Microscale HPLC enables a new paradigm for commercialization of complex chiral stationary phases

The small column size (0.3 mm i.d. x 15 cm) used in microscale HPLC contains only a small fraction (<1%) of the chromatographic packing material of a typical analytical HPLC column. Consequently, chromatographic stationary phases that are prohibitively expensive in conventional HPLC, owing either to synthetic complexity or costly starting materials, may become commercially viable in the microscale format. To illustrate this point, a previously described, synthetically complex, crown ether chiral stationary phase was prepared and evaluated in the microscale format, showing excellent separation of the enantiomers of underivatized amine analytes.     

Piezoelectric ink jet processing of materials for medical and biological applications

Many advanced medical and biological devices require microscale patterning of cells, proteins, and other biological materials. This article describes the use of piezoelectric ink jet processing in the fabrication of biosensors, cell-based assays, and other microscale medical devices. A microelectromechanical system-based piezoelectric transducer was used to develop uniform fluid flow through nozzles and to prepare well-defined microscale patterns of proteins, monofunctional acrylate ester, sinapinic acid, deoxyribonucleic acid (DNA), and DNA scaffolds on relevant substrates. Our results demonstrate that piezoelectric ink jet deposition is a powerful non-contact, non-destructive additive process for developing biosensors, cell culture systems, and other devices for medical and biological applications.     

Laminar-flow-based separations at the microscale

The natural separation maintained by microfluidic flows is employed as the basis of a particle/cell sorting device. This method of separating particulate suspensions exploits the inherent laminar nature of microscale fluid dynamics and incorporates applied fields and image cytometry to enable sorting based upon any visually identifiable difference between colloid-sized cells or particles. This technique may be used to easily isolate, separate, sort, or enrich virtually any suspension of microscale biological or colloidal particles within a microfluidic system. The entire footprint of the device described here is less than 0.01 mm(2), allowing it to be readily incorporated within highly integrated micro total analysis systems (microTAS).     

Design of Strain-Sensing Devices in Microscale by Using Surface Plasmon Polaritons

We have presented a strain-sensing device in microscale by using surface plasmon polaritons and multimode interference effects. The device is numerically investigated by the finite-difference time-domain method. Optimum depths and length of the structure are designed for sensing a strain. The size of the designed structure is several micrometers and is about a thousandth compared with a fiber Bragg grating strain sensor. The sensitivity of the designed structure is 11.34 pm/μ?? that is about ten times larger than that of a fiber Bragg grating strain sensor. The temperature sensitivity of the designed structure is 34.43 pm/ ^°C. This temperature sensitivity is three times larger than that of a fiber Bragg grating strain sensor. Therefore, temperature compensation techniques are needed for the structure. The presented structure has a simple design such as a plasmonic waveguide with a trench structure. The simple structural design device has a capability of being used in micro- and nano-electromechanical systems.     

Exploring biomolecular recognition using optical biosensors.

Understanding the basic forces that determine molecular recognition helps to elucidate mechanisms of biological processes and facilitates discovery of innovative biotechnological methods and materials for therapeutics, diagnostics, and separation science. The ability to measure interaction properties of biological macromolecules quantitatively across a wide range of affinity, size, and purity is a growing need of studies aimed at characterizing biomolecular interactions and the structural elements that drive them. Optical biosensors have provided an increasingly impactful technology for such biomolecular interaction analyses. These biosensors record the binding and dissociation of macromolecules in real time by transducing the accumulation of mass of an analyte molecule at the sensor surface coated with ligand molecule into an optical signal. Interactions of analytes and ligands can be analyzed at a microscale and without the need to label either interactant. Sensors enable the detection of bimolecular interaction as well as multimolecular assembly. Most notably, the method is quantitative and kinetic, enabling determination of both steady-state and dynamic parameters of interaction. This article describes the basic methodology of optical biosensors and presents several examples of its use to investigate such biomolecular systems as cytokine growth factor-receptor recognition, coagulation factor assembly, and virus-cell docking.     

Biodegradable Monocrystalline Silicon Photovoltaic Microcells as Power Supplies for Transient Biomedical Implants

Bioresorbable electronic materials serve as foundations for implantable devices that provide active diagnostic or therapeutic function over a timeframe matched to a biological process, and then disappear within the body to avoid secondary surgical extraction. Approaches to power supply in these physically transient systems are critically important. This paper describes a fully biodegradable, monocrystalline silicon photovoltaic (PV) platform based on microscale cells (microcells) designed to operate at wavelengths with long penetration depths in biological tissues (red and near infrared wavelengths), such that external illumination can provide realistic levels of power. Systematic characterization and theoretical simulations of operation under porcine skin and fat establish a foundational understanding of these systems and their scalability. In vivo studies of a representative platform capable of generating ≈60 µW of electrical power under 4 mm of porcine skin and fat illustrate an ability to operate blue light‐emitting diodes (LEDs) as subdermal implants in rats for 3 d. Here, the PV system fully resorbs after 4 months. Histological analysis reveals that the degradation process introduces no inflammatory responses in the surrounding tissues. The results suggest the potential for using silicon photovoltaic microcells as bioresorbable power supplies for various transient biomedical implants.     

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation,X-ray CT,FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram’s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems^1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.     

Partitioning of host and recombinant cells in aqueous two-phase polymer systems

The separation of host and recombinant Escherichia coli bacterial cells has been studied using the surface-sensitive technique of partitioning in aqueous two-phase polymer systems. Experiments were designed to probe charge-and hydrophobicity-related property differences of antibiotic-resistant recombinant cells and their antibiotic-sensitive hosts. Differential partitioning was observed in both charge-sensitive and non-charge-sensitive phase systems for three host-recombinant cell systems, but the non-charge-related effects appear to have a greater impact on partitioning behavior. This result suggests that plasmid-encoded products related to antibiotic resistance modify the surface hydrophobicity of the E. coli bacterial cell and that these differences can be exploited for cell separation.     

Complex proteome prefractionation using microscale solution isoelectrofocusing

The proteomes of mammalian cells, tissues and biologic fluids are complex and consist of proteins present over a wide dynamic range. Current protein profiling technologies do not have the capacity to overcome the sample complexity for comprehensive analysis of complex proteomes. A common strategy to substantially expand protein profiling capacities is sample prefractionation. A prefractionation method developed in the authors' laboratory, microscale solution isoelectrofocusing, has resulted in a commercial product, the ZOOM IEF Fractionator, which provides a simple and convenient method for high-resolution separation of complex proteomes based upon their isoelectric points. Complex human samples such as cancer cells and biologic fluids can be fractionated into well-resolved fractions with minimal cross-contamination of proteins between adjacent fractions. This review focuses on the application of microscale solution isoelectrofocusing prefractionation and subsequent downstream strategies in expanding protein profiling capacities and mining low-abundance proteins of complex proteomes.     

Complex proteome prefractionation using microscale solution isoelectrofocusing

The proteomes of mammalian cells, tissues and biologic fluids are complex and consist of proteins present over a wide dynamic range. Current protein profiling technologies do not have the capacity to overcome the sample complexity for comprehensive analysis of complex proteomes. A common strategy to substantially expand protein profiling capacities is sample prefractionation. A prefractionation method developed in the authors’ laboratory, microscale solution isoelectrofocusing, has resulted in a commercial product, the ZOOM^® IEF Fractionator, which provides a simple and convenient method for high-resolution separation of complex proteomes based upon their isoelectric points. Complex human samples such as cancer cells and biologic fluids can be fractionated into well-resolved fractions with minimal cross-contamination of proteins between adjacent fractions. This review focuses on the application of microscale solution isoelectrofocusing prefractionation and subsequent downstream strategies in expanding protein profiling capacities and mining low-abundance proteins of complex proteomes.     

Portable magnetic tweezers device enables visualization of the three-dimensional microscale deformation of soft biological materials

We have designed and built a magnetic tweezers device that enables the application of calibrated stresses to soft materials while simultaneously measuring their microscale deformation using confocal microscopy. Unlike previous magnetic tweezers designs, our device is entirely portable, allowing easy use on microscopes in core imaging facilities or in collaborators' laboratories. The imaging capabilities of the microscope are unimpaired, enabling the 3-D structures of fluorescently labeled materials to be precisely determined under applied load. With this device, we can apply a large range of forces (~1-1200 pN) over micron-scale contact areas to beads that are either embedded within 3-D matrices or attached to the surface of thin slab gels. To demonstrate the usefulness of this instrument, we have studied two important and biologically relevant materials: polyacrylamide-based hydrogel films typical of those used in cell traction force microscopy, and reconstituted networks of microtubules, essential cytoskeletal filaments.     

Magnetic aqueous two-phase separation in preparative applications.

Magnetic aqueous two-phase separation is a new technique to speed up the separation of aqueous two-phase systems (Anal. Biochem. 1987, 167, 331-339). It is based on the addition of magnetically susceptible material (e.g. 1-micron iron oxide particles) which induces rapid phase separation when a mixed system is placed in a magnetic field. The technique has been applied to a number of two-phase systems. The time for phase separation was decreased by a factor of 5-240,000, with the largest improvement for systems containing high concentrations of protein and for systems with viscous or nearly isopycnic phases. An apparatus for preparative multistage extraction with magnetic separation was constructed and tested on glycolytic enzymes present in a yeast extract using a dextran/Cibacron blue-polyethylene glycol system. The presence of iron oxide particles did not adversely affect the extracted enzymes. An electromagnet-based apparatus for continuous phase separation on a larger scale was also designed. A phase system containing crude dextran and unpurified cell homogenate was effectively processed. The apparatus also allowed effective separation when the phase containing iron oxide particles was only a small fraction (4%) of the total phase system.     

Aqueous two-phase systems

Biphasic systems formed by mixing of two polymers or a polymer and a salt in water can be used for separation of cells, membranes, viruses, proteins, nucleic acids, and other biomolecules. The partitioning between the two phases is dependent on the surface properties and conformation of the materials, and also on the composition of the two-phase system. The mechanism of partitioning is, however, complex and not easily predicted. Aqueous two-phase systems (ATPS) have proven to be a useful tool for analysis of biomolecular and cellular surfaces and their interactions, fractionation of cell populations, product recovery in biotechnology, and so forth. Potential for environmental remediation has also been suggested. Because ATPS are easily scalable and are also able to hold high biomass load in comparison with other separation techniques, the application that has attracted most interest so far has been the large-scale recovery of proteins from crude feedstocks. As chemicals constitute the major cost factor for large-scale systems, use of easily recyclable phase components and the phase systems generated by a single-phase chemical in water are being studied.     

Microfluidic separation of DNA

DNA separation is important for numerous applications in biotechnology and medicine. Efforts to improve DNA separation in microdevices have led to advances in capillary electrophoresis and the development of novel separation strategies. Current research on microcapillary electrophoresis materials is focused on the development of separation matrices with low injection viscosities and wall-coating capabilities. Microcapillary injector geometries are being designed to allow increased control of sample plug volumes. Novel separation strategies using entropic traps, arrays of pillars and Brownian ratchets are also being developed.     

Unraveling Charge Separation and Transport Mechanisms in Aqueous‐Processed Polymer/CdTe Nanocrystal Hybrid Solar Cells

Recently great progress has been achieved in highly effective hybrid solar cells fabricated using aqueous materials. The state‐of‐the‐art energy conversion efficiency has been close to 5% with high photocurrent. However, charge separation and transport mechanism in the aqueous‐processed hybrid solar cells are rarely reported and are usually assumed to be similar to oil‐phase processed systems; that is, self‐assembly polymers are mainly responsible for charge separation and carrier transport. To date, this assumption has prohibited further improvement of the conversion efficiency in aqueous‐processed hybrid systems by adopting any appropriate technique routes. Here, ultrafast carrier dynamics in these hybrid solar cells consisting of poly(p‐phenylenevinylene) (PPV)‐based aqueous polymers and water‐solution CdTe nanocrystals (NCs) are investigated in detail. Self‐charge separation in grown CdTe NC partly capped CdS shell layers after anneal treatment is unambiguously identified. Different from their oil‐soluble counterparts, these core/shell nanocrystals do not have the restrictions of quantum confinement and surface ligands, form effective charge transport networks, and play a dominant role in the charge separation and carrier transport processes. These findings provide a greater understanding on the fundamental photophysics in aqueous‐processed hybrid systems.     

Biomaterial films of Bombyx mori silk fibroin with poly(ethylene oxide)

Phase separation into controllable patterned microstructures was observed for Bombyx mori silkworm silk and poly(ethylene oxide) (PEO) (900000 g/mol) blends cast from solution. The evolution of the microstructures with increasing PEO volume fraction is strikingly similar to the progression of phases and microstructures observed with surfactants. The chemically patterned materials obtained provide engineerable biomaterial surfaces with predictable microscale features which can be used to create topographically patterned or chemically functionalized biomaterials. Solution blending was used to incorporate water-soluble PEO into silk to enhance elasticity and hydrophilicity. The sizes of the globule fibroin phase ranged from 2.1 +/- 0.5 to 18.2 +/- 2.1 microm depending on the ratio of silk/PEO. Optical microscopy and SEM analysis confirmed the micro-phase separation between PEO and silk. Surface properties were determined by XPS and contact angle. Methanol can be used to control the conformational transition of silk fibroin to the insoluble beta-sheet state. Subsequentially, the PEO can be easily extracted from the films with water to generate silk matrixes with definable porosity and enhanced surface roughness. These blend films formed from two biocompatible polymers provide potential new biomaterials for tissue engineering scaffolds.     

Effect of rapidity of phase separation on the efficiency of cell fractionation by partitioning in aqueous two-phase systems

Partitioning in two-polymer aqueous phase systems is an established method for the separation, purification and characterization of biomaterials. Because of the relatively slow settling rates of these phases, a consequence of the slight difference in density between them, effort has been directed to speeding up phase separation by various means (e.g., the development of a thin-layer countercurrent distribution apparatus). This has resulted in the more rapid processing of materials. Unlike soluble materials, biological particulates (e.g., cells) generally partition between one of the bulk phases and the interface. The mechanism of cell partitioning involves cell-specific adsorption to droplets of one phase suspended in the other, subsequent to phase mixing, and the delivery of adsorbed cells to the bulk interface as the droplets settle. In this communication we show, using erythrocytes as a model, that speeding up phase separation is counterproductive when partitioning cells and results in reduced efficiency of their separation or subfractionation. The most likely reason for this result is that increasing the speed of phase settling removes the droplets of one phase suspended in the other more rapidly than cells can attach to them, thereby interfering with the mechanism whereby cells partition.     

Freeze drying formulation using microscale and design of experiment approaches: a case study using granulocyte colony-stimulating factor

The lyophilization of proteins in microplates, to assess and optimise formulations rapidly, has been applied for the first time to a therapeutic protein and, in particular, one that requires a cell-based biological assay, in order to demonstrate the broader usefulness of the approach. Factorial design of experiment methods were combined with lyophilization in microplates to identify optimum formulations that stabilised granulocyte colony-stimulating factor during freeze drying. An initial screen rapidly identified key excipients and potential interactions, which was then followed by a central composite face designed optimisation experiment. Human serum albumin and Tween 20 had significant effects on maintaining protein stability. As previously, the optimum formulation was then freeze-dried in stoppered vials to verify that the microscale data is relevant to pilot scales. However, to validate the approach further, the selected formulation was also assessed for solid-state shelf-life through the use of accelerated stability studies. This approach allows for a high-throughput assessment of excipient options early on in product development, while also reducing costs in terms of time and quantity of materials required.     

FRET analysis of domain formation and properties in complex membrane systems

The application of Förster Resonance Energy Transfer (FRET) to the detection and characterization of phase separation in lipid bilayers (both in model systems and in cell membranes) is reviewed. Models describing the rate and efficiency of FRET for both uniform probe distribution and phase separation, and recently reported methods for detection of membrane heterogeneity and determination of phase boundaries, probe partition coefficients and domain size, are presented and critically discussed. Selected recent applications of FRET to one-phase lipid systems, gel/fluid phase separation, liquid ordered/liquid disordered phase separation (lipid rafts), complex systems containing ceramide and cell membranes are presented to illustrate the wealth of information that can be inferred from carefully designed FRET studies of membrane domains.     

Porous Nanomaterials for Ultrabroadband Omnidirectional Anti‐Reflection Surfaces with Applications in High Concentration Photovoltaics

Materials for nanoporous coatings that exploit optimized chemistries and self‐assembly processes offer capabilities to reach ≈98% transmission efficiency and negligible scattering losses over the broad wavelength range of the solar spectrum from 350 nm to 1.5 µm, on both flat and curved glass substrates. These nanomaterial anti‐reflection coatings also offer wide acceptance angles, up to ±40°, for both s‐ and p‐polarization states of incident light. Carefully controlled bilayer films have allowed for the fabrication of dual‐sided, gradient index profiles on plano‐convex lens elements. In concentration photovoltaics platforms, the resultant enhancements in the photovoltaics efficiencies are ≈8%, as defined by experimental measurements on systems that use microscale triple‐junction solar cells. These materials and their applications in technologies that require control over interface reflections have the potential for broad utility in imaging systems, photolithography, light‐emitting diodes, and display technologies.