Measuring Localized Redox Enzyme Electron Transfer in a Live Cell with Conducting Atomic Force Microscopy

Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of <i>E. coli</i>. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD⁺ binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD⁺ binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells

Cross-Linked Micellar Spherical Nucleic Acids from Thermoresponsive Templates

A one-pot synthesis of micellar spherical nucleic acid (SNA) nanostructures using Pluronic F127 as a thermoresponsive template is reported. These novel constructs are synthesized in a chemically straightforward process that involves intercalation of the lipid tails of DNA amphiphiles (CpG motifs for TLR-9 stimulation) into the hydrophobic regions of Pluronic F127 micelles, followed by chemical cross-linking and subsequent removal of non-cross-linked structures. The dense nucleic acid shell of the resulting cross-linked micellar SNA enhances their stability in physiological media and facilitates their rapid cellular internalization, making them effective TLR-9 immunomodulatory agents. These constructs underscore the potential of SNAs in regulating immune response and address the relative lack of stability of noncovalent constructs

Windowless Observation of Evaporation-Induced Coarsening of Au–Pt Nanoparticles in Polymer Nanoreactors

The interactions between nanoparticles and solvents play a critical role in the formation of complex, metastable nanostructures. However, direct observation of such interactions with high spatial and temporal resolution is challenging with conventional liquid-cell transmission electron microscopy (TEM) experiments. Here, a windowless system consisting of polymer nanoreactors deposited via scanning probe block copolymer lithography (SPBCL) on an amorphous carbon film is used to investigate the coarsening of ultrafine (1–3 nm) Au–Pt bimetallic nanoparticles as a function of solvent evaporation. In such reactors, homogeneous Au–Pt nanoparticles are synthesized from metal-ion precursors in situ under electron irradiation. The nonuniform evaporation of the thin polymer film not only concentrates the nanoparticles but also accelerates the coalescence kinetics at the receding polymer edges. Qualitative analysis of the particle forces influencing coalescence suggests that capillary dragging by the polymer edges plays a significant role in accelerating this process. Taken together, this work (1) provides fundamental insight into the role of solvents in the chemistry and coarsening behavior of nanoparticles during the synthesis of polyelemental nanostructures, (2) provides insight into how particles form via the SPBCL process, and (3) shows how SPBCL-generated domes, instead of liquid cells, can be used to study nanoparticle formation. More generally, it shows why conventional models of particle coarsening, which do not take into account solvent evaporation, cannot be used to describe what is occurring in thin film, liquid-based syntheses of nanostructures

Windowless Observation of Evaporation-Induced Coarsening of Au–Pt Nanoparticles in Polymer Nanoreactors

The interactions between nanoparticles and solvents play a critical role in the formation of complex, metastable nanostructures. However, direct observation of such interactions with high spatial and temporal resolution is challenging with conventional liquid-cell transmission electron microscopy (TEM) experiments. Here, a windowless system consisting of polymer nanoreactors deposited via scanning probe block copolymer lithography (SPBCL) on an amorphous carbon film is used to investigate the coarsening of ultrafine (1–3 nm) Au–Pt bimetallic nanoparticles as a function of solvent evaporation. In such reactors, homogeneous Au–Pt nanoparticles are synthesized from metal-ion precursors in situ under electron irradiation. The nonuniform evaporation of the thin polymer film not only concentrates the nanoparticles but also accelerates the coalescence kinetics at the receding polymer edges. Qualitative analysis of the particle forces influencing coalescence suggests that capillary dragging by the polymer edges plays a significant role in accelerating this process. Taken together, this work (1) provides fundamental insight into the role of solvents in the chemistry and coarsening behavior of nanoparticles during the synthesis of polyelemental nanostructures, (2) provides insight into how particles form via the SPBCL process, and (3) shows how SPBCL-generated domes, instead of liquid cells, can be used to study nanoparticle formation. More generally, it shows why conventional models of particle coarsening, which do not take into account solvent evaporation, cannot be used to describe what is occurring in thin film, liquid-based syntheses of nanostructures

Windowless Observation of Evaporation-Induced Coarsening of Au–Pt Nanoparticles in Polymer Nanoreactors

The interactions between nanoparticles and solvents play a critical role in the formation of complex, metastable nanostructures. However, direct observation of such interactions with high spatial and temporal resolution is challenging with conventional liquid-cell transmission electron microscopy (TEM) experiments. Here, a windowless system consisting of polymer nanoreactors deposited via scanning probe block copolymer lithography (SPBCL) on an amorphous carbon film is used to investigate the coarsening of ultrafine (1–3 nm) Au–Pt bimetallic nanoparticles as a function of solvent evaporation. In such reactors, homogeneous Au–Pt nanoparticles are synthesized from metal-ion precursors in situ under electron irradiation. The nonuniform evaporation of the thin polymer film not only concentrates the nanoparticles but also accelerates the coalescence kinetics at the receding polymer edges. Qualitative analysis of the particle forces influencing coalescence suggests that capillary dragging by the polymer edges plays a significant role in accelerating this process. Taken together, this work (1) provides fundamental insight into the role of solvents in the chemistry and coarsening behavior of nanoparticles during the synthesis of polyelemental nanostructures, (2) provides insight into how particles form via the SPBCL process, and (3) shows how SPBCL-generated domes, instead of liquid cells, can be used to study nanoparticle formation. More generally, it shows why conventional models of particle coarsening, which do not take into account solvent evaporation, cannot be used to describe what is occurring in thin film, liquid-based syntheses of nanostructures

Windowless Observation of Evaporation-Induced Coarsening of Au–Pt Nanoparticles in Polymer Nanoreactors

The interactions between nanoparticles and solvents play a critical role in the formation of complex, metastable nanostructures. However, direct observation of such interactions with high spatial and temporal resolution is challenging with conventional liquid-cell transmission electron microscopy (TEM) experiments. Here, a windowless system consisting of polymer nanoreactors deposited via scanning probe block copolymer lithography (SPBCL) on an amorphous carbon film is used to investigate the coarsening of ultrafine (1–3 nm) Au–Pt bimetallic nanoparticles as a function of solvent evaporation. In such reactors, homogeneous Au–Pt nanoparticles are synthesized from metal-ion precursors in situ under electron irradiation. The nonuniform evaporation of the thin polymer film not only concentrates the nanoparticles but also accelerates the coalescence kinetics at the receding polymer edges. Qualitative analysis of the particle forces influencing coalescence suggests that capillary dragging by the polymer edges plays a significant role in accelerating this process. Taken together, this work (1) provides fundamental insight into the role of solvents in the chemistry and coarsening behavior of nanoparticles during the synthesis of polyelemental nanostructures, (2) provides insight into how particles form via the SPBCL process, and (3) shows how SPBCL-generated domes, instead of liquid cells, can be used to study nanoparticle formation. More generally, it shows why conventional models of particle coarsening, which do not take into account solvent evaporation, cannot be used to describe what is occurring in thin film, liquid-based syntheses of nanostructures

Energetically Biased DNA Motor Containing a Thermodynamically Stable Partial Strand Displacement State

Current work in tuning DNA kinetics has focused on changing toehold lengths and DNA concentrations. However, kinetics can also be improved by enhancing the completion probability of the strand displacement process. Here, we execute this strategy by creating a toehold DNA motor device with the inclusion of a synthetic nucleotide, inosine, at selected sites. Furthermore, we found that the energetic bias can be tuned such that the device can stay in a stable partially displaced state. This work demonstrates the utility of energetic biases to change DNA strand displacement kinetics and introduces a complementary strategy to the existing designs