Nancy L. Allbritton

Micro total analysis systems for cell biology and biochemical assays.

Novel applications of micro total analysis systems (µTAS) are addressing fundamental biological questions, creating new biomedical reagents, and developing innovative cell and biochemical assays. These efforts impact progress in all areas of µTAS from materials to fluidic handling as well as detection and external control systems. Three areas show the greatest current and potential impact on the biomedical sciences: improvements in device fabrication and operation, development of enabling technologies, and advancements at the interface with biology (Figure 1). The range of materials from which devices can be fabricated has expanded considerably and now includes paper, fabric and thread, and a multitude of polymers as well as more conventional materials. Thus device substrates and component materials suitable for nearly all biological applications are readily available. Devices are also becoming increasingly integrated with advancements in sample handling and preparation, key first steps in any biological analysis. Another growing area focuses on modular components that can be mixed and matched on-demand and applied to many different assays, so-called programmable microfluidics. This development should enhance the rate at which new bioassays are generated as well as customize existing experimental protocols. A second area of rapid advancement has been the development new technologies that enable assays that cannot be efficiently performed by any method except µTAS. Novel analyses of single cells are enabled due to effective manipulation of picoliter-scale volumes. Synthesis and screening of large-scale libraries has become increasingly feasible due to the fast processing speeds and combinatorial mixing of reagents provided by lab-on-chip systems. Increased automation within a completely contained system has now begun to provide some of the first true µTAS diagnostic devices for clinical medicine. The third area in which µTAS has begun to yield high dividends is the interfacing of living entities with microdevices to create biological communities, including tissues and organs on-chip. Control of cell placement in multiple dimensions has produced biological systems midway between the conventional tissue-culture dish and an intact animal. Thus the complexities of living constructs can be recreated in a controlled experimental environment permitting groundbreaking biological questions to be addressed. Application of µTAS in all of these areas continues to be highly interdisciplinary, utilizing techniques and strategies from almost every scientific field. This multidisciplinary focus insures continued relevance to the biological community as well as a bright future. Figure 1 We highlight recent contributions to µTAS in three interlocking areas: fabrication & operation, enabling technologies, and interfacing with biology. Due to the rapid progress of µTAS or “lab-on-a-chip” systems, this review focuses on advances impacting cell biology and biochemistry and covers the time span from March 2010 through August 2011. The material for the review was compiled using several strategies: reviews of high impact journals such as Analytical Chemistry, Lab on a Chip, Science, Nature, and PNAS; extensive key word searches in databases such as PubMed, SciFinder, Web of Science, and Google Scholar; and screens of other recent topical reviews. Although several thousand papers were identified and over a thousand papers received a detailed examination, we focused on the most novel and exciting methods, devices, and applications in the areas of cell biology and biochemistry. We also endeavored to cover the most prominent work from a range of labs and countries. Ultimately we were limited by space constraints and our desire to craft a readable commentary on the state of the field. We apologize in advance for omitted papers and welcome feedback regarding any oversights on our part.

Electroosmotic properties of microfluidic channels composed of poly (dimethylsiloxane)

Microfluidic devices fabricated from polymers exhibit great potential in biological analyses. Poly(dimethylsiloxane) (PDMS) has shown promise as a substrate for rapid prototyping of devices. Despite this, disagreement exists in the literature as to the ability of PDMS to support electroosmotic (EO) flow and the stability of that flow over time. We demonstrate that in low ionic strength solutions near neutral in pH. oxidized PDMS had a four-fold greater EO mobility (mu(eo)) compared to native PDMS. The greater mu(eo) was maintained irrespective of whether glass or PDMS was used as a support forming one side of the channel. This enhanced mu(eo) was preserved as long as the channels were filled with an aqueous solution. Upon exposure of the channels to air, the mobility decreased by a factor of two with a half-life of 9 h. The EO properties of the air-exposed, oxidized PDMS were regenerated by exposure to strong base. High ionic strength, neutral in pH buffers compatible with living eukaryotic cells diminished the EO flow in the oxidized PDMS devices to a much greater extent than in the native PDMS devices. For analyses utilizing intact and living cells, oxidation of PDMS may not be an effective strategy to substantially increase the mu(eo).

Micro total analysis systems: Fundamental advances and applications in the laboratory, clinic, and field

Applications of micro total analysis systems (μTAS) span basic-science research, clinical medicine, and field work. Assay devices designed for these applications offer improvements to existing methods or provide fundamentally new strategies. Both mature methods and novel techniques have benefited from the increased throughput, integration and miniaturization afforded by μTAS. Traditional assays such Western blots and binding assays are recapitulated in a μTAS format but with reduced reagent usage, decreased performance times and added capabilities. An increasingly vibrant area is the performance of drug screening and toxicology assays on-chip, enabling the efficient screening of very large numbers of molecules. Similarly, recent μTAS reactors demonstrate greater chemical synthetic yields and novel product synthesis compared to macro-systems, often as a result of accurate control over reaction conditions including precision reagent dispensing. These exciting systems are now enabling on-site production of short-lived radioactive compounds for medical applications. The greatest impact of μTAS may very well be the ability to perform massively parallel laboratory experiments, for example, the use millions of reaction vessels or the analysis of hundreds of thousands of single cells. Another strength of μTAS lies in the creation of multicellular communities, for example, the combination of many cell types into an interacting system to explore intercellular communication. Devices with multiple layers of co-cultured tissues benefit from precise placement of molecules, such as extracellular matrices or growth factors, in both space and time. Similarly, the complexity and variety of organ-on-chip and organism-on-chip technologies continues to escalate rapidly. Impressively, the types of organisms cultured on-chip now range from the simplest bacteria to complex animals such as fish. Automation, reliability, and integration must all increase as a device moves from the specialist environment of a lab to usage by non-expert personnel in the outside world, for example, at a clinical point-of-care or in environmental monitoring. Key innovations in recent months result in devices that operate with minimal external equipment, error-free operation, and unambiguous readouts, all critical for operation by untrained personnel. Lightweight, portable devices are increasingly used to identify chemical and biological toxins in water, air and soil with applications in public health, defense, and homeland security. Perhaps most exciting is the development of μTAS with sufficient robustness for operation in challenging environments, such as the ocean and outer space. A central component of these systems is the ability to withstand the unexpected. These systems push the boundaries of current integration principles and spur rapid growth of new design philosophies. This review focuses on advances in the area of μTAS or “lab-on-a-chip” systems over the time span of May 2011 through September 2012 with a focus on applications in basic research, clinical medicine and field usage. A range of journals with 2011 impact factors from 2.0 to 36.3 were screened to cover publications with highly specialized content as well as those directed at multidisciplinary audiences. These publications included discipline-specific journals such as Analytical Chemistry and Lab on a Chip as well as general scientific publications, e.g. Science and Nature. To identify material beyond the individually examined journals, extensive key word searches in databases such as PubMed, SciFinder, and Web of Science were performed. Recent reviews in the area of μTAS were also examined for appropriate references. Care was taken to identify impactful and exciting work from across the globe. Well over a thousand papers in the three target areas were identified and discussed. Due to space limitations, we were unable to include all papers but instead incorporated those most fitting into the review scheme and those reporting innovations in basic microdevice technology as well as in applications to biological, physical and engineering sciences. We apologize in advance for omitted papers and welcome feedback regarding any oversights on our part.

Measurement of kinase activation in single mammalian cells

We demonstrate a new method for the simultaneous measurement of the activation of key regulatory enzymes within single cells. To illustrate the capabilities of the technique, the activation of protein kinase C (PKC), protein kinase A (PKA), calcium-calmodulin activated kinase II (CamKII), and cdc2 protein kinase (cdc2K) was measured in response to both pharmacological or physiological stimuli. This assay strategy should be applicable to a broad range of intracellular enzymes, including phosphatases, proteases, nucleases, and other kinases.

Sperm Factor Induces Intracellular Free Calcium Oscillations by Stimulating the Phosphoinositide Pathway

Abstract Injection of a porcine cytosolic sperm factor (SF) or of a porcine testicular extract into mammalian eggs triggers oscillations of intracellular free calcium ([Ca2+]i) similar to those initiated by fertilization. To elucidate whether SF activates the phosphoinositide (PI) pathway, mouse eggs or SF were incubated with U73122, an inhibitor of events leading to phospholipase C (PLC) activation and/or of PLC itself. In both cases, U73122 blocked the ability of SF to induce [Ca2+]i oscillations, although it did not inhibit Ca2+ release caused by injection of inositol 1,4,5-triphosphate (IP3). The inactive analogue, U73343, had no effect on SF-induced Ca2+ responses. To determine at the single cell level whether SF triggers IP3 production concomitantly with a [Ca2+]i rise, SF was injected into Xenopus oocytes and IP3 concentration was determined using a biological detector cell combined with capillary electrophoresis. Injection of SF induced a significant increase in [Ca2+]i and IP3 production in these oocytes. Using ammonium sulfate precipitation, chromatographic fractionation, and Western blotting, we determined whether PLCγ1, PLCγ2, or PLCδ4 and/or its splice variants, which are present in sperm and testis, are responsible for the Ca2+ activity in the extracts. Our results revealed that active fractions do not contain PLCγ1, PLCγ2, or PLCδ4 and/or its splice variants, which were present in inactive fractions. We also tested whether IP3 could be the sensitizing stimulus of the Ca2+-induced Ca2+ release mechanism, which is an important feature of fertilized and SF-injected eggs. Eggs injected with adenophostin A, an IP3 receptor agonist, showed enhanced Ca2+ responses to CaCl2 injections. Thus, SF, and probably sperm, induces [Ca2+]i rises by persistently stimulating IP3 production, which in turn results in long-lasting sensitization of Ca2+-induced Ca2+ release. Whether SF is itself a PLC or whether it acts upstream of the eggs PLCs remains to be elucidated.

Chemical Analysis of Single Cells

Chemical analysis of single cells requires methods for quickly and quantitatively detecting a diverse array of analytes from extremely small volumes (femtoliters to nanoliters) with very high sensitivity and selectivity. Microelectrophoretic separations, using both traditional capillary electrophoresis and emerging microfluidic methods, are well suited for handling the unique size of single cells and limited numbers of intracellular molecules. Numerous analytes, ranging from small molecules such as amino acids and neurotransmitters to large proteins and subcellular organelles, have been quantified in single cells using microelectrophoretic separation techniques. Microseparation techniques, coupled to varying detection schemes including absorbance and fluorescence detection, electrochemical detection, and mass spectrometry, have allowed researchers to examine a number of processes inside single cells. This review also touches on a promising direction in single cell cytometry: the development of microfluidics for integrated cellular manipulation, chemical processing, and separation of cellular contents.

Examination of laser microbeam cell lysis in a PDMS microfluidic channel using time-resolved imaging

We use time-resolved imaging to examine the lysis dynamics of non-adherent BAF-3 cells within a microfluidic channel produced by the delivery of single highly-focused 540 ps duration laser pulses at lambda = 532 nm. Time-resolved bright-field images reveal that the delivery of the pulsed laser microbeam results in the formation of a laser-induced plasma followed by shock wave emission and cavitation bubble formation. The confinement offered by the microfluidic channel constrains substantially the cavitation bubble expansion and results in significant deformation of the PDMS channel walls. To examine the cell lysis and dispersal of the cellular contents, we acquire time-resolved fluorescence images of the process in which the cells were loaded with a fluorescent dye. These fluorescence images reveal cell lysis to occur on the nanosecond to microsecond time scale by the plasma formation and cavitation bubble dynamics. Moreover, the time-resolved fluorescence images show that while the cellular contents are dispersed by the expansion of the laser-induced cavitation bubble, the flow associated with the bubble collapse subsequently re-localizes the cellular contents to a small region. This capacity of pulsed laser microbeam irradiation to achieve rapid cell lysis in microfluidic channels with minimal dilution of the cellular contents has important implications for their use in lab-on-a-chip applications.

Localized calcium spikes and propagating calcium waves

Ca2+ signals control or modulate diverse cellular processes such as cell growth, muscle contraction, hormone secretion, and neuronal plasticity. Elevations in intracellular Ca2+ concentrations can be highly localized to micron and submicron domains or propagated as intra- and intercellular waves over distances as large as 1 mm. Localized, subcellular Ca2+ spikes are thought to selectively activate effector systems such as Ca2+ activated chloride currents in pancreatic acinar cells, neurotransmitter release in synaptic nerve terminals, and morphological changes in neural growth cones. In contrast, long-ranged Ca2+ waves synchronize the activities of different cytoplasmic regions of a single cell, such as cortical granule exocytosis after egg fertilization or coordinate the activities of many cells, such as ciliary beating in pulmonary epithelium. The purpose of this review is to delineate the role of Ca2+ in the generation of localized, subcellular Ca2+ spikes and long-ranged intracellular and intercellular Ca2+ waves.

Benchtop micromolding of polystyrene by soft lithography

Polystyrene (PS), a standard material for cell culture consumable labware, was molded into microstructures with high fidelity of replication by an elastomeric polydimethylsiloxane (PDMS) mold. The process was a simple, benchtop method based on soft lithography using readily available materials. The key to successful replica molding by this simple procedure relies on the use of a solvent, for example, gamma-butyrolactone, which dissolves PS without swelling the PDMS mold. PS solution was added to the PDMS mold, and evaporation of the solvent was accomplished by baking the mold on a hotplate. Microstructures with feature sizes as small as 3 μm and aspect ratios as large as 7 were readily molded. Prototypes of microfluidic chips made from PS were prepared by thermal bonding of a microchannel molded in PS with a flat PS substrate. The PS microfluidic chip displayed much lower adsorption and absorption of hydrophobic molecules (e.g. rhodamine B) compared to a comparable chip created from PDMS. The molded PS surface exhibited stable surface properties after plasma oxidation as assessed by contact angle measurement. The molded, oxidized PS surface remained an excellent surface for cell culture based on cell adhesion and proliferation. To demonstrate the application of this process for cell biology research, PS was micromolded into two different microarray formats, microwells and microposts, for segregation and tracking of non-adherent and adherent cells, respectively. The micromolded PS possessed properties that were ideal for biological and bioanalytical needs, thus making it an alternative material to PDMS and suitable for building lab-on-a-chip devices by soft lithography methods.

Measuring enzyme activity in single cells

Seemingly identical cells can differ in their biochemical state, function and fate, and this variability plays an increasingly recognized role in organism-level outcomes. Cellular heterogeneity arises in part from variation in enzyme activity, which results from interplay between biological noise and multiple cellular processes. As a result, single-cell assays of enzyme activity, particularly those that measure product formation directly, are crucial. Recent innovations have yielded a range of techniques to obtain these data, including image-, flow- and separation-based assays. Research to date has focused on easy-to-measure glycosylases and clinically-relevant kinases. Expansion of these techniques to a wider range and larger number of enzymes will answer contemporary questions in proteomics and glycomics, specifically with respect to biological noise and cellular heterogeneity.