William L. Hwang

Droplet interface bilayers

Droplet interface bilayers (DIBs) provide a superior platform for the biophysical analysis of membrane proteins. The versatile DIBs can also form networks, with features that include built-in batteries and sensors.

Droplet Routing in the Synthesis of Digital Microfluidic Biochips

Recent advances in microfluidics are expected to lead to sensor systems for high-throughput biochemical analysis. CAD tools are needed to handle increased design complexity for such systems. Analogous to classical VLSI synthesis, a top-down design automation approach can shorten the design cycle and reduce human effort. We focus here on the droplet routing problem, which is a key issue in biochip physical design automation. We develop the first systematic droplet routing method that can be integrated with biochip synthesis. The proposed approach minimizes the number of cells used for droplet routing, while satisfying constraints imposed by throughput considerations and fluidic properties. A real-life biochemical application is used to evaluate the proposed method

Droplet networks with incorporated protein diodes show collective properties

Recently, we demonstrated that submicrolitre aqueous droplets submerged in an apolar liquid containing lipid can be tightly connected by means of lipid bilayers to form networks. Droplet interface bilayers have been used for rapid screening of membrane proteins and to form asymmetric bilayers with which to examine the fundamental properties of channels and pores. Networks, meanwhile, have been used to form microscale batteries and to detect light. Here, we develop an engineered protein pore with diode-like properties that can be incorporated into droplet interface bilayers in droplet networks to form devices with electrical properties including those of a current limiter, a half-wave rectifier and a full-wave rectifier. The droplet approach, which uses unsophisticated components (oil, lipid, salt water and a simple pore), can therefore be used to create multidroplet networks with collective properties that cannot be produced by droplet pairs.

Asymmetric Droplet Interface Bilayers

In cell membranes, the lipid compositions of the inner and outer leaflets differ. Therefore, a robust model system that enables single-channel electrical recording with asymmetric bilayers would be very useful. We and others recently developed the droplet interface bilayer (DIB), which is formed by connecting lipid monolayer-encased aqueous droplets submerged in an oil-lipid mixture. Here, we incorporate lipid vesicles of different compositions into aqueous droplets and immerse them in an oil bath to form asymmetric DIBs (a-DIBs). Both alpha-helical and beta-barrel membrane proteins insert readily into a-DIBs, and their activity can be measured by single-channel electrical recording. We show that the gating behavior of outer membrane protein G (OmpG) from Escherichia coli differs depending on the side of insertion in an asymmetric DIB with a positively charged leaflet opposing a negatively charged leaflet. The a-DIB system provides a general platform for studying the effects of bilayer leaflet composition on the behavior of ion channels and pores.

Screening Blockers Against a Potassium Channel with a Droplet Interface Bilayer Array

Droplet interface bilayers (DIBs) form between two lipid monolayer-encased aqueous droplets submerged in oil. Both major structural classes of membrane proteins, alpha-helix bundles and beta barrels, represented by channels and pores, respectively, spontaneously insert into DIBs when freshly expressed by cell-free transcription and translation. Electrodes embedded within the droplets allow the measurement of transmembrane ionic currents carried by individual channels and pores. On the basis of these findings, we have devised a chip-based approach for the rapid screening of blockers against ion channels. The technique is demonstrated here with the viral potassium channel, Kcv.

ISWI Remodelers Slide Nucleosomes with Coordinated Multi-Base-Pair Entry Steps and Single-Base-Pair Exit Steps

ISWI-family enzymes remodel chromatin by sliding nucleosomes along DNA, but the nucleosome translocation mechanism remains unclear. Here we use single-molecule FRET to probe nucleosome translocation by ISWI-family remodelers. Distinct ISWI-family members translocate nucleosomes with a similar stepping pattern maintained by the catalytic subunit of the enzyme. Nucleosome remodeling begins with a 7 bp step of DNA translocation followed by 3 bp subsequent steps toward the exit side of nucleosomes. These multi-bp, compound steps are comprised of 1 bp substeps. DNA movement on the entry side of the nucleosome occurs only after 7 bp of exit-side translocation, and each entry-side step draws in a 3 bp equivalent of DNA that allows three additional base pairs to be moved to the exit side. Our results suggest a remodeling mechanism with well-defined coordination at different nucleosomal sites featuring DNA translocation toward the exit side in 1 bp steps preceding multi-bp steps of DNA movement on the entry side.

Stepwise nucleosome translocation by RSC remodeling complexes

The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1–2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome. DOI: http://dx.doi.org/10.7554/eLife.10051.001

Defect-oriented testing and diagnosis of digital microfluidics-based biochips

Microfluidics-based biochips are soon expected to revolutionize biosensing, clinical diagnostics and drug discovery. Robust off-line and on-line test techniques are required to ensure system dependability as these biochips are deployed for safety-critical applications. Due to the underlying mixed-technology and mixed-energy domains, biochips exhibit unique failure mechanisms and defects. We first relate some realistic defects to fault models and observable errors. We next set up an experiment to evaluate the manifestations of electrode-short faults. Motivated by the experimental results, we present a testing and diagnosis methodology to detect catastrophic faults and locate faulty regions. The proposed method is evaluated using a biochip performing real-life multiplexed bioassays

Automated design of pin-constrained digital microfluidic arrays for lab-on-a-chip applications*

Microfluidics-based biochips, also referred to as lab-on-a-chip (LoC), are devices that integrate fluid-handling functions such as sample preparation, analysis, separation, and detection. This emerging technology combines electronics with biology to open new application areas such as point-of-care diagnosis, on-chip DNA analysis, and automated drug discovery. We propose a design automation method for pin-constrained LoCs that manipulate nanoliter volumes of discrete droplets on a microfluidic array. In contrast to the direct-addressing scheme that has been studied thus far in the literature, we assign a small number of independent control pins to a large number of electrodes in the LoC, thereby reducing design complexity and product cost. We apply the proposed method to a microfluidic array for a set of multiplexed bioassays

Epi-illumination through the microscope objective applied to darkfield imaging and microspectroscopy of nanoparticle interaction with cells in culture

Existing darkfield illumination schemes are incompatible with many types of samples and/or procedures. We present a darkfield epi-illumination scheme which addresses these incompatibilities by providing illumination through the imaging objective. We validate the system performance using silver nanospheres in varying refractive index environments, characterize the intensity distribution of the darkfield illumination, and demonstrate system capabilities through a preliminary study of functionalized gold nanosphere interactions with cancer cells in culture. We observe a broadened scattering spectrum from unconjugated nanoparticles, as compared with anti-EGFR conjugated nanoparticles, upon incubation with cancer cells, and discuss the implications of this observation.