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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. This study used fluorescein as a model analyte to evaluate isotachophoretic preconcentration as a function of thermal polymer concentration and temperature. Results demonstrated that increasing polymer concentration in microfluidic channels increased the apparent analyte concentration. A critical minimum of 10% (w/v) Pluronic was required to achieve efficient preconcentration with maximum focusing occurring in 20 and 25% polymer gels. Temperature of the thermal gel also impacted analyte focusing. Most efficient focusing was achieved at 25°C with diminishing analyte accumulation at higher and lower temperatures. Under optimal conditions, isotachophoretic preconcentration increased an additional threefold simply by including thermal gels in the system. This approach can be readily implemented in other applications to increase detection sensitivity and measure low-concentration analytes within simple microfluidic devices.

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. Thermal gels were employed as the gel matrix because they can be reversibly converted between liquid and solid phases as a function of temperature. Rather than fabricate costly heating elements into devices to control temperature-and thus the phase of the gel-Joule heating was used instead. Adjoining regions of liquid-phase and solid-phase gel were formed within microfluidic channels by selectively inducing localized Joule heat. Cells migrated through the liquid gel but could not enter the solid gel-accumulating at the liquid-solid gel boundary-whereas small molecule contaminants passed through to waste. Barriers were then liquified on-demand by removing Joule heat to collect the purified, non-lysed cells for downstream analyses. Using voltage-controlled Joule heating to regulate the phase of thermal gels is an innovative approach to facilitate in-gel cell enrichment in low-cost microfluidic devices.

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. To overcome these limitations, a microfluidic thermal gel electrophoresis platform was developed to provide high-sensitivity, high-resolution analyses of native protein variants. A thermally reversible gel was utilized as a separation matrix while in its solid state (30 °C). This thermal gel provided sufficient separation resolution to identify three variants of a fluorescently labeled model protein. To increase detection sensitivity, analyte preconcentration was conducted in parallel with the separation. Continuous analyte enrichment afforded detection limits of 500 fg of protein (250 pM) while simultaneous baseline separation resolution was achieved between variants. The effects of temperature on thermal gel electrophoresis were also characterized. The unique temperature-dependent outcomes illustrated how method performance can be tuned through a thermal dimension. Ultimately, the high detection sensitivity and separation resolution provided by thermal gel electrophoresis enabled rapid screening of native protein variants.

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. Here, we report the use of ice-nucleating proteins (INPs) to induce ice formation at relatively warm temperatures in microfluidic devices. Microfluidic channels were filled with buffers containing femtomolar INP concentrations from Pseudomonas syringae. The channels were cooled externally with simple, small-footprint Peltier thermoelectric coolers (TECs), and the times required for channel freezing (valve closure) and thawing (valve opening) were measured. Under optimized conditions in plastic chips, INPs made sub-10 s actuations possible at TEC temperatures as warm as -13 °C. Additionally, INPs were found to have no discernible inhibitory effects in model enzyme-linked immunosorbent assays or polymerase chain reactions, indicating their compatibility with microfluidic systems that incorporate these widely used bioassays. FTVs with INPs provide a much needed reliable valving scheme for rigid plastic devices with low complexity, low cost, and no moving parts on the device or instrument. The reduction in freeze time, accessible actuation temperatures, chemical compatibility, and low complexity make the implementation of compact INP-based FTV arrays practical and attractive for the control of integrated biochemical assays.

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. Sample preparation concerns for infant-derived samples were addressed by validating a heat-assisted extraction method for the analysis of low volume (≤100 µL) samples from a plasma matrix. It was determined that the sample matrix (plasma versus serum) did not affect the measured MA concentrations, while extracting smaller volumes of plasma that underwent heat-induced gelation afforded higher MA recoveries than larger volume samples. These methods were then applied to blood samples collected from infants housed in the neonatal intensive care unit. It was discovered that these newborns had significantly elevated concentrations of MAs at younger ages (< 6 months) while amounts were similar between infants 6 months old and adults. The data are preliminary, but demonstrate an interesting age dependence on the concentrations of these nitric oxide inhibitors, which has not been previously reported.

Methylarginines (MAs) are potent vasoconstrictors that have been reported to be present at elevated concentrations in the blood of patients suffering from cardiovascular disease (CVD). To determine the diagnostic potential of MAs for CVD, a method capable of rapidly quantifying their endogenous concentrations from serum samples is required. To that end, a heat-assisted extraction method was developed. Serum was first rapidly heated, causing it to congeal into a gel, and then subjected to solid-liquid extraction. The extraction solution was then derivatized with a fluorogenic dye and analyzed by CE-LIF to permit quantitation of the MAs. This heat-assisted extraction procedure allowed a no-net-dilution extraction of the analytes to be performed into a solvent compatible with the subsequent CE analysis. This enabled direct detection of low abundance analytes, such as MAs, without the need for a preconcentration step. This sample preparation method was compared with a commonly used solid-phase extraction method for MA analysis. Endogenous MA concentrations determined by both the heating and SPE methods were found to be in good agreement with each other and with values previously reported in the literature.

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. The analytes of interest were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA) to produce fluorescent 1-cyanobenz[f]isoindole (CBI) derivatives and then subjected to CE analysis. Baseline resolution of SDMA, ADMA, MMA, and arginine was achieved in less than 8 min. The limits of detection for SDMA, ADMA, MMA, and arginine were determined to be 15, 20, 25, and 5 nM, respectively, which are well below the expected plasma concentrations. The CE separation method was then transferred to a glass MCE device with LIF detection. MAs were baseline resolved in 3 min on-chip using a 14 cm separation channel with detection limits of approximately 10 nM for each species. To the best of the authors’ knowledge, this is the first report of the separation of MAs by MCE.

Hydrogel particles have an historic presence in the field of drug formulation and delivery. Applications of particulate forms of gel materials continue to expand as a result of enhanced control over physicochemical properties including particle size, degradability, and environmental responsiveness. Here, acid-labile poly(N-vinylformamide) (PNVF) nanogels ∼100 nm in diameter were synthesized via inverse emulsion polymerization of N-vinylformamide in the presence of a ketal-containing cross-linker. The dissolution half-life of nanogels proved to be dramatically faster at low pH. Nanogels with a monomer:cross-linker ratio of seven demonstrated a 90 min half-life at pH 5.8 compared to ∼57 h at pH 7.4. Approximately 95% of lysozyme encapsulated in nanogels released over 200 min at pH 5.8 compared to only ∼15% released at pH 7.4. The encapsulation efficiency was moderate with optimal conditions leading to ∼60% encapsulation efficiency. In addition, released lysozyme retained about 50% of the original activity. ...

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. Several methods have been developed to investigate the transport and metabolism of drugs, peptides, and endogenous compounds at the BBB. In vivo methods include intravenous injection, brain perfusion, positron emission tomography, and microdialysis sampling. Researchers have also developed in vitro cell-culture models that can be employed to investigate transport and metabolism at the BBB without the complication of systemic involvement. All these methods require sensitive and selective analytical methods to monitor the transport and metabolism of the compounds of interest at the BBB.

Summary 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 100mg/kg) in order to study its pharmacokinetics. Microdialysis samples were collected from the striatum, hippocampus, and jugular vein every 5min. The microdialysates were analyzed using high-performance liquid chromatography with electrochemical detection (HPLC-EC). The ECoG activity was monitored via screws placed onto the cortex. Noncompartmental pharmacokinetics analysis was performed to obtain the elimination constants ( K e ), the maximum concentration ( C max ), the time to achieve maximum concentration ( T max ), and the area under the concentration–time curves (AUC inf ). Results The average brain K e for the 50 and the 100mg/kg doses were 0.060 and 0.018min −1 , respectively. The brain AUC inf for the 50 and 100mg/kg doses were 353 and 2168mgmin −1 mL −1 , respectively. This led to a 67-fold increase in the observed number of seizures in the higher dose with the average seizure intensity double that of the smaller dose. These data led to the dosing scheme for the chemical seizure model of administering a 3-MPA loading dose of 60mg/kg followed by a constant infusion of 50mg/(kgmin −1 ). Conclusions This study describes, to our knowledge, the first successful attempt to combine in vivo microdialysis with electrophysiology to monitor in real-time, the concentration and effects of 3-MPA in the brain. This led to the development of a steady-state chemical seizure model.