David S Kong

Parallel gene synthesis in a microfluidic device

The ability to synthesize custom de novo DNA constructs rapidly, accurately and inexpensively is highly desired by researchers, as synthetic genes and longer DNA constructs are enabling to numerous powerful applications in both traditional molecular biology and the emerging field of synthetic biology. However, the current cost of de novo synthesis—driven largely by reagent and handling costs—is a significant barrier to the widespread availability of such technology. In this work, we demonstrate, to our knowledge, the first gene synthesis in a microfluidic environment. The use of microfluidic technology greatly reduces reaction volumes and the corresponding reagent and handling costs. Additionally, microfluidic technology enables large numbers of complex reactions to be performed in parallel. Here, we report the fabrication of a multi-chamber microfluidic device and its use in carrying out the syntheses of several DNA constructs. Genes up to 1 kb in length were synthesized in parallel at minute starting oligonucleotide concentrations (10–25 nM) in four 500 nl reactors. Such volumes are one to two orders of magnitude lower than those utilized in conventional gene synthesis. The identity of all target genes was verified by sequencing, and the resultant error rate was determined to be 1 per 560 bases.

Nanostructure fabrication by direct electron-beam writing of nanoparticles

Direct additive-layer fabrication of nanostructures is a widely sought goal, which is not possible using traditional layered resist optical and electron-beam lithographic techniques. However, recently, it has been shown that certain metallic and semiconducting nanoparticles capped with protective organic groups are promising “inklike” resist materials for patterning a variety of electronic and mechanical structures [C. A. Bulthaup et al., Appl. Phys. Lett. 79, 1525 (2001)]. Several groups have successfully patterned single-layer gold nanoparticle films by means of direct electron-beam writing [X. M. Lin, R. Parthasarathy, and H. M. Jaeger, Appl. Phys. Lett. 78, 1915 (2001); T. R. Bedson, R. E. Palmer, T. E. Jenkins, D. J. Hayton, and J. P. Wilcoxon, Appl. Phys. Lett. 78, 1921 (2001); L. Clarke et al., Appl. Phys. Lett. 71, 617 (1997)]. In this work, we apply these materials in a new lithographic mode, using an electron beam to cause direct sintering of these 2–10 nm nanoparticles, building structures of m...

DNA Assembly in 3D Printed Fluidics

The process of connecting genetic parts—DNA assembly—is a foundational technology for synthetic biology. Microfluidics present an attractive solution for minimizing use of costly reagents, enabling multiplexed reactions, and automating protocols by integrating multiple protocol steps. However, microfluidics fabrication and operation can be expensive and requires expertise, limiting access to the technology. With advances in commodity digital fabrication tools, it is now possible to directly print fluidic devices and supporting hardware. 3D printed micro- and millifluidic devices are inexpensive, easy to make and quick to produce. We demonstrate Golden Gate DNA assembly in 3D-printed fluidics with reaction volumes as small as 490 nL, channel widths as fine as 220 microns, and per unit part costs ranging from

Barriers to Drug Delivery in Interventional Oncology

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Enabling Microfluidics: from Clean Rooms to Makerspaces

5.71. A 3D-printed syringe pump with an accompanying programmable software interface was designed and fabricated to operate the devices. Quick turnaround and inexpensive materials allowed for rapid exploration of device parameters, demonstrating a manufacturing paradigm for designing and fabricating hardware for synthetic biology.

3D Printed Multimaterial Microfluidic Valve.

Although much attention has been paid to mechanisms of anticancer drug resistance that focus on intracellular processes that protect tumor cells, it has recently become increasingly evident that the unique features of the tumor microenvironment profoundly impact the efficacy of cancer therapies. The properties of this extracellular milieu, including increased interstitial pressure, decreased pH, hypoxia, and abnormal vascularity, result in limited drug efficacy; this finding is true not only for systemic chemotherapy but also for catheter-based therapies, including chemoembolization and radioembolization. The present review summarizes the barriers to drug delivery imposed by the tumor microenvironment and provides methods to overcome these hurdles.

Open-source, community-driven microfluidics with Metafluidics

The traditional requirement for clean rooms and specialized skills has inhibited many biologists from pursuing new microfluidic innovations. Makerspaces provide a growing alternative to clean rooms: they provide low-cost access to fabrication equipment such as laser cutters, plotter cutters, and 3D printers; use commercially available materials; and attract a diverse community of product designers. This Opinion discusses the materials, tools, and building methodologies particularly suited for developing novel microfluidic devices in these spaces, with insight into biological applications and leveraging the maker community. The lower barrier to access of makerspaces ameliorates the otherwise poor accessibility and scalability of microfluidic prototyping.

Conductive nanostructure fabrication by focused ion beam direct-writing of silver nanoparticles

We present a novel 3D printed multimaterial microfluidic proportional valve. The microfluidic valve is a fundamental primitive that enables the development of programmable, automated devices for controlling fluids in a precise manner. We discuss valve characterization results, as well as exploratory design variations in channel width, membrane thickness, and membrane stiffness. Compared to previous single material 3D printed valves that are stiff, these printed valves constrain fluidic deformation spatially, through combinations of stiff and flexible materials, to enable intricate geometries in an actuated, functionally graded device. Research presented marks a shift towards 3D printing multi-property programmable fluidic devices in a single step, in which integrated multimaterial valves can be used to control complex fluidic reactions for a variety of applications, including DNA assembly and analysis, continuous sampling and sensing, and soft robotics.

Community driven design of living technologies

Microfluidic devices have the potential to automate and miniaturize biological experiments, but open-source sharing of device designs has lagged behind sharing of other resources such as software. Synthetic biologists have used microfluidics for DNA assembly, cell-free expression, and cell culture, but a combination of expense, device complexity, and reliance on custom set-ups hampers their widespread adoption. We present Metafluidics, an open-source, community-driven repository that hosts digital design files, assembly specifications, and open-source software to enable users to build, configure, and operate a microfluidic device. We use Metafluidics to share designs and fabrication instructions for both a microfluidic ring-mixer device and a 32-channel tabletop microfluidic controller. This device and controller are applied to build genetic circuits using standard DNA assembly methods including ligation, Gateway, Gibson, and Golden Gate. Metafluidics is intended to enable a broad community of engineers, DIY enthusiasts, and other nontraditional participants with limited fabrication skills to contribute to microfluidic research.

Experiments and simulations on short chain fatty acid production in a colonic bacterial community

A focused ion beam has been used to directly pattern thin films of organometallic silver nanoparticles down to a resolution of 100nm. The unexposed regions were washed in hexane leaving the desired pattern, and subsequent annealing formed conductive, metallic features. Multiple-layer structures were also fabricated by spin-coating and exposing additional films of silver nanoparticles on top of already patterned structures. The sensitivity of the nanoparticles to 30keVGa+ ions was measured to be approximately 5μC∕cm2. Using this technique test structures were fabricated in two and three dimensions with resistivities as low as 288μΩcm and 13μΩcm for single- and multiple-layer structures, respectively, as compared to a value of 1.589μΩcm for bulk silver. To our knowledge, this is the highest demonstrated throughput for any electron or ion beam direct-write process utilizing metal-organic precursors.