Numerically exact modeling based on Förster theory is developed to establish the criteria for designing a highly efficient panchromatic light-harvesting unit. An approximate modeling method is also developed to greatly reduce the complexity of the modeling problem. Both the exact and approximate models are verified by designing and experimentally testing an efficient three-chromophore light-harvesting system. For the experiments, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod) as the lowest energy chromophore, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD) as an intermediate acceptor, and Marina Blue® 1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine (MB) as the highest energy absorber (donor) are selected. From chromophore concentrations and R0 values, modeling predicts an overall efficiency of energy transfer to the terminal acceptor greater than ≈0.6 across a broad excitation wavelength range of ≈250 nm. The predicted transfer efficiency is verified by the experimental results. In addition, comparison of the approximate modeling method with both the numerically exact method and experimental results confirms that the computationally efficient approximate method is sufficiently accurate to guide choices of experimental parameters such as chromophore concentration. Overall, these results show predictive design of panchromatic light-harvesting performance can be performed rapidly and efficiently using an approximate kinetic model for randomly distributed assemblies of multiple chromophores.
Biological molecular imaging and sensing requires fluorophores that are not only stable and bright, but also small enough to allow the unencumbered observation of the movement of proteins. Toward ...this goal, we have studied the formation of fluorescent metallic gold nanoclusters stabilized by small molecule ligands. The morpholine and piperazine backbones of Good’s buffers were used to template fluorescent clusters, through a process of etching of nanoparticles first formed in the reaction. The clusters are found to be subnanometer sized, with nanosecond fluorescence lifetimes and as bright, or brighter, than the commercial dye norharmane.
Multiple intervalence‐transfer (IT) absorptions (see picture) appear in the near‐IR/IR spectra of the mixed‐valence complex Cl3RuII(tppz)RuIIICl3− (tppz=tetrakis(2‐pyridyl)pyrazine), whose X‐ray ...crystal structure and electronic and vibrational spectroscopic features indicate localized‐to‐delocalized (Class II–III) behavior.
Lipid vesicles are used as the organizational structure of self-assembled light-harvesting systems. Following analysis of 17 chromophores, six were selected for inclusion in vesicle-based antennas. ...The complementary absorption features of the chromophores span the near-ultraviolet, visible, and near-infrared region. Although the overall concentration of the pigments is low (∼1 μM for quantitative spectroscopic studies) in a cuvette, the lipid-vesicle system affords high concentration (≥10 mM) in the bilayer for efficient energy flow from donor to acceptor. Energy transfer was characterized in 13 representative binary mixtures using static techniques (fluorescence–excitation versus absorptance spectra, quenching of donor fluorescence, modeling emission spectra of a mixture versus components) and time-resolved spectroscopy (fluorescence, ultrafast absorption). Binary donor–acceptor systems that employ a boron-dipyrrin donor (S0 ↔ S1 absorption/emission in the blue-green) and a chlorin or bacteriochlorin acceptor (S0 ↔ S1 absorption/emission in the red or near-infrared) have an average excitation-energy-transfer efficiency (ΦEET) of ∼50%. Binary systems with a chlorin donor and a chlorin or bacteriochlorin acceptor have ΦEET ∼ 85%. The differences in ΦEET generally track the donor-fluorescence/acceptor-absorption spectral overlap within a dipole–dipole coupling (Förster) mechanism. Substantial deviation from single-exponential decay of the excited donor (due to the dispersion of donor–acceptor distances) is expected and observed. The time profiles and resulting ΦEET are modeled on the basis of (Förster) energy transfer between chromophores relatively densely packed in a two-dimensional compartment. Initial studies of two ternary and one quaternary combination of chromophores show the enhanced spectral coverage and energy-transfer efficacy expected on the basis of the binary systems. Collectively, this approach may provide one of the simplest designs for self-assembled light-harvesting systems that afford broad solar collection and efficient energy transfer.
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