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Nancy L Allbritton

Publications and source records attributed to Nancy L Allbritton.

16 recordsLinked to original sources
PubMed2007

Micropallet arrays for the separation of single, adherent cells.

The selection and collection of single cells from within a heterogeneous population is required to produce genetically engineered cell lines, to develop new stem cell lines, and for single-cell studies. We describe a new platform for the positive selection of single live mammalian cells while the cells remain adherent to their growth surface. Cells were grown on arrays of microfabricated, releasable elements composed of SU-8 polymer termed "cell pallets". The presence of air between the elements restricted the cells to the top surfaces of the pallets. Single pallets situated within large arrays of pallets were released on demand using a single, focused, laser pulse. The laser pulses were low in energy (2-5 muJ) and did not detach nearby, nontargeted pallets. Since the SU-8 pallets and the underlying glass substrate were optically transparent, the cells on the pallets could be visualized by microscopy before and after release. Over 90% of cells remained attached to the pallet during laser-based release. The feasibility of growing the cells from the released pallets into clonal colonies was demonstrated. The pallet array system permits adherent cells to be inspected using conventional microscopy and selected cells released for further analysis. The ability to assess cells while they remain adherent to a surface will broaden the number of attributes that can be utilized for cell separation, for example, cell shape, cytoskeletal properties, and other attributes.

Animals↗
PubMed2006

Micropatterning of living cells on a heterogeneously wetted surface.

We report a simple approach to fabricate heterogeneously wetted surfaces which can be used to pattern living cells or biomolecules. An array of pedestals composed of SU-8 was fabricated on a glass surface which was then derivatized with a hydrophobic silane. Upon addition of aqueous solutions to the array, air was trapped within the hydrophobic cavities between the pedestals. The trapped air formed a "virtual wall" blocking access to these cavities. Cells cultured on the array were forced to grow only on the tops of the pedestals, i.e., the surfaces wetted by aqueous media. The virtual walls were stable during manipulation of the array and over long time periods (months).

Air↗
PubMed2006

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.

Animals↗
PubMed2006

Localized sampling, electrophoresis, and biosensor analysis of Xenopus laevis cytoplasm for subcellular biochemical assays.

The Xenopus oocyte is a widely used model cell for studies of signal transduction mechanisms. Advances in microanalytical methods have made it feasible to perform rapid, localized collection of cytoplasm from individual Xenopus oocytes. Analytes contained in the cytoplasmic sample are separated by electrophoresis in a capillary and simultaneously transferred to a detection region. The development of bioengineered cells as sensitive detectors of intracellular components made quantitative measurements of native signaling molecules within the electrophoresed sample possible. Local determination of the second messenger inositol 1,4,5-trisphosphate is described to illustrate the methods for the sampling, electrophoresis, detection, and quantification of signaling molecules in single oocytes.

Animals↗
PubMed2005

Covalent micropatterning of poly(dimethylsiloxane) by photografting through a mask.

A new photografting method to micropattern a covalent surface modification on poly(dimethylsiloxane) (PDMS) provides advantages in simplicity and efficiency. To accomplish the entire process on the benchtop, the PDMS was initially treated with benzophenone dissolved in a water/acetone mixture. This process permitted limited diffusion of the photoinitiator into the PDMS surface. Polymerization of acrylic acid was initiated by exposure of the benzophenone-implanted PDMS to UV radiation through a photomask with a thin aqueous layer of acrylic acid sandwiched between the PDMS and photomask. This procedure resulted in patterned poly(acrylic acid) (PAA) on the PDMS surface. In the modified regions, PAA and PDMS formed an interpenetrating polymer network extending 50 microm into the PDMS with an X-Y spatial resolution of 5 microm. The carboxyl groups of the PAA graft could be derivatized to covalently bond other molecules to the patterned PAA. Two bioanalytical applications of this micropatterned surface were demonstrated: (1) a guide for cell attachment and growth and (2) a substrate for immunoassays. 3T3 cells were shown to selectively localize to modified surface regions where they could be cultured for up to 7 days. Additionally, the micropatterned surface was used to immobilize either protein A or antibody for heterogeneous immunoassays.

Acrylic Resins↗
PubMed2004

Characterization of TAT-mediated transport of detachable kinase substrates.

The conjugation of peptides derived from the HIV TAT protein to membrane-impermeant molecules has gained wide acceptance as a means for intracellular delivery. Numerous studies have addressed the mechanism of uptake and kinetics of TAT translocation, but the cytosolic concentrations and bioavailability of the transported cargo have not been well-characterized. The current paper utilizes a microanalytical assay to perform quantitative single-cell measurements of the concentration and accessibility of peptide-based substrates for protein kinase B (PKB) and Ca(2+)/calmodulin-activated kinase II. The substrate peptide and TAT were conjugated through a releasable linker, either a disulfide or photolabile bond. Free substrate peptide concentrations of approximately 10(-20)-10(-18) moles were attainable in a cell when substrates were delivered utilizing these conjugates. The substrate peptides delivered as a disulfide conjugate were often present in the cytosol as several oxidized forms. Brief exposure of cells loaded with the photolabile conjugates to UVA light released free substrate peptide into the cytosol. Substrate peptide delivered by either conjugate was accessible to cytosolic kinase as demonstrated by the efficient phosphorylation of the peptide when the appropriate kinase was active. After incubation of the conjugated substrate with cells, free, kinase-accessible substrate was detectable in less than 30 min. Release of the majority of loaded substrate peptide from sequestered organelles occurred within 1 h. The utility of the photocleavable conjugates was demonstrated by measuring the activation of PKB in 3T3 cells after addition of varying concentrations of platelet-derived growth factor.

Animals↗
PubMed2004

Tailoring the surface properties of poly(dimethylsiloxane) microfluidic devices.

Poly(dimethylsiloxane) (PDMS) is an attractive material for microelectrophoretic applications because of its ease of fabrication, low cost, and optical transparency. However, its use remains limited compared to that of glass. A major reason is the difficulty of tailoring the surface properties of PDMS. We demonstrate UV grafting of co-mixed monomers to customize the surface properties of PDMS microfluidic channels in a simple one-step process. By co-mixing a neutral monomer with a charged monomer in different ratios, properties between those of the neutral monomer and those of the charged monomer could be selected. Mixtures of four different neutral monomers and two different charged monomers were grafted onto PDMS surfaces. Functional microchannels were fabricated from PDMS halves grafted with each of the different mixtures. By varying the concentration of the charged monomer, microchannels with electrophoretic mobilities between +4 x 10(-4) cm2/(V s) and -2 x 10(-4) cm2/(V s) were attainable. In addition, both the contact angle of the coated surfaces and the electrophoretic mobility of the coated microchannels were stable over time and upon exposure to air. By carefully selecting mixtures ofmonomers with the appropriate properties, it may be possible to tailor the surface of PDMS for a large number of different applications.

Dimethylpolysiloxanes↗
PubMed2004

Surface-directed, graft polymerization within microfluidic channels.

We demonstrate a simple procedure to coat the surfaces of enclosed PDMS microchannels by UV-mediated graft polymerization. In prior applications, only disassembled channels could be coated by this method. This limited the utility of the method to coatings that could easily and tightly seal with themselves. By preadsorbing a photoinitiator onto the surface of PDMS microchannels, the rate of polymer formation at the surface was greatly accelerated compared to that in solution. Thus, a gel did not form in the lumen of enclosed microchannels. We demonstrate that the photoinitiator benzophenone remained on the surface of PDMS even after extensive washing. After addition of a variety of monomer solutions (acrylic acid, poly(ethylene glycol) monomethoxyl acrylate, or poly(ethylene glycol) diacrylate) and illumination with UV light, a stable, covalently attached surface coating formed in the microchannels. The electroosmotic mobility was stable in response to air exposure and to repeated cycles of hydration-dehydration of the coating. These surfaces also supported the electrophoretic separation of two model analytes. Placement of an opaque mask over a portion of the channel permitted photopatterning of the microchannels with a resolution of approximately 100 microm. By using an appropriate mixture of monomers combined with masks, it should be possible to fabricate PDMS microfluidic devices with distinct surface properties in different regions or channels.

Journal Article↗
PubMed2004

A quantitative single-cell assay for protein kinase B reveals important insights into the biochemical behavior of an intracellular substrate peptide.

The introduction of peptides into living cells for the purpose of manipulating cellular biochemistry has become widespread throughout biology. However, little is known about the behavior of these short sequences of amino acids within cells, particularly those used as substrates or inhibitors for kinases and other enzymes. We utilized a quantitative, single-cell assay to demonstrate that an 11-amino acid peptide was efficiently phosphorylated by intracellular protein kinase B (PKB) in fibrosarcoma cell line HT1080 and in NIH-3T3 cells. The phosphorylated peptide was also readily dephosphorylated by intracellular phosphatases. Assays of the peptide's phosphorylation in single, living cells measured the balance of the activities of PKB and phosphatases in that cell. At a peptide concentration below the K(M) of PKB and the phosphatases, the ratio of phosphorylated to nonphosphorylated peptide at the steady state was independent of the peptide concentration. A single-cell assay utilizing this peptide revealed the existence of two subpopulations of cells whose unique activities had hitherto been obscured by population averaging. Additional studies of cells stimulated by PDGF demonstrated that a quantitative analysis of PKB activation in response to a physiological stimulus was possible. These studies demonstrated that short peptides can remain specific within the complex intracellular milieu and function as sensitive reporters of the activation state of native kinases within live cells.

Animals↗
PubMed2004

A wave of IP3 production accompanies the fertilization Ca2+ wave in the egg of the frog, Xenopus laevis: theoretical and experimental support.

The fertilization Ca2+ wave in Xenopus laevis is a single, large wave of elevated free Ca2+ that is initiated at the point of sperm-egg fusion and traverses the entire width of the egg. This Ca2+ wave involves an increase in inositol-1,4,5-trisphosphate (IP3) resulting from the interaction of the sperm and egg, which then results in the activation of the endoplasmic reticulum Ca2+ release machinery. The extraordinarily large size of this cell (1.2 mm diameter) together with the small surface region of sperm-receptor activation makes special demands on the IP3-dependent Ca2+ mobilizing machinery. We propose a detailed model of the fertilization Ca2+ wave in Xenopus eggs that requires an accompanying wave of IP3 production. While the Ca2+ wave is initiated by a localized increase of IP3 near the site of sperm-egg fusion, the Ca2+ wave propagates via IP3 production correlated with the Ca2+ wave-possibly via Ca(2+)-mediated PLC activation. Such a Ca(2+)-mediated IP(3) production wave has not been required previously to explain the fertilization Ca2+ wave in eggs; we argue this is necessary to explain the observed IP3 dynamics in Xenopus eggs. To test our hypothesis, we have measured the IP3 levels from 20 nl "sips" of the egg cortex during wave propagation. We were unable to detect the low IP3 levels in unfertilized eggs, but after fertilization, [IP3] ranged from 175 to 430 nM at the sperm entry point and from 120 to 700 nM 90 degrees away once the Ca2+ wave passed that region about 2 min after fertilization. Prior to the Ca2+ wave reaching that region the IP3 levels were undetectable. Since significant IP3 could not diffuse to this region from the sperm entry point within 2 min, this observation is consistent with a regenerative wave of IP3 production.

Animals↗
PubMed2003

Microfluidic devices for the high-throughput chemical analysis of cells.

A microfluidic device is reported that integrated cell handling, rapid cell lysis, and electrophoretic separation and detection of fluorescent cytosolic dyes. The device function was demonstrated using Jurkat cells that were loaded with the fluorogenic dyes - carboxyfluorescein diacetate, Oregon green carboxylic acid diacetate, or Calcein AM. The loaded cells were hydrodynamically transported from the cell-containing reservoir to a region on the microfluidic device where they were focused and then rapidly lysed using an electric field. Complete lysis was accomplished in <33 ms. The hydrolyzed, fluorescent dyes in the cell lysate were automatically injected into a separation channel on the device and detected 3 mm downstream of the injection point. The total separation time was approximately 2.2 s with absolute migration time reproducibilities of <1% and efficiencies ranging from 2300 to 4000 theoretical plates. Results from 139 cells are reported. A small fraction of these cells, approximately 9%, were found to enzymatically hydrolyze the loaded dyes in a manner significantly different from the majority of the cells. Cell analysis rates of 7-12 cells/min were demonstrated and are >100 times faster than those reported using standard bench-scale capillary electrophoresis.

Algorithms↗
PubMed2003

Fast electrical lysis of cells for capillary electrophoresis.

In the past decade, capillary electrophoresis has demonstrated increasing utility for the quantitative analysis of single cells. New applications for the analysis of dynamic cellular properties demand sampling methods with sufficient temporal resolution to accurately measure these processes. In particular, intracellular signaling pathways involving many enzymes can be modulated on subsecond time scales. We have developed a technique to rapidly lyse an adherent mammalian cell using a single electrical pulse followed by efficient loading of the cellular contents into a capillary. Microfabricated electrodes were designed to create a maximum voltage drop across the flattened cell's plasma membrane at a minimum interelectrode voltage. The influence of the interelectrode distance, pulse duration, and pulse strength on the rate of cell lysis was determined. The ability to rapidly lyse a cell and collect and separate the cellular contents was demonstrated by loading cells with Oregon Green and two isomers of carboxyfluorescein. All three fluorophores were detected with a separation efficiency comparable to that of standards. Parallel comparison of electrical lysis to that produced by a laser-based lysis system revealed that the sampling efficiencies of the two techniques were comparable. Rapid cell lysis by an electrical pulse may increase the application of capillary electrophoresis to the study of cellular dynamics requiring fast sampling times.

Animals↗
PubMed2003

Separation of mixtures of acidic and basic peptides at neutral pH.

Mixtures of acidic and basic peptides composed of the phosphorylated and nonphosphorylated forms of peptide substrates for kinases and a phosphatase were separated by capillary electrophoresis (CE) in buffer conditions compatible with live mammalian cells. The separation of such mixtures was especially challenging given the high salt and neutral pH of the requisite physiologic buffers. Due to poor peak reproducibility in bare capillaries, several strategies were implemented to improve the electrophoretic separation of the peptide mixtures. Covalent coating of the capillary with the neutral polymer poly(dimethylacrylamide) (PDMA) resulted in a 2-fold improvement in the migration time RSD, but required the use of hydrodynamic flow to overcome the differing electrophoretic mobilities (microeo) of the peptides at neutral pH. This parabolic fluid flow diminished separation efficiency almost 5-fold. Polarity switching during the CE run was used to overcome the opposed microeo, but required the retention of hydrodynamic flow and consequent reduction in separation efficiency. The most efficient separations were seen with the use of covalently-linked, charged polymer coatings to maintain electroosmotic flow and to reduce wall interactions. Two such coatings were tested in the current study. Relative to the PDMA coating, an anionic poly(acrylate) improved the average migration time RSD of six peptides from 1.3 to 0.85% and average separation efficiency from 4.8 to 18.0 (x 10(4) plates/m). Likewise, cationic poly([3-(methacryloylamino)propyl]-trimethylammonium) improved the average migration time RSD of eight peptides from 1.2 to 1.1% and average separation efficiency from 4.8 to 33.9 (x 10(4) plates/m). These findings will be of value to the growing number of applications for analytical techniques utilizing CE for cellular analysis and biochemical studies.

Amino Acid Sequence↗
PubMed2003

Single-cell kinase assays: opening a window onto cell behavior.

Recent advances in analytical techniques have made the performance of biochemical assays on individual mammalian cells possible. Of particular interest is the ability to measure the activation of kinases, enzymes with critical roles in virtually every aspect of cell physiology. Single-cell kinase assays promise to deliver a newfound understanding of the molecular mechanisms responsible for cellular control and behavior by revealing the dynamic nature of signal transduction networks in living cells. A recent exciting development is the potential to perform assays of multiple kinases simultaneously in a single cell.

Cells↗
PubMedDate not supplied

Targeted protein functionalization using His-tags.

With the impressive growth in gene sequence data that has become available, recombinant proteins represent an increasingly vast source of molecular components, with unique functional and structural properties, for use in biotechnological applications and devices. To facilitate the use, manipulation, and integration of such molecules into devices, a controllable method for their chemical modification was developed. In this approach, a trifunctional labeling reagent first recognizes and binds a His-tag on the target protein's surface. After binding, a photoreactive group on the trifunctional molecule is triggered to create a covalent linkage between the reagent and the target protein. The third moiety on the labeling reagent can be varied to bring unique chemical functionality to the target protein. This approach provides: (1) specificity in that only His-tagged targets are modified, (2) regio-specific control in that the target is modified proximal to the His-tag, the position of which can be varied, and (3) stoichiometric control in that the number modifications is limited by the binding capacity of the His-tag. Two such labeling reagents were designed, synthesized, and used to modify both N- and C-terminally His-tagged versions of the enzyme murine dihydrofolate reductase (mDHFR). The first reagent biotinylated the enzyme,while the second served to attach an oligonucleotide to yield a protein-DNA conjugate. In all cases, modification in this manner brings new functionality to the protein while leaving the enzymatic activity intact. The protein-DNA conjugate was used to specifically immobilize the active enzyme through DNA hybridization onto polystyrene microspheres, a step toward creating a functional protein microarray.

DNA↗