Samuel M. Stavis

Microfluidic Mixing and the Formation of Nanoscale Lipid Vesicles

We investigate the formation of unilamellar lipid vesicles (liposomes) with diameters of tens of nanometers by controlled microfluidic mixing and nanoparticle determination (COMMAND). Our study includes liposome synthesis experiments and numerical modeling of our microfluidic implementation of the batch solvent injection method. We consider microfluidic liposome formation from the perspective of fluid interfaces and convective-diffusive mixing, as we find that bulk fluid flow parameters including hydrodynamically focused alcohol stream width, final alcohol concentration, and shear stress do not primarily determine the vesicle formation process. Microfluidic device geometry in conjunction with hydrodynamic flow focusing strongly influences vesicle size distributions, providing a coarse method to control liposome size, while total flow rate allows fine-tuning the vesicle size in certain focusing regimes. Although microfluidic liposome synthesis is relatively simple to implement experimentally, numerical simulations of the mixing process reveal a complex system of fluid flow and mass transfer determining the formation of nonequilibrium vesicles. These results expand our understanding of the microfluidic environment that controls liposome self-assembly and yield several technological advances for the on-chip synthesis of nanoscale lipid vesicles.

Conformation, Length, and Speed Measurements of Electrodynamically Stretched DNA in Nanochannels

A method is presented to rapidly and precisely measure the conformation, length, speed, and fluorescence intensity of single DNA molecules constrained by a nanochannel. DNA molecules were driven electrophoretically from a nanoslit into a nanochannel to confine and dynamically elongate them beyond their equilibrium length for repeated detection via laser-induced fluorescence spectroscopy. A single-molecule analysis algorithm was developed to analytically model bursts of fluorescence and determine the folding conformation of each stretched molecule. This technique achieved a molecular length resolution of 114 nm and an analysis time of around 20 ms per molecule, which enabled the sensitive investigation of several aspects of the physical behavior of DNA in a nanochannel. lambda-bacteriophage DNA was used to study the dependence of stretching on the applied device bias, the effect of conformation on speed, and the amount of DNA fragmentation in the device. A mixture of lambda-bacteriophage with the fragments of its own HindIII digest, a standard DNA ladder, was sized by length as well as by fluorescence intensity, which also allowed the characterization of DNA speed in a nanochannel as a function of length over two and a half orders of magnitude.

Microfluidic Directed Self-Assembly of Liposome-Hydrogel Hybrid Nanoparticles

We present a microfluidic method to direct the self-assembly of temperature-sensitive liposome-hydrogel hybrid nanoparticles. Our approach yields nanoparticles with structural properties and highly monodisperse size distributions precisely controlled across a broad range relevant to the targeted delivery and controlled release of encapsulated therapeutic agents. We used microfluidic hydrodynamic focusing to control the convective-diffusive mixing of two miscible nanoparticle precursor solutions (a DPPC:cholesterol:DCP phospholipid formulation in isopropanol and a photopolymerizable N-isopropylacrylamide mixture in aqueous buffer) to form nanoscale lipid vesicles with encapsulated hydrogel precursors. These precursor nanoparticles were collected off-chip and were irradiated with ultraviolet (UV) light in bulk to polymerize the nanoparticle interiors into hydrogel cores. Multiangle laser light scattering in conjunction with asymmetric flow field-flow fractionation was used to characterize nanoparticle size distributions, which spanned the approximately 150 to approximately 300 nm diameter range as controlled by microfluidic mixing conditions, with a polydispersity of approximately 3% to approximately 5% (relative standard deviation). Transmission electron microscopy was then used to confirm the spherical shape and core-shell composition of the hybrid nanoparticles. This method may be extended to the directed self-assembly of other similar cross-linked hybrid nanoparticle systems with engineered size/structure-function relationships for practical use in healthcare and life science applications.

Kilohertz Rotation of Nanorods Propelled by Ultrasound, Traced by Microvortex Advection of Nanoparticles

We measure the microvortical flows around gold nanorods propelled by ultrasound in water using polystyrene nanoparticles as optical tracers. We infer the rotational frequencies of such nanomotors assuming a hydrodynamic model of this interaction. In this way, we find that nanomotors rotate around their longitudinal axes at frequencies of up to ≈ 2.5 kHz, or ≈ 150 000 rpm, in the planar pressure node of a half-wavelength layered acoustic resonator driven at ≈ 3 MHz with an acoustic energy density of <10 J·m(-3). The corresponding tangential speeds of up to ≈ 2.5 mm·s(-1) at a nanomotor radius of ≈ 160 nm are 2 orders of magnitude faster than the translational speeds of up to ≈ 20 μm·s(-1). We also find that rotation and translation are independent modes of motion within experimental uncertainty. Our study is an important step toward understanding the behavior and fulfilling the potential of this dynamic nanotechnology for hydrodynamically interacting with biological media, as well as other applications involving nanoscale transport, mixing, drilling, assembly, and rheology. Our results also establish the fastest reported rotation of a nanomotor in aqueous solution.

Suspended glass nanochannels coupled with microstructures for single molecule detection

We present a nonlithographic approach for forming free standing nanochannels, made of a variety of materials, that can be easily integrated with microfabricated structures. The approach uses a deposited polymeric fiber as a sacrificial template around which a deposited coating forms a tube. We formed suspended nanochannels of silica glass spanning a trench on a silicon wafer and used these structures for detection of single fluorescently labeled proteins. This geometry provides excellent isolation of the molecules of interest and also separates them from surrounding material that could create unwanted background fluorescence. The same geometry provides a platform for observing motion and mechanical response of the suspended nanochannel, and we measured the mechanical resonance of a glass channel of the type used for the fluorescent detection. This type of structure provides a general approach for integrating fluid carrying nanochannels with microstructures.

Nanofluidic structures with complex three-dimensional surfaces

Nanofluidic devices have typically explored a design space of patterns limited by a single nanoscale structure depth. A method is presented here for fabricating nanofluidic structures with complex three-dimensional (3D) surfaces, utilizing a single layer of grayscale photolithography and standard integrated circuit manufacturing tools. This method is applied to construct nanofluidic devices with numerous (30) structure depths controlled from approximately 10 to approximately 620 nm with an average standard deviation of <10 nm over distances of >1 cm. A prototype 3D nanofluidic device is demonstrated that implements size exclusion of rigid nanoparticles and variable nanoscale confinement and deformation of biomolecules.

Single-molecule mobility and spectral measurements in submicrometer fluidic channels

Electrophoretic mobility differences of biological molecules are frequently exploited to physically separate and subsequently identify the components of a mixture. We present a method to rapidly identify single molecules by measuring both their mobility and fluorescence emission under continuous flow without separation. Submicrometer fluidic channels were used to detect individual nucleic-acid-engineered fluorescent labels driven electrokinetically in free solution. Two separate focal volumes along the length of the fluidic channel collected spectral, spatial, and temporal information from the passage of fluorescent labels through the channel. One focal volume was defined by a focused 488-nm-wavelength laser and the other by a focused 568-nm laser. The subfemtoliter focal volumes resulted in signal-to-noise ratios sufficient for single-fluorophore detection, and the two excitation wavelengths enabled detection of multicolor fluorescent labels and discrimination of single-color detection events. Each fluor...

Single molecule analysis of bacterial polymerase chain reaction products in submicrometer fluidic channels

Laser induced fluorescence in submicrometer fluidic channels was used to characterize the synthesis of polymerase chain reaction (PCR) products from a model bacterial system in order to explore the advantages and limitations of on chip real time single molecule PCR analysis. Single oligonucleotide universal bacterial primers and PCR amplicons from the 16S rDNA of Thermobifida fusca (325 bp) were directly detected at all phases of the reaction with low sample consumption and without post-amplification purification or size screening. Primers were fluorescently labeled with single Alexa Fluor 488 or Alexa Fluor 594 fluorophores, resulting in double labeled, two color amplicons. PCR products were driven electrokinetically through a fused silica channel with a 250 nm by 500 nm rectangular cross section. Lasers with 488 nm and 568 nm wavelengths were focused and overlapped on the channel for fluorescence excitation. All molecules entering the channel were rapidly and uniformly analyzed. Photon burst analysis was used to detect and identify individual primers and amplicons, and fluorescence correlation and cross-correlation spectroscopy were used to account for analyte flow speed. Conventional gel and capillary electrophoresis were also used to characterize the PCR amplification, and the results of differences in detection sensitivity and analyte discrimination were examined. Limits were imposed by the purity and labeling efficiency of the PCR reagents, which must be improved in parallel with increases in detection sensitivity.

MEMS Kinematics by Super-Resolution Fluorescence Microscopy

Super-resolution fluorescence microscopy is used for the first time to study the nanoscale kinematics of a MEMS device in motion across a surface. A device under test is labeled with fluorescent nanoparticles that form a microscale constellation of near-ideal point sources of light. The constellation is imaged by widefield epifluorescence microscopy, and the image of each nanoparticle is fit to a Gaussian distribution to calculate its position. Translations and rotations of the device are measured by computing the rigid transform that best maps the constellation from one image to the next. This technique is used to measure the stepwise motion of a scratch drive actuator across each of 500 duty cycles with 0.13-nm localization precision, 1.85-nm displacement uncertainty, and 100-μrad orientation uncertainty for a constellation diameter of 15 μm. This novel measurement reveals acute aperiodic variations in the step size of the actuator, which have been neither previously observed nor predicted by any of the published models of the operation of the device. These unexpected results highlight the importance of super-resolution fluorescence microscopy to the measurement of MEMS kinematics, which will have broad impact in fundamental investigations of surface forces, wear, and tribology in MEMS and related applications.

Optical tracking of nanoscale particles in microscale environments

The trajectories of nanoscale particles through microscale environments record useful information about both the particles and the environments. Optical microscopes provide efficient access to this information through measurements of light in the far field from nanoparticles. Such measurements necessarily involve trade-offs in tracking capabilities. This article presents a measurement framework, based on information theory, that facilitates a more systematic understanding of such trade-offs to rationally design tracking systems for diverse applications. This framework includes the degrees of freedom of optical microscopes, which determine the limitations of tracking measurements in theory. In the laboratory, tracking systems are assemblies of sources and sensors, optics and stages, and nanoparticle emitters. The combined characteristics of such systems determine the limitations of tracking measurements in practice. This article reviews this tracking hardware with a focus on the essential functions of nanoparticles as optical emitters and microenvironmental probes. Within these theoretical and practical limitations, experimentalists have implemented a variety of tracking systems with different capabilities. This article reviews a selection of apparatuses and techniques for tracking multiple and single particles by tuning illumination and detection, and by using feedback and confinement to improve the measurements. Prior information is also useful in many tracking systems and measurements, which apply across a broad spectrum of science and technology. In the context of the framework and review of apparatuses and techniques, this article reviews a selection of applications, with particle diffusion serving as a prelude to tracking measurements in biological, fluid, and material systems, fabrication and assembly processes, and engineered devices. In so doing, this review identifies trends and gaps in particle tracking that might influence future research.