Assessing Surface Adsorption in Cyclic Olefin Copolymer Microfluidic Devices Using Two-Dimensional Nano Liquid Chromatography-Micro Free Flow Electrophoresis Separations.

Surface interactions are a concern in microscale separations, where analyte adsorption can decrease the speed, sensitivity, and resolution otherwise achieved by miniaturization. Here, we functionally characterize the surface adsorption of hot-embossed cyclic olefin copolymer (COC) micro free-flow electrophoresis (μFFE) devices using two-dimensional nLC × μFFE separations, which introduce a 3- to 5 s plug of analyte into the device and measure temporal broadening that arises from surface interactions. COC is an attractive material for microfluidic devices, but little is known about its potential for surface adsorption in applications with continuous fluid flow and temporal measurements.

Fabrication of µFFE Devices in COC via Hot Embossing with a 3D-Printed Master Mold.

The fabrication of high-performance microscale devices in substrates with optimal material properties while keeping costs low and maintaining the flexibility to rapidly prototype new designs remains an ongoing challenge in the microfluidics field. To this end, we have fabricated a micro free-flow electrophoresis (µFFE) device in cyclic olefin copolymer (COC) via hot embossing using a PolyJet 3D-printed master mold. A room-temperature cyclohexane vapor bath was used to clarify the device and facilitate solvent-assisted thermal bonding to fully enclose the channels.

Reduced surface adsorption in 3D printed acrylonitrile butadiene styrene micro free-flow electrophoresis devices.

We have 3D printed and fabricated micro free-flow electrophoresis (µFFE) devices in acrylonitrile butadiene styrene (ABS) that exhibit minimal surface adsorption without requiring additional surface coatings or specialized buffer additives. 2D, nano LC-micro free flow electrophoresis (2D nLC × µFFE) separations were used to assess both spatial and temporal broadening as peaks eluted through the separation channel. Minimal broadening due to wall adsorption was observed in either the spatial or temporal dimensions during separations of rhodamine 110, rhodamine 123, and fluorescein.

Cytoplasmic nucleic acid-based XNAs directly enhance live cardiac cell function by a Ca cycling-independent mechanism via the sarcomere.

Nucleic acid - protein interactions are critical for regulating gene activation in the nucleus. In the cytoplasm, however, potential nucleic acid-protein functional interactions are less clear. The emergence of a large and expanding number of non-coding RNAs and DNA fragments raises the possibility that the cytoplasmic nucleic acids may interact with cytoplasmic cellular components to directly alter key biological processes within the cell.

Biochemical markers of striatal desensitization in cortical-limbic hyperglutamatergic TS- & OCD-like transgenic mice.

Tics and compulsions in comorbid Tourette's syndrome (TS) and obsessive-compulsive disorder (OCD) are associated with chronic hyperactivity of parallel cortico/amygdalo-striato-thalamo-cortical (CSTC) loop circuits. Comorbid TS- & OCD-like behaviors have likewise been observed in D1CT-7 mice, in which an artificial neuropotentiating transgene encoding the cAMP-elevating intracellular subunit of cholera toxin (CT) is chronically expressed selectively in somatosensory cortical & amygdalar dopamine (DA) D1 receptor-expressing neurons that activate cortico/amygdalo-striatal glutamate (GLU) output. We've now examined in D1CT-7 mice whether the chronic GLU output from their potentiated cortical/limbic CSTC subcircuit afferents associated with TS- & OCD-like behaviors elicits desensitizing neurochemical changes in the striatum (STR).

Microfluidic methods for aptamer selection and characterization.

Nucleic acid aptamers have tremendous potential as molecular recognition elements in biomedical targeting, analytical arrays, and self-signaling sensors. However, practical limitations and inefficiencies in the process of selecting novel aptamers (SELEX) have hampered widespread adoption of aptamer technologies. Many factors have recently contributed to more effective aptamer screening, but no influence has done more to increase the efficiency, scale, and automation of aptamer selection than that of new microfluidic SELEX techniques.

Micro free flow electrophoresis.

Micro free-flow electrophoresis (μFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar separation channel. Analyte streams are deflected laterally based on their electrophoretic mobilities as they flow through the separation channel. A number of μFFE separation modes have been demonstrated, including free zone (FZ), micellar electrokinetic chromatography (MEKC), isoelectric focusing (IEF) and isotachophoresis (ITP).

High-Speed, Comprehensive, Two Dimensional Separations of Peptides and Small Molecule Biological Amines Using Capillary Electrophoresis Coupled with Micro Free Flow Electrophoresis.

Two-dimensional (2D) separations are able to generate significantly higher peak capacities than their one-dimensional counterparts. Unfortunately, current hyphenated 2D separations are limited by the speed of the second dimension separation and the consequent loss of peak capacity due to under sampling of peaks as they elute from the first dimension separation. Continuous micro free flow electrophoresis (μFFE) separations eliminate under sampling as a limitation when incorporated as the second dimension of a 2D separation.

In Vivo Monitoring of Amino Acid Biomarkers from Inguinal Adipose Tissue Using Online Microdialysis-Capillary Electrophoresis.

We have developed an online, high-throughput, microdialysis-capillary electrophoresis (MD-CE) assay for measuring the in vivo dynamics of amino acid biomarkers of metabolism in adipose tissue. Microdialysis probes were implanted into the inguinal adipose tissue depot of C57BL6 mice. The probe location and integrity were verified following each experiment, demonstrating our ability to accurately target the inguinal adipose tissue depot without damaging the probe.

Reversal of Phospholamban Inhibition of the Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA) Using Short, Protein-interacting RNAs and Oligonucleotide Analogs.

The sarco(endo)plasmic reticulum Ca-ATPase (SERCA) and phospholamban (PLN) complex regulates heart relaxation through its removal of cytosolic Ca during diastole. Dysfunction of this complex has been related to many heart disorders and is therefore a key pharmacological target. There are currently no therapeutics that directly target either SERCA or PLN.

High-Speed Microdialysis-Capillary Electrophoresis Assays for Measuring Branched Chain Amino Acid Uptake in 3T3-L1 cells.

We have developed a high-throughput microdialysis-capillary electrophoresis (MD-CE) assay for monitoring branched chain amino acid (BCAA) uptake/release dynamics in 3T3-L1 cells. BCAAs (i.e.

3D Printed Micro Free-Flow Electrophoresis Device.

The cost, time, and restrictions on creative flexibility associated with current fabrication methods present significant challenges in the development and application of microfluidic devices. Additive manufacturing, also referred to as three-dimensional (3D) printing, provides many advantages over existing methods. With 3D printing, devices can be made in a cost-effective manner with the ability to rapidly prototype new designs.

Effect of Fluorescent Labels on Peptide and Amino Acid Sample Dimensionality in Two Dimensional nLC × μFFE Separations.

Multidimensional separations present a unique opportunity for generating the high peak capacities necessary for the analysis of complex biological mixtures. We have coupled nano liquid chromatography with micro free flow electrophoresis (nLC × μFFE) to produce high peak capacity separations of peptide and amino acid mixtures. Currently, μFFE largely relies on laser-induced fluorescence (LIF) detection.

Effect of Surface Adsorption on Temporal and Spatial Broadening in Micro Free Flow Electrophoresis.

Analyte adsorption onto surfaces presents a challenge for many separations, often becoming a significant source of peak broadening and asymmetry. We have shown that surface adsorption has no effect on peak position or spatial broadening in micro free flow electrophoresis (μFFE) separations. Surface adsorption does affect the time it takes an analyte to travel through the μFFE separation channel and therefore contributes to temporal broadening.

Rheostatic Regulation of the SERCA/Phospholamban Membrane Protein Complex Using Non-Coding RNA and Single-Stranded DNA oligonucleotides.

The membrane protein complex between sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and phospholamban (PLN) is a prime therapeutic target for reversing cardiac contractile dysfunctions caused by calcium mishandling. So far, however, efforts to develop drugs specific for this protein complex have failed. Here, we show that non-coding RNAs and single-stranded DNAs (ssDNAs) interact with and regulate the function of the SERCA/PLN complex in a tunable manner.

Comprehensive multidimensional separations of peptides using nano-liquid chromatography coupled with micro free flow electrophoresis.

The throughput of existing liquid phase two-dimensional separations is generally limited by the peak capacity lost due to under sampling by the second dimension separation as peaks elute off the first dimension separation. In the current manuscript, a first dimension nanoliquid chromatography (nLC) separation is coupled directly with a second dimension micro free flow electrophoresis (μFFE) separation. Since μFFE performs continuous separations, no complicated injection or modulation is necessary to couple the two techniques.

Development of a Sensitive Assay for SERCA Activity Using FRET Detection of ADP.

Various isoforms of sarco(endo)plasmic reticulum Ca-ATPase (SERCA) regulate Ca homeostatic balance in both the heart (SERCA2a) and skeletal muscle (SERCA1a). Ca plays a key role in these tissues as an intracellular signal that controls contractility. Due to its key role in the contractility cycle, SERCA is emerging as a promising pharmacological target to modulate heart muscle function.

Analysis of individual mitochondria via fluorescent immunolabeling with Anti-TOM22 antibodies.

Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria.

Isolating single stranded DNA using a microfluidic dialysis device.

Isolating a particular strand of DNA from a double stranded DNA duplex is an important step in aptamer generation as well as many other biotechnology applications. Here we describe a microfluidic, flow-through, dialysis device for isolating single-stranded DNA (ssDNA) from double-stranded DNA (dsDNA). The device consists of two channels fabricated in polydimethylsiloxane (PDMS) separated by a track etched polycarbonate membrane (800 nm pore size).

Tracking the emergence of high affinity aptamers for rhVEGF165 during capillary electrophoresis-systematic evolution of ligands by exponential enrichment using high throughput sequencing.

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) is a powerful technique for isolating aptamers for various targets, from large proteins to small peptides with molecular weights of several kilodaltons. One of the unique characteristics of CE-SELEX is the relatively high heterogeneity of the ssDNA pools that remains even after multiple rounds of selection. Enriched sequences or highly abundant oligonucleotide motifs are rarely reported in CE-SELEX studies.

Monitoring neurochemical release from astrocytes using in vitro microdialysis coupled with high-speed capillary electrophoresis.

We have developed a novel in vitro approach for monitoring fast neurochemical dynamics in model cell systems using microdialysis sampling coupled with high-speed capillary electrophoresis (CE). Cells from an immortalized astrocyte line (C8-D1A) were cultured in direct contact with the porous membrane of a microdialysis probe. Confocal microscopy was used to confirm cell viability and confluency over the microdialysis sampling region.

Effect of cross sectional geometry on PDMS micro peristaltic pump performance: comparison of SU-8 replica molding vs. micro injection molding.

Two different fabrication methods were employed to fabricate micropumps with different cross-sectional channel geometries. The first was to fabricate rectangular cross-sectional microchannel geometries using the well known fabrication method of replica molding (REM). The second, and far less utilized fabrication technique, was to create microchannel molds using an in-house fabricated handheld micro injection molding apparatus.

Capillary electrophoresis-SELEX selection of catalytic DNA aptamers for a small-molecule porphyrin target.

Capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) has previously been used to select aptamers for large-molecule targets such as proteins, lipopolysaccharides, and peptides. For the first time, we have performed CE-SELEX selection for a small-molecule target, N-methyl mesoporphyrin (NMM), with a molecular weight of only 580 g/mol. DNA aptamers with high-nanomolar to low-micromolar dissociation constants were achieved after only three rounds of selection.