Nanoscale protein patterning.

<p>Submicron squares were patterned with biotin and then labeled with strep-647. From left to right the squares have 1 μm, 500 nm, 300 nm and 250 nm sides, respectively; 250 nm is the resolution limit of the lithography tool. The inhomogeneity of fluorescent signal was most likely caused by the nature of super-resolution fluorescent imaging.</p

Sidewall coverage depends on how conformal the deposition process is.

<p>Biotin microarrays were generated by either Method 1 (A) or Method 2 (B) illustrated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195062#pone.0195062.g001" target="_blank">Fig 1</a>. Strep-647 was then used to label biotinylated surfaces. Sidewall conjugation is shown in A) pointed out by red arrows but not in B) due to the higher conformal deposition of LPCVD (B) vs. E-beam evaporation (A). Intensity profiles along the yellow dash lines in A) and B), are shown in C) and D) respectively. Red arrow in C) correspond to the side wall intensity labeled by the red arrows in A). Any misalignment between conjugated regions and wells was too small (<100nm) to measure with our fluorescent microscopy.</p

Germanium dissolution.

<p>A) SEM image of 50 nm Ge films deposited by LPCVD before dissolution. Red arrow indicates the edge of a 25 μm by 25 μm square well, etched 500nm deep. B) Higher magnification of red box in A). C&D) SEM images of Ge layers after 10min C) and overnight D) incubation in 0.35% H₂O₂ at room temperature. Ge was completely dissolved in 10min with no residues left. Faint square outlines are due to the over-etch process in the Ge etching recipe and thus the adhesive amorphous Si layer between Ge and HTO is etched away.</p

Biotin and streptavidin activity after H₂O₂ incubation.

<p>Microarrays were fabricated and conjugated with biotin as per Method 2. Streptavidin conjugated chips were obtained by incubating biotin conjugated chips with 100ug/ml non-fluorescent streptavidin solution overnight. Both biotin and streptavidin conjugated chips were then incubated in 0.35% H₂O₂ for 10min, 30min, 60min, 3h and 6h at room temperature. After H₂O₂ incubation, all chips were blocked in 1%BSA. Biotin chips were then labeled with 10ug/ml Strep-647 while streptavidin chips were labeled with 1ug/ml Biotin-488. Intensity was normalized by using control group (0min) as 100% intensity.</p

A Synthetic Chemomechanical Machine Driven by Ligand–Receptor Bonding

The ability to create synthetic chemomechanical machines with engineered functionality promises large technological rewards. However, current efforts in molecular chemistry are restrained by the formidable challenges faced in molecular structure and function prediction. An alternative approach to engineering machines with tailorable chemomechanical functionality is to design Brownian ratchet devices using molecular assemblies. We demonstrate this through the creation of autonomous molecular machines that sense, mechanically react, and extract energy from ligand–receptor binding. We present a specific instantiation, measuring approximately 100 nm in length, which actuates upon detection of a streptavidin ligand. Machines were designed through the tailoring of energy landscapes on 3D DNA origami motifs. We also analyzed the response over a logarithmic concentration ratio (device:ligand) range from 1:10¹ to 1:10⁵

A portable bioelectronic sensing system (BESSY) for environmental deployment incorporating differential microbial sensing in miniaturized reactors

<div><p>Current technologies are lacking in the area of deployable, <i>in situ</i> monitoring of complex chemicals in environmental applications. Microorganisms metabolize various chemical compounds and can be engineered to be analyte-specific making them naturally suited for robust chemical sensing. However, current electrochemical microbial biosensors use large and expensive electrochemistry equipment not suitable for on-site, real-time environmental analysis. Here we demonstrate a miniaturized, autonomous <u>b</u>io<u>e</u>lectronic <u>s</u>ensing <u>sy</u>stem (BESSY) suitable for deployment for instantaneous and continuous sensing applications. We developed a 2x2 cm footprint, low power, two-channel, three-electrode electrochemical potentiostat which wirelessly transmits data for on-site microbial sensing. Furthermore, we designed a new way of fabricating self-contained, submersible, miniaturized reactors (m-reactors) to encapsulate the bacteria, working, and counter electrodes. We have validated the BESSY’s ability to specifically detect a chemical amongst environmental perturbations using differential current measurements. This work paves the way for <i>in situ</i> microbial sensing outside of a controlled laboratory environment.</p></div

A miniaturized BESSY for deployable electrochemical monitoring.

<p>(a) Circuit diagram of a commercial, off the shelf (COTS) potentiostat featuring three on chip operational amplifiers on a MSP430FG437 microcontroller sending serial data to Redbear Labs Wifi Micro board for wireless data transmission. Only circuitry for one three-electrode channel is on board, but switching results in multiple channel potentiostat function. (b) Final potentiostat PCB (green) footprint is 2x2 cm and is connected to WiFi micro board (red); penny is shown for size comparison. (c) Contrast corrected photograph of the BESSY for differential sensing and deployment in aqueous environments.</p

Lactate sensing platform.

<p>Average lactate responsive (blue) and lactate null (red) current response to 40mM lactate injection at t = 16h (<b>o</b>), with standard deviations (yellow) for three technical repeats (repeated at the same time) of one of two similar biological repeats (bacteria grown on different days). The ~19 μA increase in current demonstrates the microbe’s ability to reduce the carbon fibers while allowing environmental chemicals to diffuse in and out of the silica coated electrode. It also demonstrates sensing of multiple channels using one device without crosstalk.</p

M-reactor for microbial integration.

<p>(a) Conceptual schematic of electrode detailing how the scaffold stabilizes the bacteria containing, carbon felt, working electrode (WE) compartment (black) with the nonconductive, agarose, counter electrode (CE) compartment (gray) then coated with a silica layer. Titanium electrodes are partially embedded in agarose for contact with the carbon felt and partially emerge from the reactor complex for attachment to the electronics. (b) Realization of m-reactor with working electrode compartment (black) contained in a 1% agarose mixture stabilized with a 3D printed PLA scaffold (orange); penny is shown for size comparison.</p

Fumarate BESSY responds amongst chemical and temperature perturbations.

<p>(a) Current in response to temperature fluctuations over time, simulated by immersing in an ice bath (◆) and removal from ice bath (<b>■</b>), shows both strains decrease and increase current production similarly. (b) The ratio of the two strains as a function of time does not change significantly, with RMS noise ~0.05. In this way, the sensor can filter out environmental variables such as temperature when deployed for environmental sensing. (c) Current in response to chemical perturbations over time generates no significant response when 10 mM lactate is added (<b>▲</b>), and a significant differential response when 1mM fumarate is added (⚫). (d) The ratio of the two currents shown above as a function of time. As expected, there is no significant fluctuation in the ratio upon addition of lactate, which contrasts the fumarate response. Figures shown here are representative of two biological repeats, each with three technical repeats (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184994#pone.0184994.s002" target="_blank">S2 Fig</a>). Response time, defined by time to reach two standard deviations above the average environmental noise, is 1.8±0.7 hours, and the maximum ratio of I_responsive/I_null is 2.1±0.4.</p