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PubMed1995

Monitoring an oxidative stress mechanism at a single human fibroblast.

Easily oxidizable substances inside human diploid fibroblast cell strains were monitored amperometrically with a platinized carbon-fiber microelectrode. The experiment involved positioning a microelectrode over a single biological cell, forcing the electrode tip into the cell via micromanipulator control, and measuring the transient current corresponding to the complete electrolysis of electroactive species released by the cell. A second series of experiments involved puncturing a hole into the cell with a micropipet and measuring the transient current corresponding to the complete electrolysis of electroactive species emitted by the cell with an electrode positioned above the cell. The selectivity of both amperometric measurements was demonstrated through the use of known hydrogen peroxide scavengers (added catalase or intracellular peroxidase + added o-dianisidine) to the media bathing the cells. The abolition of the amperometric signal under these conditions suggested that hydrogen peroxide was the primary substance detected. The magnitude and the time course of the transient current measured implied that the hydrogen peroxide detected was not only that initially present in the cell before its membrane was pierced but represented mostly an oxidative stress response of the cell to its injury.

Cells, Cultured↗
PubMed2002

Laser ablation construction of on-column reagent addition devices for capillary electrophoresis.

A simple and reproducible technique for constructing perfectly aligned gaps in fused-silica capillaries has been developed for postcolumn reagent addition with capillary electrophoresis. This technique uses laser ablation with the second harmonic of a Nd:YAG laser (532 nm) at 13.5 mJ/pulse and a repetition rate of 15 Hz to create these gaps. A capillary is glued to a microscope slide and positioned at the focal point of a cylindrical lens using the focused beam from a laser pointer as a reference. Gaps of 14.0 +/- 2.2 microm (n = 33) at the bore of the capillary are produced with a success rate of 94% by ablation with 400 pulses. This simple method of gap construction requires no micromanipulation under a microscope, hydrofluoric acid etching, or use of column fittings. These structures have been used for reagent addition for postcolumn derivatization with laser-induced fluorescence detection and have been tested for the separation of proteins and amino acids. Detection limits of 6 x 10(-7) and 1 x 10(-8) M have been obtained for glycine and tranferrin, respectively. Separation efficiencies obtained using these gap reactors range from 38,000 to 213,000 theoretical plates.

Electrophoresis, Capillary↗
PubMed2002

Electroanalysis of metabolic flux from single cells in simple picoliter-volume microsystems.

A picoliter-volume electrochemical analytical chamber has been developed for detecting the metabolic flux resulting from the stress responses of a single plant cell. Electrochemical cells, with volumes as small as 100 pL, were fabricated by controlled electrochemical dissolution of a gold wire sealed in glass (the back-etching of the metal realizing an ultralow-volume titer chamber). In the first instance, the electrode contained within the chamber was characterized by the microinjection of standard aliquots of either ascorbic acid or hydrogen peroxide. In all cases, experimental currents obtained correlated well with theoretical calculations. Subsequently, single plant cells were micromanipulated into the chambers and were exposed to amounts of the detergent SDS (which permeabilized the cell membrane and released the intracellular contents). The flux of metabolite released from a single cell was estimated by using electrochemical-linked assays based upon the enzymes catalase, ascorbate oxidase, and horseradish peroxidase (in each case), in the presence of a mediator. In so doing, we investigated the activity of the cellular protection mechanisms through the determination of peroxides, while the individual cell was "stressed". The technique was found to provide a reliable and reproducible method for making single-cell measurements, using fabrication procedures that are both simple and do not require photolithographic methods.

Journal Article↗
PubMed2004

Direct sampling from muscle cross sections for electrophoretic analysis of individual mitochondria.

Muscle is a highly heterogeneous tissue. Practical approaches to sample selectively small regions of muscle cross sections would help to effectively utilize analytical techniques on muscle studies while taking into account tissue heterogeneity. In this report, semimembranosus muscle tissue cross sections were directly sampled and analyzed by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Prior to CE-LIF analysis, a small region in the muscle cross section was stained with 10-nonyl acridine orange (NAO) which is a mitochondrion-selective fluorescent probe known to form a stable complex with cardiolipin, a phospholipid found only in mitochondria. By micromanipulation, the injection end of the capillary was brought into contact with the tissue exhibiting fluorescently labeled mitochondria. Sampling from a region similar in size to the cross section of a single fiber was carried out by applying 11 kPa of negative pressure for 3 s. When an electric field of -200V/cm was applied, fluorescently labeled mitochondria electromigrated and were individually detected by postcolumn LIF detection. For each sample, the electropherogram displays a migration time window with a collection of narrow peaks. The collection of individual peak measurements is represented as a distribution of individual intensities related to cardiolipin content of mitochondria and a distribution of individual electrophoretic mobilities. Positioning the capillary injection end was sufficiently spatially accurate to deplete mitochondria in the sampled region upon repetitive injections. Treatment of a muscle cross section with a protease (trypsin) prior to mitochondria sampling resulted in a higher number of detected mitochondria, suggesting that one of the effects of this enzyme is a partial digestion of the muscles myofibrils, which eases the release of interfibrillar mitochondria entangled within these fibers. The protease treatment also resulted in changes to the electrophoretic mobility distribution of individual mitochondria, which may imply that partial digestion of proteins bound to the mitochondria contributes to the alteration in the electrophoretic mobility of mitochondria. The ability to sample a region as small as a single muscle fiber cross section and its direct CE-LIF analysis opens exciting possibilities for the direct analysis of muscle biopsies and mapping the mitochondrial electrophoretic properties in highly heterogeneous tissues.

Aminoacridines↗
PubMed2005

Multiple sampling in single-cell enzyme assays using CE-laser-induced fluorescence to monitor reaction progress.

A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 microm in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to alpha-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, alpha1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the alpha-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of alpha-glucosidase I. Embryos were simultaneously assayed for a second enzyme, beta-galactosidase, illustrating that the mutants were viable except for their lack of alpha-glucosidase I activity.

Animals↗
PubMed2005

Single-cell microarray for analyzing cellular response.

Detection of cellular response by measuring intracellular calcium, (Ca2+)i with Ca2+-dependent fluorescent dye are standard approaches to detect ligand-stimulated cells and to study signaling through ligand/receptor interaction. We describe a single-cell microarray system to analyze cellular response of individual cells such as lymphocytes using microchamber array chips. The single-cell microarray chip is made from polystyrene with over 30,000 microchambers, which can accommodate only single cells. Lymphocytes derived from mouse spleen or human blood were spread on the microarray, and over 80% of the microchambers achieved single-cell status. Stimulation of B-cells through antigen receptors on the microarray allowed us to detect activated B-cells by comparing the states of single B-cells before and after stimulation with antigen, which is disabled for flow cytometry. In addition, this novel method demonstrated retrieval of positive single B-cells from microchambers by a micromanipulator and achieved antibody DNA analysis. The system is suitable for high-throughput analysis of intracellular Ca2+ response at the single-cell level and is applicable to screen antigen-specific lymphocytes for making specific monoclonal antibody.

Amino Acid Sequence↗
PubMed1997

Ultrathin slab gel separations of DNA using a single capillary sample introduction system.

We demonstrate here a novel method for DNA separations which combines the parallel processing capabilities of slab gels with the advantages of sample introduction obtained with a single capillary. This sample introduction format allows rapid sequential separations or continuous analysis to be carried out on ultrathin slab gels with efficient heat dissipation. Ultrathin slab gels have been fabricated by using 57-micron spacers between quartz plates, and a single capillary has been used to introduce plugs of dsDNA fragments into the ultrathin gel. These fragment plugs were deposited along the entrance to the ultrathin gel at spatially discrete locations by micromanipulation of the capillary. Spatially resolved detection has been accomplished with an argon ion laser focused to a line for excitation and a CCD for collection of fluorescence. Double-stranded DNA separations are demonstrated in a plug injection format. This approach allows multiple unique samples to be rapidly deposited on the ultrathin slab gels for separation.

DNA↗
PubMed1999

Fine chemical manipulations of microscopic liquid samples. 1. Droplet loading with chemicals.

Physically and chemically stable microscopic aqueous droplets of nano-, pico-, and femtoliter volumes were made and kept under heptane. Such droplets can contain chemicals of interest in pico-, femto-, and attomole amounts or less. Their fine chemical manipulation was achieved by a diffusional microburet (DMB), which consists of a pulled glass capillary whose microscopic tip is filled with a tiny diffusion membrane of agar or polyacrylamide gel. Once this tip is moved into a target droplet by a fine micromanipulator under a microscope, diffusional reagent delivery from the DMB body begins, driven by the concentration gradient within the tip. This system was tested in this work by delivering an inert fluorescent dye, Lucifer Yellow CH, into buffer droplets while fluorescence intensity in each droplet was recorded. An exponential decay of delivery rate was observed corresponding to an exponential saturation process for the accumulation of the delivered chemical inside the target droplet.

Chemistry Techniques, Analytical↗
PubMed2006

In vitro degradation behavior of microspheres based on cross-linked dextran.

The aim of this study was to investigate the in vitro degradation of hydroxyl ethyl methacrylated dextran (dex-HEMA) microspheres. Dextran microspheres were incubated in phosphate buffer pH 7.4 at 37 degrees C, and the dry mass, mechanical strength, and chemical composition of the microspheres were monitored in time. The amount and nature of the formed degradation products were established for microspheres with different cross-link densities by FT-IR (Fourier transformed infrared spectroscopy), NMR, mass spectrometry, SEC analysis, and XPS (X-ray photoelectron microscopy). The dex-HEMA microspheres DS 12 (degree of HEMA substitution; the number of HEMA groups per 100 glucose units) incubated at pH 7.4 and 37 degrees C showed a continuous mass loss, leaving after 6 months a residue of about 10% (w/w) of water-insoluble products. NMR, mass spectrometry, and SEC showed that the water-soluble degradation products consisted of dextran, low molecular weight pHEMA (M(n) approximately 15 kg/mol), and small amounts of unreacted HEMA and HEMA-DMAP (intermediate reaction product of the Baylis-Hillman reaction of HEMA with DMAP (4-dimethyl aminopyridine)). Microscopy revealed that the water-insoluble residue consisted of particles with shape and size similar to that of nondegraded microspheres. However, these particles had lost their mechanical strength as evidenced from micromanipulation experiments. FT-IR and XPS (X-ray photoelectron microscopy) revealed that these particles consisted of pHEMA, of which a small fraction was soluble in methanol (M(n) ranging between 27 and 82 kg/mol). The insoluble material likely consisted of lightly cross-linked pHEMA. In conclusion, in vitro degradation of dex-HEMA microspheres results in the formation of water-soluble degradation products (mainly dextran), leaving a small water-insoluble residue mainly consisting of pHEMA.

Chromatography, Gel↗
PubMed2002

Observation of topical catalysis by sphingomyelinase coupled to microspheres.

Sphingomyelinase, SMase (EC 3.1.4.12), was coupled onto amino-derivatized acrylate microspheres and was shown to retain its catalytic activity. The immobilized enzyme allows one to carry out topical enzymatic reaction in a controlled manner. Accordingly, these spheres were held with a micropipet and using micromanipulator brought into contact with a giant liposome membrane composed of phosphatidylcholine and sphingomyelin (SOPC/C16:0-SM, 0.75:0.25, molar ratio), representing the substrate for the immobilized enzyme. The macroscopic consequences of the enzyme reaction were visualized using fluorescence microscopy as well as differential interference contrast microscopy. The surface contact of the giant vesicle and immobilized enzyme causes membrane microdomain formation and domain clustering (capping) in the membrane and subsequent shedding of small vesicles from the membrane into the interior of the giant liposome. The method described represents a novel approach to study enzymatic reactions and allows manipulating giant vesicles as well as cultured cells in a spatially controlled manner.

Catalysis↗
PubMed2003

Long-range electron transfer through monolayers and bilayers of alkanethiols in electrochemically controlled Hg[bond]neling junctions.

The rates of electron tunneling through monolayers and bilayers of alkanethiols self-assembled in a potentiostatically controlled Hg-Hg junction are reported. An alkanethiolate monolayer is formed in situ on one or both Hg drops via oxidative adsorption at the controlled potential. Subsequently, the Hg drops are brought into contact using micromanipulators. The junction formation is instantly followed by the flow of a steady-state tunneling current between the two electrodes. A plot of the logarithm of the tunneling current density vs the total number of carbon atoms in each junction yields identical tunneling coefficients, beta = 1.06 +/- 0.04/-CH(2)- and beta = 1.02 +/- 0.07/-CH(2)-, for monolayers and bilayers of alkanethiols, respectively. Careful examination of the tunneling data indicates that the solvent and ions are ejected from the junction area. The tunneling current recorded for a bilayer of 1-octanethiol or 1-nonanethiol is ca. 2-fold larger than a corresponding tunneling current recorded for monolayers of 1-hexadecanethiol or 1-octadecanethiol, respectively. This result is explained in terms of weak electronic coupling across the noncovalent molecule/electrode interface.

Journal Article↗
PubMed2005

The binding energy of two nitrilotriacetate groups sharing a nickel ion.

Among the various molecular interactions used to construct supramolecular self-assembling systems, homoliganded metallic NTA-Ni-NTA complexes have received little attention despite their considerable potential applications, such as the connection of different biochemical functions. The stability of this complex is investigated here by using two concordant nanotechniques (surface forces apparatus and vesicle micromanipulation) that allow direct measurements of adhesion energies due to the chelation of nickel ions by nitrilotriacetate (NTA) groups grafted on surfaces. We show that two NTA groups can share a nickel ion, and that the association of a Ni-NTA complex with an NTA group has a molecular binding energy of 1.4 kcal/mol. Binding measurements in bulk by isothermal titration calorimetry experiments give the same value and, furthermore, indicate that the Ni-NTA chelation bond is about five times stronger than the NTA-Ni-NTA one. This first direct proof and quantification of the simultaneous chelation of a nickel ion by two NTA groups sheds new light on association dynamics involving chelation processes and offers perspectives for the development of new supramolecular assemblies and anchoring strategies.

Journal Article↗
PubMed2004

Directed assembly and rupture mechanics of colloidal aggregates.

The macroscopic rheological behavior of colloidal gels arises from the micromechanical properties of the gel backbone, which are governed by nanoscale particle interactions. These colloidal interactions have been commonly understood in terms of the Derjaguin-Landau-Verwey-Overbeek (DLVO) theory. Recent work has shown, however, that nonidealities, such as surface roughness and charge nonuniformity, may cause the particle interactions to significantly deviate from DLVO predictions at near-contact separations. Here we present novel techniques for directing the assembly of colloidal aggregates that mimic the gel backbone, based on optical micromanipulation of multiple particles using laser tweezers. This also provides an in situ method for measuring near-contact interactions via single-bond rupture forces. We find that PMMA particles aggregated in the presence of nonorganic salts exhibit interparticle bond strengths more than 10 times greater than those predicted by DLVO theory. However, good agreement is found with DLVO predictions when the anionic surfactant sodium dodecyl sulfate (SDS) is used as the flocculant.

Journal Article↗
PubMed2005

Single microgel particle studies demonstrate the influence of hydrophobic interactions between charged micelles and oppositely charged polyions.

The binding of two cationic surfactants, dodecyltrimethylammonium bromide (DoTAB) and N-(1,1,2,2-tetrahydroperfluorodecanyl)pyridinium bromide (HFDePB), to covalently cross-linked sodium poly(styrenesulfonate) (PSS) microgels has been investigated by means of micromanipulator-assisted time-resolved light microscopy on single gels. It is demonstrated that repeated measurements on the same microgel under conditions of controlled liquid flow give highly reproducible results. The two surfactants are found to behave very differently with respect to degree of swelling, surfactant distribution in the gels, both during shrinking and at equilibrium, and kinetics of volume changes induced by them. The main difference is attributed to the presence of a hydrophobic interaction between PSS and the DoTAB micelles, absent in the case of HFDePB. Kinetic shrinking curves are recorded and analyzed using a model for steady-state transport of surfactant between the solution and the gels. Aggregation numbers for DoTAB in PSS solutions obtained from fluorescence quenching measurements are presented. A strong dependence on the surfactant-to-polyion concentration ratio is observed. Relations between surfactant binding isotherms, phase diagrams for linear polyelectrolyte/surfactant/water systems, and the binding to gels are discussed.

Binding Sites↗
PubMed2006

Visualizing the interaction between poly-L-lysine and poly(acrylic acid) microgels using microscopy techniques: effect of electrostatics and peptide size.

The interaction between lightly cross-linked poly(acrylic acid) (pAA) microgels (50-150 microm in diameter) and poly-L-lysine (pLys) was studied as a function of pH, ionic strength, peptide size, and concentration. The swelling response and distribution of polypeptides within microgel particles was monitored by micromanipulator-assisted light microscopy and confocal laser scanning microscopy, while binding isotherms of pLys in the microgels were determined spectrophotometrically. Conformational changes of pLys were investigated by circular dichroism. The molecular weight of pLys was found to influence the degree of peptide-induced microgel deswelling, largely due to limitation of peptides larger than the effective network mesh size to penetrate the entire gel. Large peptides were concentrated within a surface layer of the gel particles, and at low ionic strength this dense surface layer was shown to act as a largely steric barrier for further penetration of compounds into the gel core. Small peptides, however, distributed evenly throughout the microgel particles and were able to create large microgel volume reductions. The deswelling of microgels increased with decreasing pH, while the uptake of pLys was significantly reduced at low pH. The effect of ionic strength on the interactions of pLys and oppositely charged pAA microgels was moderate and only pronounced for deswelling of gels at high pH. A significant increase in the alpha-helix content of pLys interacting with the oppositely charged microgels was observed for high molecular weight peptides, and the extent of alpha-helix formation was as expected more pronounced at high pH, i.e., at high charge density of the microgels but reduced charge density of the peptides.

Acrylic Resins↗
PubMed2006

Direct reconstitution of plasma membrane lipids and proteins in nanotube-vesicle networks.

We demonstrate here that nanotube-vesicle networks can be constructed directly from plasma membranes of cultured cells. We used a combination of dithiothreitol (DTT) and formaldehyde to produce micron-sized plasma membrane vesicles that were subsequently shaped into networks using micromanipulation methods previously used on purely synthetic systems. Only a single cell is required to derive material sufficient to build a small network. This protocol covers the advantages of reconstitution in vesicles, such as full control over the solution environment, while keeping the proteins in their original surroundings with the proper orientation. Furthermore, control of membrane protein and lipid content in the networks is achievable by employing different cell types, for example, by overexpression of a desired protein or the use of specialized cell-types as sources for rare proteins and lipids. In general, the method provides simple accessibility for functional studies of plasma membrane constituents. Specifically, it provides a direct means to functionalize nanotube-vesicle networks with desired proteins and lipids for studies of transport activity both across membranes (protein-mediated) and across nanotubes (diffusion), and substrate conversion down to the single-molecule limit. Nanotube-vesicle networks can adopt different geometries and topologies and undergo shape changes at will, providing a flexible system for changing the physical and chemical environment around, for example, a membrane protein. Furthermore, the method offers unique possibilities for extracting membrane and protein material for nanotechnological sensor and analytical devices based on lipid membrane networks.

Animals↗
PubMed1998

Detection of identical Hodgkin-Reed Sternberg cell specific immunoglobulin gene rearrangements in a patient with Hodgkin's disease of mixed cellularity subtype at primary diagnosis and in relapse two and a half years later.

BACKGROUND: The malignant nature of Hodgkin-Reed Sternberg (H-RS) cells has been questioned due to their scarcity in lymphoma tissues. Recently, using micromanipulation of H-RS cells and single cell PCR evidence was obtained that H-RS cells represent a clonal B-cell population. In these studies H-RS cells were isolated from each one lymph node for a given case. In classical Hodgkin's disease (HD) it thus could not be ruled out that H-RS cell clonality reflected a locally restricted clonal proliferation. We analysed biopsy specimens from a patient suffering from HD for the presence of clonally related H-RS cells at primary diagnosis and during relapse of the disease. MATERIALS AND METHODS: In 1994 the H-RS cell line L1236 was generated from the peripheral blood of a patient suffering from a disseminating relapse of HD of mixed cellularity subtype. The patient had relapsed despite intensive treatment including high dose chemotherapy and autologous bone marrow transplantation. The clonal identity of this cell line with H-RS cells in situ was proven by amplifying identical Ig gene rearrangements of the cell line as well as of single H-RS cells picked from the patients bone marrow. Primers covering the CDR3 region were chosen from the H-RS cell specific VH1 gene rearrangement to detect H-RS cells of the identical clone by amplifying the rearranged VH1 genes in tissue samples obtained during disseminating relapsing disease and at primary diagnosis of HD in 1991. RESULTS: The H-RS cell specific DNA sequence was detected in all affected tissues analysed including the cervical lymph node which has been exstirpated at primary diagnosis. CONCLUSION: This finding indicates the existence of a clonal H-RS cell population during the first manifestation of HD and persistence and dissemination of this clone despite aggressive treatment. Thus, in the described case the malignant nature of H-RS cells defined by dissemination and recurrence of the identical H-RS cell clone in relapsing disease is proven.

Adult↗
PubMed1999

An easy and reliable procedure of microdissection technique for the analysis of chromosomal breakpoints and marker chromosomes.

Microdissection in combination with reverse painting fluorescence in-situ hybridization (FISH) is a very effective method to identify breakpoints and rearrangements of derived chromosomes and reveal the chromosomal origin of marker chromosomes. We describe an innovation that allows a convenient, fast and safe isolation of microdissected fragments as currently available protocols. The microdissected chromosomes are harvested in a collection drop located in a movable micropipette adjusted to a second micromanipulator under microscopic observation. We used this technique to analyze several cytogenetic aberrations. In order to evaluate the efficiency of our microdissection procedure, we compared the results obtained with microdissection probes made from only one fragment with those obtained with more than six microdissected fragments. In all cases, the single-fragment microdissections were sufficient to provide probes.

Chromosome Painting↗