W. Hampton Henley

Distinct pathways mediate axon degeneration during apoptosis and axon-specific pruning.

Neurons can activate pathways that destroy the whole cell via apoptosis or selectively degenerate only the axon (pruning). Both apoptosis and axon degeneration require Bax and caspases. Here we demonstrate that despite this overlap, the pathways mediating axon degeneration during apoptosis versus axon pruning are distinct. While caspase-6 is activated in axons following nerve growth factor (NGF) deprivation, microfluidic chamber experiments reveal that caspase-6 deficiency only protects axons during axon-specific but not whole-cell (apoptotic) NGF deprivation. Strikingly, axon-selective degeneration requires the apoptotic proteins Caspase-9 and Caspase-3 but, in contrast to apoptosis, not Apaf-1. Additionally, cell bodies of degenerating axons are protected from caspase activation by protea some activity and XIAP. Also, mature neurons restrict apoptosis but remain permissive for axon degeneration, further demonstrating the independent regulation of these two pathways. These results reveal insight into how neurons allow for precise control over apoptosis and axon-selective degeneration pathways, thereby permitting long-term plasticity without risking neurodegeneration.

Monolithic Integration of Two-Dimensional Liquid Chromatography-Capillary Electrophoresis and Electrospray Ionization on a Microfluidic Device

A microfluidic device capable of two-dimensional reversed-phase liquid chromatography-capillary electrophoresis with integrated electrospray ionization (LC-CE-ESI) for mass spectrometry (MS)-based proteomic applications is described. Traditional instrumentation was used for the LC sample injection and delivery of the LC mobile phase. The glass microfabricated device incorporated a sample-trapping region and an LC channel packed with reversed-phase particles. Rapid electrokinetic injections of the LC effluent into the CE dimension were performed at a cross-channel intersection. The CE separation channel terminated at a corner of the square device, which functioned as an integrated electrospray tip. In addition to LC-CE-ESI, this device was used for LC-ESI without any instrumental modifications. To evaluate the system, LC-MS and LC-CE-MS analyses of protein digests were performed and compared.

A microfluidic chip integrating DNA extraction and real-time PCR for the detection of bacteria in saliva

A microfluidic chip integrating DNA extraction, amplification, and detection for the identification of bacteria in saliva is described. The chip design integrated a monolithic aluminum oxide membrane (AOM) for DNA extraction with seven parallel reaction wells for real-time polymerase chain reaction (rtPCR) amplification of the extracted DNA. Samples were first heated to lyse target organisms and then added to the chip and filtered through the nanoporous AOM to extract the DNA. PCR reagents were added to each of the wells and the chip was thermocycled. Identification of Streptococcus mutans in a saliva sample is demonstrated along with the detection of 300 fg (100-125 copies) of both methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA) genomic DNA (gDNA) spiked into a saliva sample. Multiple target species and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests, as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device.

An automated integrated platform for rapid and sensitive multiplexed protein profiling using human saliva samples

During the last decade, saliva has emerged as a potentially ideal diagnostic biofluid for noninvasive testing. In this paper, we present an automated, integrated platform useable by minimally trained personnel in the field for the diagnosis of respiratory diseases using human saliva as a sample specimen. In this platform, a saliva sample is loaded onto a disposable microfluidic chip containing all the necessary reagents and components required for saliva analysis. The chip is then inserted into the automated analyzer, the SDReader, where multiple potential protein biomarkers for respiratory diseases are measured simultaneously using a microsphere-based array via fluorescence sandwich immunoassays. The results are read optically, and the images are analyzed by a custom-designed algorithm. The fully automated assay requires as little as 10 μL of saliva sample, and the results are reported in 70 min. The performance of the platform was characterized by testing protein standard solutions, and the results were comparable to those from the 3.5 h lab bench assay that we have previously reported. The device was also deployed in two clinical environments where 273 human saliva samples collected from different subjects were successfully tested, demonstrating the devices potential to assist clinicians with the diagnosis of respiratory diseases by providing timely protein biomarker profiling information. This platform, which combines noninvasive sample collection and fully automated analysis, can also be utilized in point-of-care diagnostics.

Microfluidic Amperometric Sensor for Analysis of Nitric Oxide in Whole Blood

Standard photolithographic techniques and a nitric oxide (NO) selective xerogel polymer were utilized to fabricate an amperometric NO microfluidic sensor with low background noise and the ability to analyze NO levels in small sample volumes (~250 μL). The sensor exhibited excellent analytical performance in phosphate buffered saline, including a NO sensitivity of 1.4 pA nM(-1), a limit of detection (LOD) of 840 pM, and selectivity over nitrite, ascorbic acid, acetaminophen, uric acid, hydrogen sulfide, ammonium, ammonia, and both protonated and deprotonated peroxynitrite (selectivity coefficients of -5.3, -4.2, -4.0, -5.0, -6.0, -5.8, -3.8, -1.5, and -4.0, respectively). To demonstrate the utility of the microfluidic NO sensor for biomedical analysis, the device was used to monitor changes in blood NO levels during the onset of sepsis in a murine pneumonia model.

Fabrication of Microfluidic Devices Containing Patterned Microwell Arrays

A rapid fabrication and prototyping technique to incorporate microwell arrays with sub-10 μm features within a single layer of microfluidic circuitry is presented. Typically, the construction of devices that incorporate very small architecture within larger components has required the assembly of multiple elements to form a working device. Rapid, facile production of a working device using only a single layer of molded polydimethylsiloxane (PDMS) and a glass support substrate is achieved with the reported fabrication technique. A combination of conventional wet-chemical etching for larger (≥20 μm) microchannel features and focused ion beam (FIB) milling for smaller (≤10 μm) microwell features was used to fabricate a monolithic glass master mold. PDMS/glass hybrid chips were then produced using simple molding and oxygen plasma bonding methods. Microwell structures were loaded with 3 μm antibody-functionalized dye-encoded polystyrene spheres, and a sandwich immunoassay for common cytokines was performed to demonstrate proof-of-principle. Potential applications for this device include highly parallel multiplexed sandwich immunoassays, DNA/RNA hybridization analyses, and enzyme linked immunosorbent assay (ELISA). The fabrication technique described can be used for rapid prototyping of devices wherever submicrometer- to micrometer-sized features are incorporated into a microfluidic device.

Ultra-high voltage capillary electrophoresis >300 kV: Recent advances in instrumentation and analyte detection

Instrumentation has been developed for the implementation of ultra-high voltage capillary electrophoresis (UHVCE) with potentials up to and exceeding 300 kV. Several separations have been used to demonstrate the utility of higher applied voltages for improving the resolution of peptide, protein, and nucleic acid separations. Previously reported instrumentation was limited to 120 kV and required submersion in a bath of transformer oil to prevent corona and high voltage arcing between the components of the instrument [Hutterer, 1999, 2000, 2005] [1-3]. A modular design that uses plastic dielectric materials to overcome these obstacles enabling simplified operation of the instrument in air is described here in detail. A forced air system developed to control the temperature of the instrument to within a few degrees over a range of 25-60 °C for use with ultra-high voltage capillary gel electrophoresis is also described. UHVCE instrumentation and its applications with UV absorption and laser induced fluorescence detection are further developed, and the first demonstration of UHVCE coupled to electrospray ionization-mass spectrometry is shown.

High electric field strength two-dimensional peptide separations using a microfluidic device.

New instrumentation has been developed to improve the resolution, efficiency, and speed of microfluidic 2D separations using MEKC coupled to high field strength CE. Previously published 2D separation instrumentation [Ramsey, J. D. et al., Anal. Chem. 2003, 75, 3758–3764] from our group was limited to a maximum potential difference of 8.4 kV, resulting in an electric field strength of only approximately 200 V/cm in the first dimension. The circuit described in this report has been designed to couple a higher voltage supply with a rapidly switching, lower voltage supply to utilize the best features of each. Voltages applied in excess of 20 kV lead to high electric field strength separations in both dimensions, increasing the separation resolution, efficiency, and peak capacity while reducing the required analysis time. Detection rates as high as six peptides per second (based on total analysis time) were observed for a model protein tryptic digest separation. Additionally, higher applied voltages used in conjunction with microfluidic chips with longer length channels maintained higher electric field strengths and produced peak capacities of over 4000 for some separations. Total separation time in these longer channel devices was comparable to that obtained in short channels at low field strength; however, resolving power improved approximately threefold.

Laser-based directed release of array elements for efficient collection into targeted microwells

A cell separation strategy capable of the systematic isolation and collection of moderate to large numbers (25-400) of single cells into a targeted microwell is demonstrated. An array of microfabricated, releasable, transparent micron-scale pedestals termed pallets and an array of microwells in poly(dimethylsiloxane) (PDMS) were mated to enable selective release and retrieval of individual cells. Cells cultured on a pallet array mounted on a custom designed stage permitted the array to be positioned independently of the microwell locations. Individual pallets containing cells were detached in a targeted fashion using a pulsed Nd:YAG laser. The location of the laser focal point was optimized to transfer individual pallets to designated microwells. In a large-scale sort (n = 401), the accuracy, defined as placing a pallet in the intended well, was 94% and the collection efficiency was 100%. Multiple pallets were observed in only 4% of the targeted wells. In cell sorting experiments, the technique provided a yield and purity of target cells identified by their fluorescence signature of 91% and 93%, respectively. Cell viability based on single-cell cloning efficiency at 72 h post collection was 77%.

High resolution separations of charge variants and disulfide isomers of monoclonal antibodies and antibody drug conjugates using ultra-high voltage capillary electrophoresis with high electric field strength

Ultra-high voltage capillary electrophoresis with high electric field strength has been applied to the separation of the charge variants, drug conjugates, and disulfide isomers of monoclonal antibodies. Samples composed of many closely related species are difficult to resolve and quantify using traditional analytical instrumentation. High performance instrumentation can often save considerable time and effort otherwise spent on extensive method development. Ideally, the resolution obtained for a given CE buffer system scales with the square root of the applied voltage. Currently available commercial CE instrumentation is limited to an applied voltage of approximately 30kV and a maximum electric field strength of 1kV/cm due to design limitations. The instrumentation described here is capable of safely applying potentials of at least 120kV with electric field strengths over 2000V/cm, potentially doubling the resolution of the best conventional CE buffer/capillary systems while decreasing analysis time in some applications. Separations of these complex mixtures using this new instrumentation demonstrate the potential of ultra-high voltage CE to identify the presence of previously unresolved components and to reduce analysis time for complex mixtures of antibody variants and drug conjugates.