Single-cell sphingosine kinase activity measurements in primary leukemia

Sphingosine kinase (SK) is a promising therapeutic target in a number of cancers, including leukemia. Traditionally, SK has been measured in bulk cell lysates, but this technique obscures the cellular heterogeneity present in this pathway. For this reason, SK activity was measured in single cells loaded with a fluorescent sphingosine reporter. An automated capillary electrophoresis (CE) system enabled rapid separation and quantification of the phosphorylated and nonphosphorylated sphingosine reporter in single cells. SK activity was measured in tissue-cultured cells derived from chronic myelogenous leukemia (K562), primary peripheral blood mononuclear cells (PBMCs) from three patients with different forms of leukemia, and enriched leukemic blasts from a patient with acute myeloid leukemia (AML). Significant intercellular heterogeneity existed in terms of the degree of reporter phosphorylation (as much as an order of magnitude difference), the amount of reporter uptake, and the metabolites formed. In K562 cells, the average amount of reporter converted to the phosphorylated form was 39?±?26 % per cell. Of the primary PBMCs analyzed, the average amount of phosphorylated reporter was 16?±?25 %, 11?±?26 %, and 13?±?23 % in a chronic myelogenous leukemia (CML) patient, an AML patient, and a B-cell acute lymphocytic leukemia (B-ALL) patient, respectively. These experiments demonstrated the challenge of studying samples comprised of multiple cell types, with tumor blasts present at 5 to 87 % of the cell population. When the leukemic blasts from a fourth patient with AML were enriched to 99 % of the cell population, 19?±?36 % of the loaded sphingosine was phosphorylated. Thus, the diversity in SK activity remained even in a nearly pure tumor sample. These enriched AML blasts loaded significantly less reporter (0.12?±?0.2 amol) relative to that loaded into the PBMCs in the other samples (≥1 amol). The variability in SK signaling may have important implications for SK inhibitors as therapeutics for leukemia and demonstrates the value of single-cell analysis in characterizing the nature of oncogenic signaling in cancer. Figure

Phosphorylation of a fluorescent sphingosine kinase reporter was used to measure single-cell SK activity in primary cells from leukemic patients. Peripheral blood mononuclear cells as well as enriched leukemic blasts were analyzed.     

Microtable arrays for culture and isolation of cell colonies

Cell microarrays with culture sites composed of individually removable microstructures or micropallets have proven benefits for isolation of cells from a mixed population. The laser energy required to selectively remove these micropallets with attached cells from the array depends on the microstructure surface area in contact with the substrate. Laser energies sufficient to release micropallets greater than 100 μm resulted in loss of cell viability. A new three-dimensional culture site similar in appearance to a table was designed and fabricated using a simple process that relied on a differential sensitivity of two photoresists to UV-mediated photopolymerization. With this design, the larger culture area rests on four small supports to minimize the surface area in contact with the substrate. Microtables up to 250 × 250 μm were consistently released with single 10-μJ pulses to each of the four support structures. In contrast, microstructures with a 150 × 150-μm surface area in contact with the substrate could not be reliably released at pulse energies up to 212 μJ. Cassie–Baxter wetting is required to provide a barrier of air to localize and sequester cells to the culture sites. A second asset of the design was an increased retention of this air barrier under conditions of decreased surface tension and after prolonged culture of cells. The improved air retention was due to the hydrophobic cavity created beneath the table and above the substrate which entrapped air when an aqueous solution was added to the array. The microtables proved an efficient method for isolating colonies from the array with 100% of selected colonies competent to expand following release from the array.     

Contact printing of arrayed microstructures

A novel contact printing method utilizing a sacrificial layer of polyacrylic acid (PAA) was developed to selectively modify the upper surfaces of arrayed microstructures. The method was characterized by printing polystyrene onto SU-8 microstructures to create an improved substrate for a cell-based microarray platform. Experiments measuring cell growth on SU-8 arrays modified with polystyrene and fibronectin demonstrated improved growth of NIH 3T3 (93% vs. 38%), HeLa (97% vs. 77%), and HT1080 (76% vs. 20%) cells relative to that for the previously used coating method. In addition, use of the PAA sacrificial layer permitted the printing of functionalized polystyrene, carboxylate polystyrene nanospheres, and silica nanospheres onto the arrays in a facile manner. Finally, a high concentration of extracellular matrix materials (ECM), such as collagen (5 mg/mL) and gelatin (0.1%), was contact-printed onto the array structures using as little as 5 μL of the ECM reagent and without the formation of a continuous film bridge across the microstructures. Murine embryonic stem cells cultured on arrays printed with this gelatin hydrogel remained in an undifferentiated state indicating an adequate surface gelatin layer to maintain these cells over time.     

Label-free biosensor by protein grating coupler on planar optical waveguides

We report a diffraction-based, label-free biosensor on the surface of a planar optical waveguide. By selectively immobilizing proteins on an optical waveguide, the proteins can form the ridge/valley of a latent image optical grating. Binding events with patterned surface proteins dynamically increase grating height and cause an increase in light coupling out of the waveguide. We demonstrate the efficacy of this strategy for protein detection and present the design and fabrication of a device.     

Separation of fluorescent phosphatidyl inositol phosphates by CE

Phosphatidyl inositol 4,5-bisphosphate (PIP2) and phosphatidyl inositol 3,4,5-trisphosphate (PIP3) labeled with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) on the acyl chain or a phosphatidyl ethanolamine head group were separated by CE with LIF detection. Several methods and capillary-coating procedures were tested for the separation of these phosphatidyl inositol phosphates (PIPs) at 20 degrees C. Separation of the PIPs in less than 20 min with excellent resolution was achieved using a buffer containing sodium deoxycholate (SDC), 1-propanol, MgCl2 and the polymer coating reagent, EOTrol LR. The efficiency of the optimized method was as high as 1.3x10(5) plates. The dependence of the separation on the concentration of 1-propanol, SDC, and MgCl2 was determined. The separation of PIP2 and PIP3 was primarily due to differential binding of the lipids to Mg2+ rather than to different solubilities in the micellar phase. The role of the SDC was to prevent adsorption of the hydrophobic lipids to the capillary wall and thus enhance the efficiency. The fluorescent PIPs are of value for both in vitro and in vivo assays of phospholipid metabolism. In particular, the use of these lipids with the optimized capillary-based separation will be of utility for drug screening as well as cell-based assays.     

Simple photografting method to chemically modify and micropattern the surface of SU-8 photoresist

SU-8 has gained widespread acceptance as a negative photoresist. It is also finding increasing use as a structural material in microanalytical devices. Consequently, methods to tailor the surface properties of SU-8 as well as to micropattern coatings on the surface of SU-8 are needed. The SU-8 photoresist consists of EPON SU-8 resin mixed with the photoacid generator triarylsulfonium hexafluoroantimonate. This photoacid generator can also serve as a photoinitiator generating free radicals when illuminated with UV light. Under the appropriate conditions, sufficient triarylsulfonium hexafluoroantimonate remains within cured SU-8 to act as a source of free radicals and initiate UV-mediated grafting of polymers onto the surface of the SU-8. UV-mediated grafting was used to coat SU-8 surfaces with poly(acrylic acid) and other water-soluble monomers. The SU-8 surface was chemically micropatterned by placing a mask between the UV light and SU-8. The X-Y spatial resolution of micropatterned poly(acrylic acid) on the SU-8 surface was 2 mum. Three applications of these chemically modified SU-8 surfaces were demonstrated. In the first, poly(ethylene glycol) was used to protect the SU-8 from interactions with proteins, yielding a surface resistant to biofouling. In the second demonstration, the SU-8 surface was micropatterned with a cell-resistant layer to guide cellular attachment and growth. In the final application, SU-8 micropallets were encoded with polymer lines. The bar codes were read by either absorbance or fluorescence measurements. Thus, UV-mediated graft polymerization is an efficient and effective method to micropattern coatings onto the surface of SU-8.     

Shape resonances in slow electron scattering by aromatic molecules. I. Anthraquinone derivatives

A series of anthraquinone (C(14)O(2)H(8)) derivatives has been studied by means of electron capture negative ion mass spectrometry (ECNI-MS), photoelectron spectroscopy (PES), and AM1 quantum chemical calculations. Mean lifetimes of molecular negative ions M(-.) (MNI) have been measured. The mechanism of long-lived MNI formation in the epithermal energy region of incident electrons has been investigated. A simple model of a molecule (a spherical potential well with the repulsive centrifugal term) has been applied for the analysis of the energy dependence of cross sections at the first stage of the electron capture process. It has been shown that a temporary resonance of MNI at the energy approximately 0.5 eV corresponds to a shape resonance with lifetime 1-2.10(-13) s in the f-partial wave (l = 3) of the incident electron. The next resonant state of MNI at the energy approximately 1.7 eV has been associated with the electron excited Feshbach resonance (whose parent state is a triplet npi* transition). In all cases the initial electron state of the MNI relaxes into the ground state by means of a radiationless transition, and the final state of the MNI is a nuclear excited resonance with a lifetime measurable on the mass spectrometry timescale. Copyright 1999 John Wiley & Sons, Ltd.     

Femtomole SHAPE reveals regulatory structures in the authentic XMRV RNA genome

Higher-order structure influences critical functions in nearly all noncoding and coding RNAs. Most single-nucleotide resolution RNA structure determination technologies cannot be used to analyze RNA from scarce biological samples, like viral genomes. To make quantitative RNA structure analysis applicable to a much wider array of RNA structure-function problems, we developed and applied high-sensitivity selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) to structural analysis of authentic genomic RNA of the xenotropic murine leukemia virus-related virus (XMRV). For analysis of fluorescently labeled cDNAs generated in high-sensitivity SHAPE experiments, we developed a two-color capillary electrophoresis approach with zeptomole molecular detection limits and subfemtomole sensitivity for complete SHAPE experiments involving hundreds of individual RNA structure measurements. High-sensitivity SHAPE data correlated closely (R = 0.89) with data obtained by conventional capillary electrophoresis. Using high-sensitivity SHAPE, we determined the dimeric structure of the XMRV packaging domain, examined dynamic interactions between the packaging domain RNA and viral nucleocapsid protein inside virion particles, and identified the packaging signal for this virus. Despite extensive sequence differences between XMRV and the intensively studied Moloney murine leukemia virus, architectures of the regulatory domains are similar and reveal common principles of gammaretrovirus RNA genome packaging.     

Sample transport and electrokinetic injection in a microchip device for chemical cytometry

Sample transport and electrokinetic injection bias are well characterized in capillary electrophoresis and simple microchips, but a thorough understanding of sample transport on devices combining electroosmosis, electrophoresis, and pressure-driven flow is lacking. In this work, we evaluate the effects of electric fields from 0 to 300 V/cm, electrophoretic mobilities from 10(-4) to 10(-6) cm(2)/Vs, and pressure-driven fluid velocities from 50 to 250 μm/s on sample injection in a microfluidic chemical cytometry device. By studying a continuous sample stream, we find that increasing electric field strength and electrophoretic mobility result in improved injection and that COMSOL simulations accurately predict sample transport. The effects of pressure-driven fluid velocity on injection are complex, and relative concentration values lie on a surface defined by pressure-driven flow rates. For high-mobility analytes, this surface is flat, and sample injection is robust despite fluctuations in flow rate. For lower mobility analytes, the surface becomes steeper, and injection depends strongly on pressure-driven flow. These results indicate generally that device design must account for analyte characteristics and specifically that this device is suited to high-mobility analytes. We demonstrate that for a suitable pair of peptides fluctuations in injection volume are correlated; electrokinetic injection bias is minimized; and electrophoretic separation is achieved.