Biochemically Programmable Isothermal PCR.

Isothermal PCR can be performed by imposing a static temperature gradient that continuously circulates reagents through denaturing, annealing, and extension conditions inside a PCR tube. But despite early promise, these systems have yet to demonstrate performance and repeatability sufficient for adoption in validated laboratory tests because the rate-limiting extension step is inherently short and cannot be increased independently of the other stages in a temperature cycle. Here, a discovery that enables isothermal PCR to be achieved with statistically robust repeatability that meets or exceeds diagnostic assay requirements (false positive/negative rate <8% at 95% confidence) by manipulating the interplay between the DNA replication biochemistry (via the amplicon GC content) and the microscale circulatory flow inside a PCR tube is reported.

Microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine.

Exosomes, the smallest sized extracellular vesicles (∽30-150 nm) packaged with lipids, proteins, functional messenger RNAs and microRNAs, and double-stranded DNA from their cells of origin, have emerged as key players in intercellular communication. Their presence in bodily fluids, where they protect their cargo from degradation, makes them attractive candidates for clinical application as innovative diagnostic and therapeutic tools. But routine isolation and analysis of high purity exosomes in clinical settings is challenging, with conventional methods facing a number of drawbacks including low yield and/or purity, long processing times, high cost, and difficulties in standardization.

Smartphone-Enabled Detection Strategies for Portable PCR-Based Diagnostics.

Incredible progress continues to be made toward development of low-cost nucleic acid-based diagnostic solutions suitable for deployment in resource-limited settings. Detection components play a vitally important role in these systems, but have proven challenging to adapt for operation in a portable format. Here we describe efforts aimed at leveraging the capabilities of consumer-class smartphones as a convenient platform to enable detection of nucleic acid products associated with DNA amplification via the polymerase chain reaction (PCR).

Synchronized chaotic targeting and acceleration of surface chemistry in prebiotic hydrothermal microenvironments.

Porous mineral formations near subsea alkaline hydrothermal vents embed microenvironments that make them potential hot spots for prebiotic biochemistry. But, synthesis of long-chain macromolecules needed to support higher-order functions in living systems (e.g.

Tunable in-situ electro-polymerization of hydrogel films for microchip-based bioanalysis.

Electro-polymerization phenomena have been previously investigated at the macroscale in the context of producing polymeric coatings over extended surface areas. But electrical actuation also offers exquisite local control of the polymerized films' position, morphology, and thickness, suggesting compelling advantages in microfluidic-based analysis systems. Here, we introduce a microfabricated platform incorporating arrays of individually addressable on-chip electrodes capable of generating discretely positioned electro-polymerized hydrogel films inside microchannels in timescales of ∼5 min.

Characterization of enzymatic micromachining for construction of variable cross-section microchannel topologies.

The ability to harness enzymatic activity as an etchant to precisely machine biodegradable substrates introduces new possibilities for microfabrication. This flow-based etching is straightforward to implement, enabling patterning of microchannels with topologies that incorporate variable depth along the cross-sectional dimension. Additionally, unlike conventional small-molecule formulations, the macromolecular nature of enzymatic etchants enables features to be precisely positioned.

Lab-on-a-Drone: Toward Pinpoint Deployment of Smartphone-Enabled Nucleic Acid-Based Diagnostics for Mobile Health Care.

We introduce a portable biochemical analysis platform for rapid field deployment of nucleic acid-based diagnostics using consumer-class quadcopter drones. This approach exploits the ability to isothermally perform the polymerase chain reaction (PCR) with a single heater, enabling the system to be operated using standard 5 V USB sources that power mobile devices (via battery, solar, or hand crank action). Time-resolved fluorescence detection and quantification is achieved using a smartphone camera and integrated image analysis app.

Instantaneous physico-chemical analysis of suspension-based nanomaterials.

High-throughput manufacturing of nanomaterial-based products demands robust online characterization and quality control tools capable of continuously probing the in-suspension state. But existing analytical techniques are challenging to deploy in production settings because they are primarily geared toward small-batch ex-situ operation in research laboratory environments. Here we introduce an approach that overcomes these limitations by exploiting surface complexation interactions that emerge when a micron-scale chemical discontinuity is established between suspended nanoparticles and a molecular tracer.

Noise-enhanced gel electrophoresis.

Macromolecules confined within a nanoporous matrix experience entropic trapping when their dimensions approach the average pore size, leading to emergence of anomalous transport behavior that can be beneficial in separation applications. But the ability to exploit these effects in practical settings (e.g.

Entropic stochastic resonance enables trapping under periodic confinement: a Brownian-dynamics study.

Entropically mediated phenomena are of emerging interest as a driving force for microscale and nanoscale transport, but their underlying stochastic nature makes them challenging to rationally manipulate and control. Stochastic resonance offers an intriguing avenue to overcome these difficulties by establishing a clear connection between the system response (the output) and an externally imposed driving force (the input). Previous studies have generally adopted a signal-processing viewpoint to classify the output in terms of a signal-to-noise ratio, but this link does not convey information that is immediately useful to infer parameters relevant to transport.

Using microchip gel electrophoresis to probe DNA-drug binding interactions.

Binding of small molecules with DNA plays an important role in many biological functions such as DNA replication, repair, and transcription. These interactions also offer enormous potential as targets for diagnostics and therapeutics, leading to intense interest in development of methods to probe the underlying binding events. In this chapter, we present a new approach to investigate the structural changes that accompany binding of DNA and small molecules.

Microscale chaotic advection enables robust convective DNA replication.

The ability of chaotic advection under microscale confinement to direct chemical processes along accelerated kinetic pathways has been recognized for some time. However, practical applications have been slow to emerge because optimal results are often counterintuitively achieved in flows that appear to possess undesirably high disorder. Here we present a 3D time-resolved analysis of polymerase chain reaction (PCR)-mediated DNA replication across a broad ensemble of geometric states.

Embedding synthetic microvascular networks in poly(lactic acid) substrates with rounded cross-sections for cell culture applications.

Synthetic microvascular networks are essential to enable in vitro studies of cell biology, biophysics, hemodynamics, and drug discovery, as well as in applications involving tissue engineering and artificial vasculature. But current limitations make it challenging to construct networks incorporating a hierarchy of microchannel diameters that possess cell-favored circular cross-sectional topographies. We report a new approach that overcomes these limitations by employing pressure-assisted expansion of biocompatible degradable poly(lactic acid) (PLA) substrates.

A nanometre-scale resolution interference-based probe of interfacial phenomena between microscopic objects and surfaces.

Interferometric techniques have proven useful to infer proximity and local surface profiles of microscopic objects near surfaces. But a critical trade-off emerges between accuracy and mathematical complexity when these methods are applied outside the vicinity of closest approach. Here we introduce a significant advancement that enables reflection interference contrast microscopy to provide nearly instantaneous reconstruction of an arbitrary convex object's contour next to a bounding surface with nanometre resolution, making it possible to interrogate microparticle/surface interaction phenomena at radii of curvature 1,000 times smaller than those accessible by the conventional surface force apparatus.

Smartphone-based detection of unlabeled DNA via electrochemical dissolution.

We describe a novel approach that enables unlabeled biomolecules and chemical analytes to be detected using ordinary smartphone optics. Electrochemical reactivity of chromium, ordinarily considered detrimental, is harnessed here to generate a signature that can be easily seen by monitoring electrode dissolution under ordinary white-light illumination. The simplicity and robustness of this approach eliminates the need for labeling and/or pre-programming with specific receptors (e.

Graphene-enhanced nanorefrigerants.

Recent reports that a host liquid's thermal properties can be augmented by dispersal of small quantities of nanoparticles have stimulated intense interest as an intriguing avenue to produce advanced heat transfer fluids. But effects are challenging to exploit in practical settings because it is difficult to prepare refrigerant-based dispersions displaying sufficient long-term stability. Moreover, the most dramatic enhancements in thermal conductivity obtained using anisotropic nanomaterials (e.

Education: DNA replication using microscale natural convection.

There is a need for innovative educational experiences that unify and reinforce fundamental principles at the interface between the physical, chemical, and life sciences. These experiences empower and excite students by helping them recognize how interdisciplinary knowledge can be applied to develop new products and technologies that benefit society. Microfluidics offers an incredibly versatile tool to address this need.

A simple microfluidic probe of nanoparticle suspension stability.

We describe a simple experimental tool that enables stability of multicomponent nanoparticle suspensions to be readily assessed by establishing a confinement-imposed chemical discontinuity at the interface between co-flowing laminar streams in a microchannel. When applied to examine Al(2)O(3) nanoparticle suspensions, this method readily reveals compositions that are susceptible to aggregation even when conventional bulk measurements (zeta potential, dynamic light scattering, bulk viscosity) suggest only subtle differences between formulations. This microfluidic stability test enables simple and rapid assessment of quality and variability in complex multicomponent mixtures for which few, if any, comparable data exist.

Rapid PCR thermocycling using microscale thermal convection.

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed(1). Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.

Tailoring the nanoporous architecture of hydrogels to exploit entropic trapping.

Macromolecules embedded in a nanoporous matrix display anomalous transport behavior in the entropic trapping regime. But these phenomena have not been widely explored in hydrogel matrices because it has not been clear how to link them to the underlying heterogeneous nanopore morphology. Here we introduce a theoretical model that establishes this connection and describe microchip DNA electrophoresis experiments that demonstrate how entropic trapping effects can be exploited to yield a trend of increasing resolving power with DNA size (the opposite of what is conventionally observed).