Multiplexed detection and applications for separations on parallel microchips

Much work has been performed since the development of the lab-on-a-chip concept that has brought microfabricated systems to the forefront of bioanalytical research. The success of using these microchips for performing complicated biological assays faster and cheaper than conventional methods has facilitated their emerging popularity among researchers. A recently exploited advantage of microfabricated technology has led to the creation of single wafers with multiple channel manifolds for high-throughput experiments. Efforts toward parallel microchip development have yielded fascinating new devices for chemical separations showing the potential for replacing conventional multiplexing techniques. This review will focus on recent work toward multiplexed separations on microdevices and complementary detection instrumentation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/60902/1/3296_ftp.pd

DNA multi-bit non-volatile memory and bit-shifting operations using addressable electrode arrays and electric field-induced hybridization.

DNA has been employed to either store digital information or to perform parallel molecular computing. Relatively unexplored is the ability to combine DNA-based memory and logical operations in a single platform. Here, we show a DNA tri-level cell non-volatile memory system capable of parallel random-access writing of memory and bit shifting operations. A microchip with an array of individually addressable electrodes was employed to enable random access of the memory cells using electric fields. Three segments on a DNA template molecule were used to encode three data bits. Rapid writing of data bits was enabled by electric field-induced hybridization of fluorescently labeled complementary probes and the data bits were read by fluorescence imaging. We demonstrated the rapid parallel writing and reading of 8 (23) combinations of 3-bit memory data and bit shifting operations by electric field-induced strand displacement. Our system may find potential applications in DNA-based memory and computations

Parallel Separations on Microfluidic Chips for High Throughput Monitoring of Insulin Secretion from Single Islets of Langerhans.

Microfluidic devices for the simultaneous characterization of insulin release from four and 15 isolated pancreatic islets were developed. Quantification of released insulin from islet samples was performed using parallel immunoassays coupled to capillary electrophoresis with fluorescence detection. Assays for insulin were completed in a serial fashion on each channel every 6 – 10 s, giving fast temporal resolution used for investigations into insulin secretion dynamics. Assay limits of detection were between 0.5 – 10 nM insulin. Individual islets were housed on the chips while perfusion streams carrying glucose or other secretagogues were used to stimulate insulin release. Secreted insulin was then mixed with fluorescently-labeled insulin and anti-insulin antibody in reaction channels for a competitive immunoassay. Portions of the continuously flowing reaction streams were injected onto separation channels where bound fluorescent insulin:antibody and free fluorescent insulin complexes were separated electrophoretically and detected via fluorescence. Relative amounts of these products were used to determine the amount of released insulin. The 15-islet microchip was used to investigate possible roles of leptin signaling on insulin secretion. Through a collaborative effort, islets from mice lacking leptin receptors only in the pancreas were compared to control islets through insulin release studies. Specifically, the effects of leptin, glibenclamide, glucagon-like peptide-1, and palmitic acid on glucose-stimulated insulin secretion were investigated. It was observed that leptin produces an inhibitory effect on insulin release and that lack of leptin signaling in islets enhances insulin release stimulated with glucose. Modifications were made to the 15-islet chip to ensure more uniform sampling of insulin from islets. The new islet sampling method was used to characterize oscillatory insulin release under various conditions. It was found that when treated appropriately, islets from individual mice displayed similar insulin secretion and Ca2+ flux oscillation frequencies. These frequencies were shown to be different from mouse to mouse, complementing previous studies. Additionally, the effects of free fatty acid-induced liptoxicity on pulsatile insulin release were investigated. Results from these experiments demonstrate the usefulness of single islet data not previously available at this level of throughput.Ph.D.ChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/61556/1/johndish_1.pd

Exploring Gradients in Electrophoretic Separation and Preconcentration on Miniaturized Devices

abstract: Over the last two decades, miniaturization, integration, and automation have made microfluidic systems popular. Core to advances in microfluidics are numerous electrophoretic separation and preconcentration strategies, some finding their origins on bench-top systems. Among them, gradient-based strategies are especially effective in addressing sensitivity challenges. This review introduces several gradient-based techniques according to a broad definition, including conductivity, field, and concentration, organized by the method of gradient generation. Each technique is introduced and described, and recent seminal advances explored

Single-Molecule Detection of Unique Genome Signatures: Applications in Molecular Diagnostics and Homeland Security

Single-molecule detection (SMD) offers an attractive approach for identifying the presence of certain markers that can be used for in vitro molecular diagnostics in a near real-time format. The ability to eliminate sample processing steps afforded by the ultra-high sensitivity associated with SMD yields an increased sampling pipeline. When SMD and microfluidics are used in conjunction with nucleic acid-based assays such as the ligase detection reaction coupled with single-pair fluorescent resonance energy transfer (LDR-spFRET), complete molecular profiling and screening of certain cancers, pathogenic bacteria, and other biomarkers becomes possible at remarkable speeds and sensitivities with high specificity. The merging of these technologies and techniques into two different novel instrument formats has been investigated. (1) The use of a charge-coupled device (CCD) in time-delayed integration (TDI) mode as a means for increasing the throughput of any single molecule measurement by simultaneously tracking and detecting single-molecules in multiple microfluidic channels was demonstrated. The CCD/TDI approach allowed increasing the sample throughput by a factor of 8 compared to a single-assay SMD experiment. A sampling throughput of 276 molecules s-1 per channel and 2208 molecules s-1 for an eight channel microfluidic system was achieved. A cyclic olefin copolymer (COC) waveguide was designed and fabricated in a pre-cast poly(dimethylsiloxane) stencil to increase the SNR by controlling the excitation geometry. The waveguide showed an attenuation of 0.67 dB/cm and the launch angle was optimized to increase the depth of penetration of the evanescent wave. (2) A compact SMD (cSMD) instrument was designed and built for the reporting of molecular signatures associated with bacteria. The optical waveguides were poised within the fluidic chip at orientation of 90° with respect to each other for the interrogation of single-molecule events. Molecular beacons (MB) were designed to probe bacteria for the classification of Gram +. MBs were mixed with bacterial cells and pumped though the cSMD which allowed S. aureus to be classified with 2,000 cells in 1 min. Finally, the integration of the LDR-spFRET assay on the cSMD was explored with the future direction of designing a molecular screening approach for stroke diagnostics

Nanotools for Neuroscience and Brain Activity Mapping

Neuroscience is at a crossroads. Great effort is being invested into deciphering specific neural interactions and circuits. At the same time, there exist few general theories or principles that explain brain function. We attribute this disparity, in part, to limitations in current methodologies. Traditional neurophysiological approaches record the activities of one neuron or a few neurons at a time. Neurochemical approaches focus on single neurotransmitters. Yet, there is an increasing realization that neural circuits operate at emergent levels, where the interactions between hundreds or thousands of neurons, utilizing multiple chemical transmitters, generate functional states. Brains function at the nanoscale, so tools to study brains must ultimately operate at this scale, as well. Nanoscience and nanotechnology are poised to provide a rich toolkit of novel methods to explore brain function by enabling simultaneous measurement and manipulation of activity of thousands or even millions of neurons. We and others refer to this goal as the Brain Activity Mapping Project. In this Nano Focus, we discuss how recent developments in nanoscale analysis tools and in the design and synthesis of nanomaterials have generated optical, electrical, and chemical methods that can readily be adapted for use in neuroscience. These approaches represent exciting areas of technical development and research. Moreover, unique opportunities exist for nanoscientists, nanotechnologists, and other physical scientists and engineers to contribute to tackling the challenging problems involved in understanding the fundamentals of brain function

Microfabricated Sampling Probes for Monitoring Brain Chemistry at High Spatial and Temporal Resolution

Monitoring neurochemical dynamics has played a crucial role in elucidating brain function and related disorders. An essential approach for monitoring neurochemicals is to couple sampling probes to analytical measurements; however, this approach is inherently limited by poor spatial and temporal resolution. In this work, we have developed miniaturized sampling probes and analytical technology to overcome these limitations. Conventional sampling probes were handmade and have several disadvantages, including large sizes (over 220 µm in diameter) and limited design flexibility. To address these disadvantages, we have used microfabrication to manufacture sampling probes. By bulk micromachining of Si, microchannels and small sampling regions can be fabricated within a probe, with an overall dimension of ~100 µm. For development of a dialysis probe, nanoporous anodic aluminum oxide was adapted for monolithically embedding a membrane. Coupling the probe to liquid chromatography-mass spectrometry, multiple neurochemicals were measured at basal conditions, including dopamine and acetylcholine. Comparing to conventional dialysis probes, the microfabricated dialysis probe provided at least 6-fold improvement in spatial resolution and potentially had lower tissue disruption. Furthermore, we have continued the development of a microfabricated push-pull probe. We enhanced functionality of the probe by integrating an additional channel into the probe for chemical delivery. Further, we demonstrated that the probe can feasibly be coupled to droplet microfluidic devices for improved temporal resolution. Nanospray ionization mass spectrometry was used for multiplexed measurements of neurochemicals in nanoliter droplet samples. Utility of the integrated system was demonstrated by monitoring in vivo dynamics during potassium stimulation of 4 neurochemicals, including glutamate and GABA. The probe provided unprecedented spatial resolution and temporal resolution as high as ~5 s. Additionally, we highlighted versatility of the method by coupling the probe to another high-throughput assay, i.e., droplet-based microchip capillary electrophoresis for rapid separation (less than 3 s) and measurement of multiple amino acid neurochemicals. This collection of work illustrates that development of the microfabricated sampling probes and their compatible microfluidic systems are highly beneficial for studying brain chemistry. The integrated miniaturized analytical technology can potentially be useful for solving other problems of biological significance.PHDChemistryUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/144094/1/nonngern_1.pd