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., polypeptides, proteins, and nucleic acids) cannot occur without enrichment of chemical precursors before initiating polymerization, and identifying a suitable mechanism has become a key unanswered question in the origin of life. Here, we apply simulations and in situ experiments to show how 3D chaotic thermal convection-flows that naturally permeate hydrothermal pore networks-supplies a robust mechanism for focused accumulation at discrete targeted surface sites. This interfacial enrichment is synchronized with bulk homogenization of chemical species, yielding two distinct processes that are seemingly opposed yet synergistically combine to accelerate surface reaction kinetics by several orders of magnitude. Our results suggest that chaotic thermal convection may play a previously unappreciated role in mediating surface-catalyzed synthesis in the prebiotic milieu.

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. Standard sample preparation is enabled by leveraging the drone's motors as centrifuges via 3D printed snap-on attachments. These advancements make it possible to build a complete DNA/RNA analysis system at a cost of ∼$50 ($US). Our instrument is rugged and versatile, enabling pinpoint deployment of sophisticated diagnostics to distributed field sites. This capability is demonstrated by successful in-flight replication of Staphylococcus aureus and λ-phage DNA targets in under 20 min. The ability to perform rapid in-flight assays with smartphone connectivity eliminates delays between sample collection and analysis so that test results can be delivered in minutes, suggesting new possibilities for drone-based systems to function in broader and more sophisticated roles beyond cargo transport and imaging.

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., electrophoretic separation of DNA) has been hindered by additional dispersion introduced as a consequence of the uncorrelated process by which the embedded macromolecules discretely hop from pore to pore. Here, we show how both the source and solution to these difficulties are intimately linked to the inherent dynamics of the underlying activated transport mechanism. By modulating the applied electric field at a frequency tuned to the characteristic activation timescale, a resonance condition can be established that synergistically combines accelerated mobility and reduced diffusion. This resonance effect can be precisely manipulated by adjusting the magnitude and period of the driving electric field, enabling enhanced separation performance and bi-directional transport of different-sized species to be achieved. Notably, these phenomena are readily accessible in ordinary hydrogels (as opposed to idealized nanomachined topologies) suggesting broad potential to apply them in a host of useful settings.

Intein-triggered artificial protein hydrogels that support the immobilization of bioactive proteins.

Protein hydrogels have important applications in tissue engineering, drug delivery, and biofabrication. We present the development of a novel self-assembling protein hydrogel triggered by mixing two soluble protein block copolymers, each containing one half of a split intein. Mixing these building blocks initiates an intein trans-splicing reaction that yields a hydrogel that is highly stable over a wide range of pH (6-10) and temperature (4-50 °C), instantaneously recovers its mechanical properties after shear-induced breakdown, and is compatible with both aqueous and organic solvents. Incorporating a "docking station" peptide into the hydrogel building blocks enables simple and stable immobilization of docking protein-fused bioactive proteins in the hydrogel. This intein-triggered protein hydrogel technology opens new avenues for both in vitro metabolic pathway construction and functional/biocompatible tissue engineering scaffolds and provides a convenient platform for immobilizing enzymes in industrial biocatalysis.

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. The resulting parametric map reveals an unexpectedly wide operating regime where reaction rates remain constant over 2 orders of magnitude of the Rayleigh number, encompassing virtually any realistic PCR condition (temperature, volume, gravitational alignment), a level of robustness previously thought unattainable in the convective format.

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.g., oligonucleotide probes), making it feasible to greatly expand availability of a host of assays where detection complexity is a limiting constraint.

Interfacial complexation explains anomalous diffusion in nanofluids.

A recent report describing dramatic anomalous enhancement in mass transport properties of nanofluids (>1000% increase in tracer dye diffusivity) has excited intense interest, but the findings have yet to be conclusively confirmed or explained. Here we investigate these phenomena using a microfluidic approach to directly probe tracer diffusion so that interactions between the suspension's principle components (nanoparticles, surfactant, and dye) can be clearly identified. Under conditions matching previously reported studies, we unexpectedly observe spontaneous formation of highly focused and intensely fluorescent plumes at the interface between fluid streams, suggesting strong complexation interactions between the dye and nanoparticles. These phenomena, driven by competition between the rates at which free tracer molecules are transported into the interfacial zone subsequently consumed by dye-nanoparticle complexation, have likely been incorrectly interpreted as anomalous diffusion enhancement. These interactions are important to consider when devising tracer-based studies of nanoparticle suspensions and may lay a foundation for new adsorption-based analytical methods.

Vortex-assisted DNA delivery.

Electroporation is one of the most widely used methods to deliver exogenous DNA payloads into cells, but a major limitation is that only a small fraction of the total membrane surface is permeabilized. Here we show how this barrier can be easily overcome by harnessing hydrodynamic effects associated with Dean flows that occur along curved paths. Under these conditions, cells are subjected to a combination of transverse vortex motion and rotation that enables the entire membrane surface to become uniformly permeabilized. Greatly improved transfection efficiencies are achievable with only a simple modification to the design of existing continuous flow electroporation systems.

Miniaturized system for rapid field inversion gel electrophoresis of DNA with real-time whole-gel detection.

Pulsed field gel electrophoresis (PFGE) methods have become standard tools in a wide range of DNA analysis applications. But many aspects of DNA migration phenomena under pulsed field conditions are not well understood as compared with the more conventional situation where the electric field is held constant. A key reason for this deficiency is that PFGE experiments are cumbersome to perform due to extremely long separation times (approximately 10-15 h) and the need to perform gel analysis by poststaining after completion of the run. Here we introduce an easy to build miniaturized slab gel apparatus that addresses these issues by enabling large DNA fragments up to 35 kb in length to be separated using field inversion gel electrophoresis (FIGE) in 60-90 min. The compact size of the device also allows the entire gel to be continuously monitored so that the separation processes can be imaged in real time using a high-resolution CCD camera. Arbitrary control over the applied voltage waveforms is achieved using a function generator interfaced with a high voltage amplifier. These capabilities allow us to probe the size dependence of fundamental physical parameters associated with DNA migration (mobility, diffusion, and separation resolution). These data reveal a surprising regime where separation resolution increases with DNA fragment size owing to a favorable interplay between mobility and diffusion scalings and highlight the important role of diffusion (a seldom quantified parameter). In addition to the practical benefit of separation times that are an order of magnitude faster than conventional instruments, the results of these studies provide a previously unavailable rational basis to identify optimal separation conditions and contribute new insights toward understanding the underlying physical processes that govern DNA electrophoresis in pulsed fields.

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).

A direct probe of the interplay between bilayer morphology and surface reactivity in polymersomes.

Bilayer vesicles self-assembled from amphiphilic poly(ethylene oxide)-b-polybutadiene (PEO-b-PBd) copolymers are cell-like structures whose high stability and tunable membrane properties make them ideal for use as potential drug carriers and cell mimicry templates. Understanding how the surface interactions (reaction, binding, etc.) are governed by the bilayer structure is critical to enable construction of polymersomes with tailored colloidal behavior. Here, we adapt a previously established chemical labeling method by incorporating coumarin functionalized copolymer into the vesicular structure. This allows us to probe the effect of poly(ethylene glycol) (PEG) brush and surface architecture on the bimolecular quenching reaction occurring at the polymersome surface. Using these measurements, we have tracked quenching in free solution, on bare particles, and on two types of vesicle surfaces: one where the functionalized copolymer groups are longer than the surrounding unfunctionalized copolymer, and one where both functionalized and unfunctionalized groups are the same length. We find that quenching in the presence of the PEG brush proceeds at less than half the free solution rate in both vesicle architectures. However, the quenching rate is further reduced when the functionalized and unfunctionalized groups are the same length. The surface reaction appears to be dominated by quencher diffusion, a conclusion supported by conductivity measurements and ion partition studies indicating that these effects arise as a consequence of retarded ion mobility in the presence of the PEG brush rather than ion exclusion effects. These studies reveal the interplay between the vesicle bilayer architecture (copolymer composition, chain length, local concentration surrounding the active site) and the surface reaction rate, thereby providing useful insights that can help guide the design of polymersomes with desired functional properties.

Reflection interference contrast microscopy of arbitrary convex surfaces.

Current accurate applications of reflection interference contrast microscopy (RICM) are limited to known geometries; when the geometry of the object is unknown, an approximated fringe spacing analysis is usually performed. To complete an accurate RICM analysis in more general situations, we review and improve the formulation for intensity calculation based on nonplanar interface image formation theory and develop a method for its practical implementation in wedges and convex surfaces. In addition, a suitable RICM model for an arbitrary convex surface, with or without a uniform layer such as a membrane or ultrathin coating, is presented. Experimental work with polymer vesicles shows that the coupling of the improved RICM image formation theory, the calculation method, and the surface model allow an accurate reconstruction of the convex bottom shape of an object close to the substrate by fitting its experimental intensity pattern.

DNA focusing using microfabricated electrode arrays.

Focusing methods are a key component in many miniaturized DNA analysis systems because they enable dilute samples to be concentrated to detectable levels while being simultaneously confined within a specified volume inside the microchannel. In this chapter, we describe a focusing method based on a device design incorporating arrays of addressable on-chip microfabricated electrodes that can locally increase the concentration of DNA in solution by electrophoretically sweeping it along the length of a microchannel. By applying a low voltage (approximately 1-2 V) between successive pairs of neighboring electrodes, the intrinsically negatively charged DNA fragments are induced to migrate toward and collect at each anode, thereby allowing the quantity of accumulated DNA to be precisely metered. We have characterized the kinetics of this process, and found the response to be robust over a range of different sample compositions and buffer environments.

Microchip DNA electrophoresis with automated whole-gel scanning detection.

Gel electrophoresis continues to play an important role in miniaturized bioanalytical systems, both as a stand alone technique and as a key component of integrated lab-on-a-chip diagnostics. Most implementations of microchip electrophoresis employ finish-line detection methods whereby fluorescently labeled analytes are observed as they migrate past a fixed detection point near the end of the separation channel. But tradeoffs may exist between the simultaneous goals of maximizing resolution (normally achieved by using longer separation channels) and maximizing the size range of analytes that can be studied (where shorter separation distances reduce the time required for the slowest analytes to reach the detector). Here we show how the miniaturized format can offer new opportunities to employ alternative detection schemes that can help address these issues by introducing an automated whole-gel scanning detection system that enables the progress of microchip-based gel electrophoresis of DNA to be continuously monitored along an entire microchannel. This permits flexibility to selectively observe smaller faster moving fragments during the early stages of the separation before they have experienced significant diffusive broadening, while allowing the larger slower moving fragments to be observed later in the run when they can be better resolved but without the need for them to travel the entire length of the separation channel. Whole-gel scanning also provides a continuous and detailed picture of the electrophoresis process as it unfolds, allowing fundamental physical parameters associated with DNA migration phenomena (e.g., mobility, diffusive broadening) to be rapidly and accurately measured in a single experiment. These capabilities are challenging to implement using finish-line methods, and make it possible to envision a platform capable of enabling separation performance to be rapidly screened in a wide range of gel matrix materials and operating conditions, even allowing separation and matrix characterization steps to be performed simultaneously in a single self-calibrating experiment.

A buoyancy-driven compact thermocycler for rapid PCR.

The authors have designed a novel convective flow-based thermocycling system capable of performing high-speed DNA amplification via the polymerase chain reaction in a simplified and inexpensive format. Successful amplification of a 191 bp influenza-A target is demonstrated within 25 minutes using a 10 muL reaction volume with no modification to standard laboratory protocols. The system is simple to assemble and can be readily integrated with existing laboratory instrumentation for automated operation.

Development of a miniature calorimeter for identification and detection of explosives and other energetic compounds.

The development of versatile systems capable of providing rapid, portable, and inexpensive detection of explosives and energetic compounds are critically needed to offer enhanced levels of protection against current and future threats to homeland security, as well as satisfying a wide range of applications in the fields of forensic analysis, emergency response, and industrial hazards analysis. Calorimetric techniques have been largely overlooked in efforts to develop advanced chemical analysis technology, largely because of limitations associated with the physical size of the instruments and the relatively long timescales (>30 min) required to obtain a result. This miniaturized calorimeter circumvents these limitations, thereby creating a first-of-its-kind system allowing thermal analysis to be performed in a portable format that can be configured for use in a variety of field operations with a significantly reduced response time (approximately 2 min). Unlike current explosives detectors, this system is based on calorimetric techniques that are inherently capable of providing direct measurements of energy release potential and therefore do not depend on prior knowledge of familiar compounds.

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). First, we show how fluorescence-based detection can be incorporated into a portable convective thermocycling system controlled by a smartphone app. Raw images captured by the phone's camera are processed to yield real-time amplification data comparable to benchtop instruments. Next, we leverage smartphone imaging to achieve label-free detection of PCR products by monitoring changes in electrochemical reactivity of embedded metal electrodes as the target DNA concentration increases during replication. These advancements make it possible to construct rugged inexpensive nucleic acid detection components that can be readily embedded in a variety of portable bioanalysis instruments.