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. Thermal gels can reversibly convert between low-viscosity liquids and high-viscosity solid gels using temperature as an adjustable parameter. The properties of thermal gels provide unmatched flexibility as a dynamic separations matrix to measure analytes ranging from small molecules to cells. This review article describes the physical and chemical properties of Pluronic thermal gels to provide a fundamental overview of polymer behavior. The performance of thermal gels is then reviewed to highlight their applications as a gel matrix for electrokinetic separations in capillary, microfluidic, and slab gel formats. The use of dynamic temperature-responsive gels in bioanalytical separations is an underexplored area of research but one that holds exciting potential to achieve performance unattainable with conventional static polymers.

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. This report describes the development of a high-temperature thermal gel electrophoresis (TGE) method to rapidly quantify miRNAs with single-nucleotide resolution using low-cost microfluidic devices. Fluorescent probes were designed for three miRNAs that differed in sequence by one or two nucleotides. A microfluidic analysis was optimized to enrich miRNA-probe hybrids into a high-concentration band and then automatically initiate a separation to resolve each species. Analyses conducted at 30 °C exhibited significant off-target hybridization, as the different-yet-structurally-similar miRNAs bound to each probe, which biased measurements. To overcome this problem, the stability of thermal gels at elevated temperatures was exploited to conduct analyses. At 50 °C, off-target hybrids melted to prevent their detection without impeding the enrichment or separation of on-target hybrids. Selectivity studies validated that high-temperature TGE prevented off-target hybrids from interfering with the quantitative responses of the target miRNAs. This work demonstrates that TGE affords rapid, highly selective analyses of structurally similar miRNAs in low-complexity microfluidic devices, which is expected to facilitate diverse biomedical research.

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). Fluorescent probes were designed complementarily in sequence to four target miRNAs that also contained variable DNA overhangs to alter their electrophoretic mobilities. Samples and probes were directly added into thermal gel and loaded throughout a microchannel. Applying voltage resulted in an inline preconcentration and separation of the miRNAs that did not require a sample injection nor user intervention to switch between modes. Baseline resolution was achieved between four double-stranded miRNA-probe hybrids and four excess single-stranded probes. Analytical performance was then improved by designing an innovative microfluidic device with a tapered channel geometry. This device exhibited superior detection limits and separation resolution compared to standard channel devices without increasing the complexity of microfabrication or device operation. A proof-of-concept demonstration was then performed, showing that target miRNAs could be detected from cell extracts. These results demonstrate that TGE provides a simple, inexpensive means of conducting multiplexed miRNA measurements, with the potential for automation to facilitate future clinical and pharmaceutical analyses.

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. 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.

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. 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.

Fundamental Studies of New Ionization Technologies and Insights from IMS-MS.

Exceptional ion mobility spectrometry mass spectrometry (IMS-MS) developments by von Helden, Jarrold, and Clemmer provided technology that gives a view of chemical/biological compositions previously not achievable. The ionization method of choice used with IMS-MS has been electrospray ionization (ESI). In this special issue contribution, we focus on fundamentals of heretofore unprecedented means for transferring volatile and nonvolatile compounds into gas-phase ions singly and multiply charged. These newer ionization processes frequently lead to different selectivity relative to ESI and, together with IMS-MS, may provide a more comprehensive view of chemical compositions directly from their original environment such as surfaces, e.g., tissue. Similarities of results using solvent- and matrix-assisted ionization are highlighted, as are differences between ESI and the inlet ionization methods, especially with mixtures such as bacterial extracts. Selectivity using different matrices is discussed, as are results which add to our fundamental knowledge of inlet ionization as well as pose additional avenues for inquiry. IMS-MS provides an opportunity for comparison studies relative to ESI and will prove valuable using the new ionization technologies for direct analyses. Our hypothesis is that some ESI-IMS-MS applications will be replaced by the new ionization processes and by understanding mechanistic aspects to aid enhanced source and method developments this will be hastened.

Natural variants of photosystem II subunit D1 tune photochemical fitness to solar intensity.

Photosystem II (PSII) is composed of six core polypeptides that make up the minimal unit capable of performing the primary photochemistry of light-driven charge separation and water oxidation in all oxygenic phototrophs. The D1 subunit of this complex contains most of the ligating amino acid residues for the Mn(4)CaO(5) core of the water-oxidizing complex (WOC). Most cyanobacteria have 3-5 copies of the psbA gene coding for at least two isoforms of D1, whereas algae and plants have only one isoform. Synechococcus elongatus PCC 7942 contains two D1 isoforms; D1:1 is expressed under low light conditions, and D1:2 is up-regulated in high light or stress conditions. Using a heterologous psbA expression system in the green alga Chlamydomonas reinhardtii, we have measured growth rate, WOC cycle efficiency, and O(2) yield as a function of D1:1, D1:2, or the native algal D1 isoform. D1:1-PSII cells outcompete D1:2-PSII cells and accumulate more biomass in light-limiting conditions. However, D1:2-PSII cells easily outcompete D1:1-PSII cells at high light intensities. The native C. reinhardtii-PSII WOC cycles less efficiently at all light intensities and produces less O(2) than either cyanobacterial D1 isoform. D1:2-PSII makes more O(2) per saturating flash than D1:1-PSII, but it exhibits lower WOC cycling efficiency at low light intensities due to a 40% faster charge recombination rate in the S(3) state. These functional advantages of D1:1-PSII and D1:2-PSII at low and high light regimes, respectively, can be explained by differences in predicted redox potentials of PSII electron acceptors that control kinetic performance.