Integrating Particle Motion Tracking into Thermal Gel Electrophoresis for Label-Free Sugar Sensing.

Bioanalytical sensors are adept at quantifying target analytes from complex sample matrices with high sensitivity, but their multiplexing capacity is limited. Conversely, analytical separations afford great multiplexing capacity but typically require analyte labeling to increase sensitivity. Here, we report the development of a separation-based sensor to sensitively quantify unlabeled polysaccharides using particle motion tracking within a microfluidic electrophoresis platform.

Multiplexed miRNA and Protein Analysis Using Digital Quantitative PCR in Microwell Arrays.

Proteins and microRNAs (miRNAs) act in tandem within biological pathways to regulate cellular functions, and their misregulation has been correlated to numerous diseases. Because of their interconnectedness, both miRNAs and proteins must be evaluated together to obtain accurate insights into the molecular pathways of pathogenesis. However, few analytical techniques can measure both classes of biomolecules in parallel from a single biological sample.

A review of electrophoretic separations in temperature-responsive Pluronic thermal gels.

Gel electrophoresis is a ubiquitous bioanalytical technique used in research laboratories to validate protein and nucleic acid samples. Polyacrylamide and agarose have been the gold standard gel materials for decades, but an alternative class of polymer has emerged with potentially superior performance. Pluronic thermal gels are water-soluble polymers that possess the unique ability to undergo a change in viscosity in response to changing temperature.

Selective miRNA quantitation with high-temperature thermal gel electrophoresis.

MicroRNAs (miRNAs) are short non-coding RNAs that control gene expression and correlate to the prognosis of numerous diseases. To support research efforts elucidating the roles of miRNAs in pathogenesis, rapid and inexpensive analytical methods are required to quantify miRNAs from biological samples. The challenge of developing new analyses with these time and cost constraints is compounded by the short sequence lengths and high degrees of homology between miRNAs that hinder detection selectivity.

Reverse transcription-free digital-quantitative-PCR for microRNA analysis.

MicroRNAs (miRNAs) are non-coding RNA sequences that regulate many biological processes and have become central targets of biomedical research. However, their naturally low abundances in biological samples necessitates the development of sensitive analytical techniques to conduct routine miRNA measurements in research laboratories. Digital PCR has the potential to meet this need because of its single-molecule detection capabilities, but PCR analyses of miRNAs are slowed by the ligation and reverse transcription steps first required to prepare samples.

Controlling the separation of native proteins with temperature in thermal gel transient isotachophoresis.

Polyacrylamide gel electrophoresis (PAGE) is a ubiquitous technique used in biochemical research laboratories to characterize protein samples. Despite its popularity, PAGE is relatively slow and provides limited separation resolution, especially for native proteins. This report describes the development of a microfluidic thermal gel transient isotachophoresis (TG-tITP) method to rapidly separate native proteins with high resolution.

Microfluidic Digital Quantitative PCR to Measure Internal Cargo of Individual Liposomes.

Lipid nanoparticles serve as drug delivery vehicles for biopharmaceutical products. The lipid membrane shields internal nucleic-acid drug cargo from enzymatic degradation and facilitates cellular uptake of the drug. However, existing methods to assess drug loading within liposomes are limited to averaged bulk measurements, which obscures heterogeneity of the biopharmaceutical formulation.

Multiplexed miRNA Quantitation Using Injectionless Microfluidic Thermal Gel Electrophoresis.

MicroRNAs (miRNAs) are a class of biomolecules that have high clinical and pharmaceutical significance because of their ability to regulate protein expression. Better methods are needed to quantify target miRNAs, but their similar sequence lengths and low concentrations in biomedical samples impede analysis. This report aimed to develop a simple, rapid method to directly quantify multiple miRNAs using microfluidic thermal gel electrophoresis (TGE).

Harnessing Joule heating in microfluidic thermal gel electrophoresis to create reversible barriers for cell enrichment.

Gel electrophoresis is a ubiquitous bioanalytical technique used to characterize the components of cell lysates. However, analyses of bulk lysates sacrifice detection sensitivity because intracellular biomolecules become diluted, and the liberation of proteases and nucleases can degrade target analytes. This report describes a method to enrich cells directly within a microfluidic gel as a first step toward online measurement of trace intracellular biomolecules with minimal dilution and degradation.

Influence of microfabrication on digital PCR performance in bead-based microwell array assays.

Digital PCR (dPCR) is a highly sensitive analytical technique used to quantify DNA targets. Detection sensitivity can be further enhanced by capturing target sequences onto beads for preconcentration and sample cleanup prior to analysis in microfluidic microwell arrays. However, robust digital analysis requires individual beads to be interrogated within individual wells.

Simultaneous Preconcentration and Separation of Native Protein Variants Using Thermal Gel Electrophoresis.

Proteins must maintain proper folding conformations and express the correct post-translational modifications (PTMs) to exhibit appropriate biological activity. However, assessing protein folding and PTMs is difficult because routine polyacrylamide gel electrophoresis (PAGE) methods lack the separation resolution necessary to identify variants of a single protein. Additionally, standard PAGE denatures proteins prior to analysis precluding determinations of folding states or PTMs.

Characterizing the impact of thermal gels on isotachophoresis in microfluidic devices.

Thermally reversible Pluronic gels have been employed as separation matrices in microfluidic devices in the analysis of biological macromolecules. The phase of these gels can be tuned between liquid and solid states using temperature to vary fluidic resistance and alter peak resolution. Although separations in thermal gels have been characterized, their effect on isotachophoresis has not.

Systematic LC/MS/MS Investigations for the IND-Enabling Extended Characterization of Antibody-Drug Conjugate Modifications.

We hypothesized that systematic liquid chromatography-tandem mass spectrometry investigations of an antibody-drug conjugate (ADC), its small and large molecular components, and surrogate small-molecule conjugates might comprise a simple and efficient approach for the extended characterization of ADCs. Furthermore, we envisioned that results from this work might allow us to assign specific composition changes in the ADC based on monoisotopic mass shifts of conjugatable modifications as detected in the surrogate small-molecule conjugates. We tested our hypothesis with a case study using an aldehyde-tag-based ADC conjugated to a noncleavable linker bearing a maytansine payload.

Use of Ice-Nucleating Proteins To Improve the Performance of Freeze-Thaw Valves in Microfluidic Devices.

Currently, reliable valving on integrated microfluidic devices fabricated from rigid materials is confined to expensive and complex methods. Freeze-thaw valves (FTVs) can provide a low cost, low complexity valving mechanism, but reliable implementation of them has been greatly hindered by the lack of ice nucleation sites within the valve body's small volume. Work to date has required very low temperatures (on the order of -40 °C or colder) to induce freezing without nucleation sites, making FTVs impractical due to instrument engineering challenges.

Alpha-particle radiotherapy: For large solid tumors diffusion trumps targeting.

Diffusion limitations on the penetration of nanocarriers in solid tumors hamper their therapeutic use when labeled with α-particle emitters. This is mostly due to the α-particles' relatively short range (≤100 μm) resulting in partial tumor irradiation and limited killing. To utilize the high therapeutic potential of α-particles against solid tumors, we designed non-targeted, non-internalizing nanometer-sized tunable carriers (pH-tunable liposomes) that are triggered to release, within the slightly acidic tumor interstitium, highly-diffusive forms of the encapsulated α-particle generator Actinium-225 (Ac) resulting in more homogeneous distributions of the α-particle emitters, improving uniformity in tumor irradiation and increasing killing efficacies.

Photobleaching kinetics-based bead encoding for multiplexed bioassays.

Multiplexing bead-based bioassays requires that each type of microsphere be uniquely encoded to distinguish one type from another. Microspheres are typically encoded using fluorescent dyes with different spectral properties and varying concentrations. However practical limits exist on the number of dyes that can be spectrally resolved or the number of distinguishable intensity levels with each dye.

Determination of Methylarginines in Infant Plasma by CE-LIF.

Methylarginines (MAs) are a class of nitric oxide synthase inhibitors that have been implicated in respiratory complications of critically ill infants. This paper describes the development of an analytical method to measure these compounds in the plasma of newborns using capillary electrophoresis (CE). The CE separation method was optimized to enable complete baseline resolution of the four MA analogues of interest.

Heat-assisted extraction for the determination of methylarginines in serum by CE.

Methylarginines (MAs) are potent vasoconstrictors that have been reported to be present at elevated concentrations in the blood of patients suffering from cardiovascular disease. To determine the diagnostic potential of MAs for cardiovascular disease, a method capable of rapidly quantifying their endogenous concentrations from serum samples is required. To that end, a heat-assisted extraction method was developed.

Analytical and biological methods for probing the blood-brain barrier.

The blood-brain barrier (BBB) is an important interface between the peripheral and central nervous systems. It protects the brain against the infiltration of harmful substances and regulates the permeation of beneficial endogenous substances from the blood into the extracellular fluid of the brain. It can also present a major obstacle in the development of drugs that are targeted for the central nervous system.

Optimization of the separation of NDA-derivatized methylarginines by capillary and microchip electrophoresis.

The methylated arginines (MAs) monomethylarginine (MMA), asymmetric dimethylarginine (ADMA), and symmetric dimethylarginine (SDMA) have been shown to be independent predictors of cardiovascular disease. This article describes progress regarding the development of an analytical method capable of rapidly analyzing MAs using capillary electrophoresis (CE) and microchip electrophoresis (MCE) with laser-induced fluorescence (LIF) detection. Several parameters including buffer composition and separation voltage were optimized to achieve an ideal separation.

An investigation into the pharmacokinetics of 3-mercaptopropionic acid and development of a steady-state chemical seizure model using in vivo microdialysis and electrophysiological monitoring.

Objectives: The goal of the present study was to develop a chemical seizure model using the convulsant, 3-mercaptopropionic acid (3-MPA). A pharmacodynamics approach was taken, combining in vivo microdialysis sampling with electrophysiological methods to simultaneously monitor, in real-time, the 3-MPA concentration in the brain and the corresponding electrocorticographic (ECoG) activity.

Methods: The 3-MPA was administered in two doses (50 and 100 mg/kg) in order to study its pharmacokinetics.