Characterization of freestanding photoresist films for biological and MEMS applications.

Photoresists are light-sensitive resins used in a variety of technological applications. In most applications, however, photoresists are generally used as sacrificial layers or a structural layer that remains on the fabrication substrate. Thin layers of patterned 1002F photoresist were fabricated and released to form a freestanding film. Films of thickness in the range of 4.5-250 µm were patterned with through-holes to a resolution of 5 µm and an aspect ratio of up to 6:1. Photoresist films could be reliably released from the substrate after a 12-hour immersion in water. The Young's modulus of a 50 µm-thick film was 1.43 ± 0.20 GPa. Use of the films as stencils for patterning sputtered metal onto a surface was demonstrated. These 1002F stencils were used multiple times without deterioration in feature quality. Furthermore, the films provided biocompatible, transparent surfaces of low autofluorescence on which cells could be grown. Culture of cells on a film with an isolated small pore enabled a single cell to be accessed through the underlying channel and loaded with exogenous molecules independently of nearby cells. Thus 1002F photoresist was patterned into thin, flexible, free-standing films that will have numerous applications in the biological and MEMS fields.

Scalable synthesis of a biocompatible, transparent and superparamagnetic photoresist for microdevice fabrication.

The functionalization of photoresists with colloids has enabled the development of novel active and passive components for microfabricated devices. Incorporation of colloidal particles often results in undesirable reductions in photolithographic fidelity and device transparency. We present a novel photoresist composite incorporating poly(methyl methacrylate-co-methacrylic acid) (PMMA/MMA), the epoxy resin 1002F and colloidal maghemite nanoparticles to produce a stable, transparent and biocompatible photoresist. The composite photoresist was prepared in a scalable fashion in batches up to 1 kg with the particles remaining dispersed during room-temperature storage for at least 6 months. Following photolithography to form films, the nanoparticle size remained well below that of visible-light wavelengths as demonstrated by electron microscopy. Structures fabricated from the photoresist by conventional photolithography displayed aspect ratios greater than ten. When grown on the photoresist, the metabolic rate of HeLa cells was unchanged relative to cells grown on glass. Primary murine mesenchymal stem cells also displayed a normal morphology on the resist surface. The ability to manipulate microstructures formed from the composite was demonstrated by magnetically collecting clonal colonies of HeLa cells from a micropallet array. The transparency, biocompatibility, scalable synthesis and superparamagnetic properties of the novel composite address key limitations of existing magnetic composites.

Evidence for a conformational change in a class II major histocompatibility complex molecule occurring in the same pH range where antigen binding is enhanced.

Many class II histocompatibility complex molecules bind antigenic peptides optimally at low pH, consistent with their exposure to antigen in acidic endosomal compartments. While it has been suggested that a partially unfolded state serves as an intermediate involved in peptide binding, very little evidence for such a state has been obtained. In this report, we show that the murine class II molecule IE becomes increasingly less stable to sodium dodecyl sulfate-induced dissociation since the pH is decreased in the same range that enhances antigenic peptide binding. Furthermore, at mildly acidic pH levels, IEk binds the fluorescent dye 1-anilino-naphthalene-8-sulfonic acid (ANS), a probe for exposed nonpolar sites in proteins, suggesting that protonation produces a molten globule-like state. The association of IEk with a single high-affinity peptide had only a small effect in these two assays, indicating that the changes that occur are distal to the peptide-binding groove. Circular dichroism analysis shows that a pH shift from neutral to mildly acidic pH causes subtle changes in the environment of aromatic residues but does not grossly disrupt the secondary structure of IEk. We propose a model in which perturbations in interdomain contacts outside the peptide-binding domain of IEk occur at acidic pH, producing a partially unfolded state that facilitates optimal antigen binding.

Calcium ion concentrations and DNA fragmentation in target cell destruction by murine cloned cytotoxic T lymphocytes.

To investigate the destruction of target cells by murine CTLs, we examined intracellular Ca2+ concentrations ([Ca2+]i) and DNA fragmentation in target cells. Changes in [Ca2+]i were followed by flow cytometry by loading the cells with indo-1, a Ca2+-binding fluorescent dye, and determining the ration of fluorescence intensities at 405 nm (emission maximum for Ca2+-bound dye) over 480 nm (emission maximum for the free dye). Within minutes after interacting with the cytolytic granule fraction that had been isolated from CTLs, [Ca2+]i in target cells was strikingly increased. A pronounced increase in [Ca2+]i was also observed in target cells when they were specifically recognized by intact CTLs. Since ionomycin, a Ca2+ ionophore, caused a similar increase in [Ca2+]i and lysed cells (provided that extracellular Ca2+ was present), it appears that a sustained high level of [Ca2+]i is cytolytic. In contrast with other cells, CTLs, which have been shown to be refractory to granule-mediated lysis and to be poor targets for other CTLs, did not manifest an elevation in [Ca2+]i when they were similarly loaded with indo-1 and treated with isolated granules. The characteristic cleavage of target cell DNA into nucleosome-sized fragments was also induced by isolated granules as well as by valinomycin, a K+ ionophore, but not by ionomycin. The results support the view that lysis of most target cells by cloned CTLs is due primarily to target cell membrane changes that are fundamentally equivalent to the formation of nonspecific ion channels. The resulting large increase in [Ca2+]i is probably responsible for target cell lysis; and changes in intracellular ion concentrations also appear to be responsible for DNA fragmentation, probably by activating endogenous target cell endonucleases.

Range of messenger action of calcium ion and inositol 1,4,5-trisphosphate.

The range of messenger action of a point source of Ca2+ or inositol 1,4,5-trisphosphate (IP3) was determined from measurements of their diffusion coefficients in a cytosolic extract from Xenopus laevis oocytes. The diffusion coefficient (D) of [3H]IP3 injected into an extract was 283 microns 2/s. D for Ca2+ increased from 13 to 65 microns 2/s when the free calcium concentration was raised from about 90 nM to 1 microM. The slow diffusion of Ca2+ in the physiologic concentration range results from its binding to slowly mobile or immobile buffers. The calculated effective ranges of free Ca2+ before it is buffered, buffered Ca2+, and IP3 determined from their diffusion coefficients and lifetimes were 0.1 micron, 5 microns, and 24 microns, respectively. Thus, for a transient point source of messenger in cells smaller than 20 microns, IP3 is a global messenger, whereas Ca2+ acts in restricted domains.

Single cells as biosensors for chemical separations.

A biosensor system based on the response of living cells was demonstrated that can detect specific components of a complex mixture fractionated by a microcolumn separation technique. This system uses ligand-receptor binding and signal-transduction pathways to biochemically amplify the presence of an analyte after electrophoretic separation. The transduced signal was measured by means of two approaches: (i) fluorescence determination of intracellular calcium concentrations in one or more rat PC-12 cells and (ii) measurement of transmembrane current in a Xenopus laevis oocyte microinjected with messenger RNA that encodes a specific receptor. This analysis system has the potential to identify biologically active ligands present in a complex mixture with exceptional sensitivity and selectivity.

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.

Localized measurement of kinase activation in oocytes of Xenopus laevis.

We have combined a rapid cytoplasmic sampling technique with capillary electrophoresis to measure the activation of protein kinase C (PKC) in a small region (approximately 60 microm) of a Xenopus oocyte. The phosphorylation of a fluorescent PKC substrate was measured following addition of a pharmacological or physiological stimulus to an oocyte. When substrates for cdc2 kinase (cdc2K), PKC, and protein kinase A (PKA) were comicroinjected into an oocyte, all three substrates could be identified on the electropherogram after cytoplasmic sampling. With this new method, it should be possible to measure simultaneously the activation of multiple different kinases in a single cell, enabling the quantitative dissection of signal transduction pathways.

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.

Metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate by the oocytes of Xenopus laevis.

The pathway and kinetics of inositol 1,4,5-trisphosphate (IP3) metabolism were measured in Xenopus laevis oocytes and cytoplasmic extracts of oocytes. Degradation of microinjected IP3 in intact oocytes was similar to that in the extracts containing comparable concentrations of IP3 ([IP3]). The rate and route of metabolism of IP3 depended on the [IP3] and the intracellular free Ca2+ concentration ([Ca2+]). At low [IP3] (100 nM) and high [Ca2+] (>/=1 microM), IP3 was metabolized predominantly by inositol 1,4, 5-trisphosphate 3-kinase (3-kinase) with a half-life of 60 s. As the [IP3] was increased, inositol polyphosphate 5-phosphatase (5-phosphatase) degraded progressively more IP3. At a [IP3] of 8 microM or greater, the dephosphorylation of IP3 was the dominant mode of IP3 removal irrespective of the [Ca2+]. At low [IP3] and low [Ca2+] (both

Characterization of a biological detector cell for quantitation of inositol 1,4,5-trisphosphate.

Prior strategies to measure inositol 1,4,5-trisphosphate (IP(3)) in single cells either have been qualitative or have had a limited spatial resolution. Capillary electrophoresis combined with a biological detector cell has been used to quantitate IP(3) in small regions of a Xenopus oocyte. To improve the detection limits of this method, we elucidated the experimental parameters which influenced the sensitivity and reliability of the IP(3)-detector cell coupled to capillary electrophoresis. The variables which influenced the detector cell were the magnitude of the voltage drop across the detector cell, the duration of this voltage drop, the direction of fluid flow in the capillary, the concentration of free Ca(2+) around the detector cell, and the presence of protease inhibitors during permeabilization of the detector cell. For the sample volumes imposed by the capillary diameter, the detector cell acted primarily as an IP(3) mass detector rather than a concentration detector. Characterization of the experimental variables influencing the sensitivity and reliability of this detector cell has the potential to enhance other analyte measurements performed by mating capillary electrophoresis with a biological detector cell.

Regulation of nuclear calcium concentration.

Transient increases in nuclear calcium concentration have been shown to activate gene expression and other nuclear processes. It has been suggested that nuclear calcium signals are controlled by a mechanism that is independent of calcium signalling in the cytosol. This would be possible if calcium diffusion is slow and a separate calcium release mechanism is localized to the nuclear region. Alternatively, the nuclear envelope could act as a diffusion barrier for calcium ions released either inside or outside the nucleus. It has also been proposed that inositol 1,4,5-trisphosphate (InsP3) can be generated inside the nucleus and that there are calcium release channels in the inner membrane of the nuclear envelope. Most of the experimental evidence supporting these hypotheses is based on the calibration of nuclear and cytosolic calcium concentrations. However, recent studies suggest that the local calibration of calcium indicators may not be accurate. We propose that nuclear calcium signals can be investigated by a different approach that does not rely on accurate calibration of indicators. We have developed calcium indicators that minimize facilitated calcium diffusion and are localized to either the nucleus or the cytosol. Using the diffusion coefficient of calcium ions, and measuring the delay between cytosolic and nuclear calcium increases, we show that the nuclear envelope is not a substantial barrier for calcium ions in PC12 (phaeochromocytoma) cells. This suggests that nuclear and cytosolic calcium signals equilibrate rapidly in these cells.

Localized sampling of cytoplasm from Xenopus oocytes for capillary electrophoresis.

Continued progress in cellular physiology requires new measurement strategies which can be applied to solitary cells. Since many cellular signaling pathways act on time scales of a few seconds, there is a critical need for single-cell techniques with subsecond time resolution. Capillary electrophoresis shows great promise as a tool for the analysis of individual cells. In the present work, we describe a technique to load a capillary with picoliter to nanoliter volumes of cytoplasm and initiate electrophoresis in less than 500 ms. When cytoplasm was sampled from a Xenopus laevis oocyte previously loaded with fluorescein, calcium green, or a mixture of the two fluorophores, their fluorescent peaks were readily identifiable on the electropherogram. Since the volume of cytoplasm (< or = 30 nL) loaded into the capillary was much smaller than the 1 microL oocyte volume, spatially localized biochemical measurements were also possible. To demonstrate the utility of this new technique, the activity of the enzyme beta-galactosidase was measured in small regions of the Xenopus oocyte. Subcellular, subsecond sampling of oocyte cytoplasm will enable biochemical measurements with the resolution required to understand many cellular signal transduction pathways.

Improved capillary electrophoresis conditions for the separation of kinase substrates by the laser micropipet system.

Phosphorylated and nonphosphorylated forms of peptide substrates for protein kinase C (PKC) and calcium-calmodulin activated kinase II (CamKII) were separated by capillary zone electrophoresis. Electrophoresis of the peptide substrates and products in biologic buffer solutions in uncoated capillaries yielded asymmetric analyte peaks with substantial peak tailing. Some of the peptides also exhibited broad peaks with unstable migration times. To improve the electrophoretic separation of the peptides, several strategies were implemented: extensive washing of the capillary with a base, adding betaine to the electrophoretic buffer, and coating the capillaries with polydimethylacrylamide (PDMA). Prolonged rinsing of the capillaries with a base substantially improved the migration time reproducibility and decreased peak tailing. Addition of betaine to the electrophoretic buffer enhanced both the migration time stability as well as the theoretical plate numbers of the peaks. Finally PDMA-coated capillaries brought about significant improvements in the resolving power of the separations. These modifications all utilized an electrophoretic buffer that was compatible with a living biologic cell. Consequently they should be adaptable for the new capillary electrophoresis-based methods to measure kinase activation in single cells.

Spatial control of cellular measurements with the laser micropipet.

Continued progress in understanding cellular physiology requires new strategies for biochemical measurements in solitary cells, multiple cells, and subcompartments of cells. Large spatial gradients in the concentrations of molecules and presumably the activities of enzymes can occur in cells. Consequently, there is a critical need for measurement techniques for mammalian cells with control over the numbers or regions of cells interrogated. In the present work, we developed a strategy to rapidly load the cytoplasmic contents of either multiple cells or a subregion of a single cell into a capillary. A single, focused pulse from a laser created a mechanical shock wave which disrupted a group of cells or a portion of a cell in the path of the shock wave. Simultaneously, the cytoplasm was loaded into a capillary for electrophoretic separation. The size of the region of cellular disruption (and therefore the volume of cytoplasm collected) was controlled by the amount of energy in the laser pulse. Higher energies could be used to sample groups of cells while much lower energies could be utilized to selectively sample the tip of a neuronal process. The feasibility of performing measurements on subcellular compartments was also demonstrated by targeting reporter molecules to these compartments. A reporter localized to the nucleus was detected on the electropherogram following laser-mediated disruption of the cell and the nucleus. Finally, we demonstrate that this method terminated cellular reactions with sufficient rapidity that cellular membrane repair mechanisms were not activated during cytoplasmic collection. The combined ability to preselect a spatial region of a cell or cells and to rapidly load that region into a capillary will greatly enhance the utility of CE in the biochemical analysis of cells.

Source of nuclear calcium signals.

Transient increases of Ca2+ concentration in the nucleus regulate gene expression and other nuclear processes. We investigated whether nuclear Ca2+ signals could be regulated independently of the cytoplasm or were controlled by cytoplasmic Ca2+ signals. A fluorescent Ca2+ indicator that is targeted to the nucleus was synthesized by coupling a nuclear localization peptide to Calcium Green dextran, a 70-kDa Ca2+ indicator. Stimulation of rat basophilic leukemia cells by antigen or by photolytic uncaging of inositol 1,4,5-trisphosphate induced transient increases in nuclear and cytosolic Ca2+ concentrations. Elevations in the nuclear Ca2+ concentration followed those in the nearby perinuclear cytosol within 200 ms. Heparin-dextran, an inhibitor of the inositol 1,4,5-trisphosphate receptor that is excluded from the nucleus, was synthesized to specifically block the release of Ca2+ from cytosolic stores. Addition of this inhibitor suppressed Ca2+ transients in the nucleus and the cytosol. We conclude that the Ca2+ level in the nucleus is not independently controlled. Rather, nuclear Ca2+ increases follow cytosolic Ca2+ increases with a short delay most likely due to Ca2+ diffusion from the cytosol through the nuclear pores.

A comparison of the cytolytic properties of murine primary CD8+ cytotoxic T lymphocytes and cloned cytotoxic T cell lines.

Lysates of many highly cytolytic murine primary CD8+ cytotoxic T lymphocytes (CTLs) have no detectable hemolytic activity and only traces of serine esterase activity, indicating a striking paucity or absence of the perforin-rich secretory granules that are abundant in the cytoplasm of murine cloned CTL cell lines. Nevertheless, the primary CTLs are almost as resistant to granule-mediated lysis as CTL cell lines. Moreover, target cells that are lysed by all CTLs so far tested, whether primary or cell lines, show similar rapid and marked increases in intracellular calcium and breakdown of DNA into nucleosome-sized fragments. A parsimonious explanation for all of these findings is that primary CTLs, like the CTL cell lines, exercise their cytolytic activity by means of perforin, but the amounts needed are extremely small and below the level of detection by the current relatively insensitive hemolytic assays.

The physiologic concentration of inositol 1,4,5-trisphosphate in the oocytes of Xenopus laevis.

To measure the concentration of inositol 1,4,5-trisphosphate ([IP3]) in small regions of single Xenopus oocytes, a biological detector cell was combined with capillary electrophoresis. This method is 10, 000 times more sensitive than all existing assays enabling subcellular measurement of [IP3] in Xenopus oocytes. Upon addition of lysophosphatidic acid to an oocyte, [IP3] increased from 40 to 650 nM within 2 min. IP3 concentrations as high as 1.8 microM were measured after activation with lysophosphatidic acid, suggesting that the physiologic concentration of IP3 ranges from the tens of nanomolar to a few micromolar in Xenopus oocytes. Since the IP3 receptor in Xenopus oocytes is nearly identical to the type I receptor of mammalian cells, the range of [IP3] in most mammalian cells is likely to be similar to that in the oocyte. By selecting or engineering the appropriate detector cell, this strategy should be applicable to cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate, and to the discovery of new Ca2+-releasing second messengers.

Characterization of cellular optoporation with distance.

We have developed and characterized cellular optoporation with visible wavelengths of light using standard uncoated glass cover slips as the absorptive media. A frequency-doubled Nd:YAG laser pulse was focused at the interface of the glass surface and aqueous buffer, creating a stress wave and transiently permeabilizing nearby cells. Following optoporation of adherent cells, three spatial zones were present which were distinguished by the viability of the cells and the loading efficiency (or number of extracellular molecules loaded). The loading efficiency also depended on the concentration of the extracellular molecules and the molecular weight of the molecules. In the zone farthest from the laser beam (> 60 microns under these conditions), nearly all cells were both successfully loaded and viable. To illustrate the wider applicability of this optoporation method, cells were loaded with a substrate for protein kinase C and the cellular contents then analyzed by capillary electrophoresis. In contrast to peptides loaded by microinjection, optoporated peptide showed little proteolytic degradation, suggesting that the cells were minimally perturbed. Also demonstrating the potential for future work, cells were optoporated and loaded with a fluorophore in the enclosed channels of microfluidic devices.