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Biomedical subjects

Paul W Bohn

Publications and source records attributed to Paul W Bohn.

At least 19 recordsLinked to original sources
PubMed2006

Nanofluidic injection and heterogeneous kinetics of organomercaptan surface displacement reactions on colloidal gold in a microfluidic stream.

Colloidal gold is developed as a molecular capture reagent in hybrid nanofluidic-microfluidic devices for mass-limited sample analysis. Two fluorescent organomercaptans are injected through a nanocapillary array membrane and subsequently captured at the surface of 19-nm-diameter colloidal Au nanoparticles. The surface displacement kinetics are monitored via quenching of the organomercaptan fluorescence by the metallic particles coupled to a distance-time conversion based on fluid velocity in the microfluidic channel using the point of mixing as the zero of time. The adsorbate concentration, colloid concentration, and fluid velocity are varied to determine the surface displacement rate constants for these heterogeneous reactions in the microfluidic device. Surface displacement rate constants are approximately 10(4) M(-1) s(-1) for a small organic molecule and for an octapeptide. These values are similar to values determined in macroscale measurements made with a traditional fluorometer and are 1 order of magnitude larger than values reported for adsorption of organomercaptans on planar Au, indicating faster kinetics in the colloid-adsorbate system. These results highlight the utility of colloidal Au nanoparticles as molecular carriers for the sequestration of analytes, allowing the manipulation of mass-limited samples and ultimately the capture and delivery of selected analytes from a microfabricated device to an off-line detector.

Journal Article↗
PubMed2006

Design and fabrication of a multilayered polymer microfluidic chip with nanofluidic interconnects via adhesive contact printing.

The design and fabrication of a multilayered polymer micro-nanofluidic chip is described that consists of poly(methylmethacrylate) (PMMA) layers that contain microfluidic channels separated in the vertical direction by polycarbonate (PC) membranes that incorporate an array of nanometre diameter cylindrical pores. The materials are optically transparent to allow inspection of the fluids within the channels in the near UV and visible spectrum. The design architecture enables nanofluidic interconnections to be placed in the vertical direction between microfluidic channels. Such an architecture allows microchannel separations within the chip, as well as allowing unique operations that utilize nanocapillary interconnects: the separation of analytes based on molecular size, channel isolation, enhanced mixing, and sample concentration. Device fabrication is made possible by a transfer process of labile membranes and the development of a contact printing method for a thermally curable epoxy based adhesive. This adhesive is shown to have bond strengths that prevent leakage and delamination and channel rupture tests exceed 6 atm (0.6 MPa) under applied pressure. Channels 100 microm in width and 20 microm in depth are contact printed without the adhesive entering the microchannel. The chip is characterized in terms of resistivity measurements along the microfluidic channels, electroosmotic flow (EOF) measurements at different pH values and laser-induced-fluorescence (LIF) detection of green-fluorescent protein (GFP) plugs injected across the nanocapillary membrane and into a microfluidic channel. The results indicate that the mixed polymer micro-nanofluidic multilayer chip has electrical characteristics needed for use in microanalytical systems.

Journal Article↗
PubMed2006

Length scale heterogeneity in lateral gradients of poly(N-isopropylacrylamide) polymer brushes prepared by surface-initiated atom transfer radical polymerization coupled with in-plane electrochemical potential gradients.

We report the preparation and characterization of poly(N-isopropylacrylamide) (PNIPAAm) polymer brushes exhibiting controlled lateral variations in the patchiness of polymer chains. These gradients were achieved through an atom transfer radical polymerization (ATRP) grafting-from approach utilizing surfaces on which the spatial profile of the initiator density was carefully controlled. Initiator density gradients were formed on Au by first preparing a hexadecanethiol (HDT) density gradient, by reductive desorption using a laterally anisotropic electrochemical gradient. The bare areas in the original HDT gradient were then back-filled with a disulfide initiator, (BrC(CH3)2COO(CH2)11S)2. The initiator coverage was characterized by X-ray photoelectron spectroscopy (XPS). Then, surface-initiated ATRP was utilized to transfer the initiator density gradient into gradients of PNIPAAm chain density. Ellipsometry, surface plasmon resonance (SPR), and atomic force microscopy (AFM) were used to characterize these PNIPAAm density gradients. The defining characteristic of the PNIPAAm gradients is the evolution of the morphology from discontinuous mushroom structures at extremely low grafting densities to heterogeneous patchy structures at intermediate grafting densities. The size of the patchy domains gradually increases, until at a high grafting density region, the morphology evolves to a smoother, presumably more extended, structure.

Journal Article↗
PubMed2005

Incorporation of a DNAzyme into Au-coated nanocapillary array membranes with an internal standard for Pb(ii) sensing.

A Pb(ii)-specific DNAzyme has been successfully incorporated into Au-coated polycarbonate track-etched (PCTE) nanocapillary array membranes (NCAMs) by thiol-gold immobilization. Incorporation of the DNAzyme into the membrane provides a substrate-bound sensor using a novel internal control methodology for fluorescence-based detection of Pb(ii). A non-cleavable substrate strand, identical to the cleavable DNAzyme substrate strand except the RNA-base is replaced by the corresponding DNA-base, is used for ratiometric comparison of intensities. The cleavable substrate strand is labeled with fluorescein, and the non-cleavable strand is labeled with a red fluorophore (Cy5 or Alexa 546) for detection after release from the membrane surface. This internal standard based ratiometric method allows for real-time monitoring of Pb(ii)-induced cleavage, as well as standardizing variations in substrate size, solution detection volume, and monolayer density. The result is a Pb(ii)-sensing structure that can be stored in a prepared state for 30 days, regenerated after reaction, and detect Pb(ii) concentrations as low as 17 nM (3.5 ppb).

Biosensing Techniques↗
PubMed2005

Profiling pH gradients across nanocapillary array membranes connecting microfluidic channels.

Nanocapillary array membranes (NCAMs), comprised of thin (d approximately 5-10 microm) nuclear track-etched polycarbonate sheets containing approximately 10(8) cm(-2) nearly parallel nanometer-diameter capillaries, may act to gate fluid transport between microfluidic channels to effect, for example, sample collection. There is interest in H+-transport across these NCAMs because there is significant practical interest in being able to process analyte-containing samples under different pH conditions in adjacent layers of an integrated microfluidic circuit and because protons, with their inherently high mobility, present a challenge in separating microfluidic environments with different properties. To evaluate the capability of NCAMs to support pH gradients, the proton transport properties of NCAMs were studied using laser scanning confocal fluorescence microscopy (LSCFM). Spatiotemporal maps of [H+] in microfluidic channels adjacent to the NCAMs yield information regarding diffusive and electrokinetic transport of protons. The NCAMs studied here are characterized by a positive zeta potential, zeta > 0, so at small nanocapillary diameters, the overlap of electrical double layers associated with opposite walls of the nanocapillary establish an energy barrier for either diffusion or electrokinetic transport of cations through the nanometer-diameter capillaries due to the positive charge on the nanocapillary surface. Proton transfer through an NCAM into microchannels is reduced for pore diameters, d < or = 50 nm and ionic strengths I < or = 50 mM, while for large pore diameters or solution ionic strengths, the incomplete overlap of electric double layer allows more facile ionic transfer across the membranes. These results establish the operating conditions for the development of multilevel integrated nanofluidic/microfluidic architectures which can support multidimensional chemical analysis of mass-limited samples requiring sequential operations to be implemented at different pH values.

Electrochemistry↗
PubMed2005

Self-assembled monolayers of alkanethiols on gold modulate electrophysiological parameters and cellular morphology of cultured neurons.

Self-assembled monolayers (SAMs) of omega-substituted alkanethiols on gold have been explored as well defined in vitro model surfaces for the investigation of neuronal growth and function. When used as cell culture substrates, surfaces with monolayers functionalized with terminal -COOH groups support neuron attachment and growth even without an intermediate protein layer. Addition of a poly-L-lysine layer (PLL) to the -COOH terminated monolayers significantly increases total neurite outgrowth. Mixed monolayers containing -COOH and -CH3 terminal groups in 1:10 and 1:100 ratios poorly support neuron adhesion and preclude neurite extension. A layer of PLL improves the ability of mixed monolayer surfaces to support neuronal growth in culture. The morphology of cultured neurons depends on the chemical composition of SAMs on the support surface. Using glass microelectrode intracellular recording, the properties of cell culture substrates modulate the dynamic properties of action potentials of cultured neurons. These findings provide insight into the cellular responses of excitable cells to the chemical details of a surface and, thus, may help direct the rational design of biologically active materials.

Alkanes↗
PubMed2005

Anisotropic hydrogel thickness gradient films derivatized to yield three-dimensional composite materials.

We report the preparation of in-plane density gradients of amino-terminated molecules and gold particles through derivatization of laterally varying thickness gradients of poly(acrylic acid) (PAA) or poly(acrylamide) (PAAm) films. PAA and PAAm gradients were formed by Zn(II)-catalyzed electropolymerization of acrylic acid (AA) or acrylamide (AAm) in the presence of an in-plane electrochemical potential gradient applied to Au or indium-tin-oxide (ITO) working electrodes. PAA thickness gradients were converted into density gradients of fluorocarbons or biocompatible groups by derivatizing with NH(2)CH(2)(CF(2))(6)CF(3) or an Arg-Gly-Asp (RGD)-containing peptide, respectively. X-ray photoelectron spectroscopy (XPS) and XPS imaging were used to characterize the modified PAA gradients. Transition regions as narrow as 104 mum were achieved for fluorocarbon gradients. PAAm gradients were treated with gold particles to form a density gradient of gold particles. Surface plasmon resonance imaging and scanning electron microscopy (SEM) as well as UV-visible absorption measurements were used to characterize the gold particle density gradients. It is likely that the gold particles were attached both on the surface and inside the PAAm film.

Journal Article↗
PubMed2005

Miniaturized lead sensor based on lead-specific DNAzyme in a nanocapillary interconnected microfluidic device.

A miniaturized lead sensor has been developed by combining a lead-specific DNAzyme with a microfabricated device containing a network of microfluidic channels that are fluidically coupled via a nanocapillary array interconnect. A DNAzyme construct, selective for cleavage in the presence of Pb2+ and derivatized with fluorophore (quencher) at the 5' (3') end of the substrate and enzyme strands, respectively, forms a molecular beacon that is used as the recognition element. The nanocapillary array membrane interconnect is used to manipulate fluid flows and deliver the small-volume sample to the beacon in a spatially confined detection window where the DNAzyme is interrogated using laser-induced fluorescence detection. A transformed log plot of the fluorescent signal exhibits a linear response (r2 = 0.982) over a Pb2+ concentration range of 0.1 - 100 microM, and a detection limit of 11 nM. The sensor has been applied to the determination of Pb2+ in an electroplating sludge reference material, the result agreeing with the certified value within 4.9%. Quantitative measurement of Pb2+ in this complex sample demonstrates the selectivity of this sensor scheme and points favorably to the application of such technologies to analysis of environmental samples. The unique combination of a DNAzyme with a microfluidic-nanofluidic hybrid device makes it possible to change the DNAzyme to select for other compounds of interest, and to incorporate multiple sensing systems within a single device for greater flexibility. This work represents the initial steps toward creation of a robust field sensor for lead in groundwater or drinking water.

Biosensing Techniques↗
PubMed2005

Voltage-tunable volume transitions in nanoscale films of poly(hydroxyethyl methacrylate) surfaces grafted onto gold.

Surface grafting of a polymerizable monomer onto Au was used to produce nanometer-scale planar hydrogel films with controllable volume. A self-assembled monolayer of 11-mercaptoundecanoic acid on a planar Au surface was activated through water-soluble carbodiimide and N-hyroxysuccinimide followed by reaction with 2-aminomethacrylate to produce a methacrylate-terminated surface layer, which readily polymerized under UV radiation in the presence of hydroxyethyl methacrylate monomer, ethylene glycol dimethacrylate cross-linker, and a photoinitiator. The reaction steps were characterized by external reflection mode Fourier transform IR spectroscopy. Under controlled UV exposure, thin (3 nm < d < 10 nm) hydrogel films were obtained from 1:1 ethanol/H(2)O. Surface plasmon resonance measurements were used to characterize both the synthesis of the hydrogel and the potential-induced volume changes. The nanometer-scale hydrogels thus produced undergo reproducible changes in thickness, when a potential is applied across the film. Thickness changes increasing with applied potential were obtained for both voltages in the range |V(appl)| </= 600 mV. In NaCl, electrolyte films swell with application of negative potentials and shrink with positive potentials, due to the imbibing or extrusion of hydrated Na(+) ions, respectively. Thickness changes as large as 50% can be achieved. An increase in the cross-linker content results in thicker films, but at the cost of dramatically restricted swelling. Response times are generally faster for smaller applied potentials, as expected if the volume change results from mass transport of electrolyte.

Journal Article↗
PubMed2005

Immobilization of a catalytic DNA molecular beacon on Au for Pb(II) detection.

A Pb(II)-specific DNAzyme fluorescent sensor has been modified with a thiol moiety in order to immobilize it on a Au surface. Self-assembly of the DNAzyme is accomplished by first adsorbing the single-thiolated enzyme strand (HS-17E-Dy) followed by adsorption of mercaptohexanol, which serves to displace any Au-N interactions and ensure that DNA is bound only through the S-headgroup. The preformed self-assembled monolayer is then hybridized with the complementary fluorophore-containing substrate strand (17DS-Fl). Upon reaction with Pb(II), the substrate strand is cleaved, releasing a fluorescent fragment for detection. Fluorescence intensity may be correlated with original Pb(II) concentration, and a linear calibration was obtained over nearly four decades: 10 microM > or = [Pb(II)] > or = 1 nM. The immobilized DNAzyme is a robust system; it may be regenerated after cleavage, allowing multiple sensing cycles. In addition, drying of fully assembled DNAzyme before reaction with Pb(II) does not significantly affect analytical performance. These results demonstrate that, in comparison with solution-based schemes, immobilization of the DNAzyme sensor onto a Au surface lowers the detection limit (from 10 to 1 nM), maintains activity and specificity, and allows sensor regeneration and long-term storage. Realization of Pb(II) detection through an immobilized DNAzyme is the first important step toward creation of a stand-alone, portable Pb(II) detection device such as those immobilizing DNAzyme recognition motifs in the nanofluidic pores of a microfluidic-nanofluidic hybrid multilayer device.

Biosensing Techniques↗
PubMed2005

Interfacial scattering at electrochemically fabricated atom-scale junctions between thin gold film electrodes in a microfluidic channel.

Atom-scale junctions were formed between two Au thin-film electrodes by a combination of lithography, microfluidics, and electrochemistry. Two Au thin-film electrodes with a small (0.25-25 microm) gap between them were lithographically defined such that the gap fell in the center of a 100-microm-wide microfluidic channel in poly(dimethylsiloxane). Directional electrodeposition between the Au thin-film electrodes, accomplished by applying a potential between the thin-film electrodes, caused Au to etch from the anode and deposit on the cathode, thereby closing the gap. Current through the gap was monitored continuously, and the directional electrodeposition was terminated when a current near that corresponding to the conductance quantum, G(0) = 2e(2)/h, was reached. To regenerate the device, the atom-scale junction was broken with a potential sweep, the microfluidic channel was rinsed, and the junction was re-formed with a subsequent comparator-terminated directional electrodeposition. Alternating current impedance was measured while hexadecanethiol (HDT) was chemisorbed onto the atom-scale junction. The interfacial scattering from chemisorption of the Lewis base HDT on the atom-scale junction caused a normalized impedance change of 71 +/- 1%, the noise level being equivalent to a population fluctuation of five HDT molecules.

Journal Article↗
PubMed2005

A multilayer poly(dimethylsiloxane) electrospray ionization emitter for sample injection and online mass spectrometric detection.

An ESI emitter made of poly(dimethylsiloxane) interfaces on-chip sample preparation with MS detection. The unique multilayer design allows both the analyte and the spray solutions to reside on the device simultaneously in discrete microfluidic environments that are spatially separated by a polycarbonate track-etched, nanocapillary array membrane (NCAM). In direct spray mode, voltage is applied to the microchannel containing a spray solution delivered via a syringe pump. For injection, the spray potential is lowered and a voltage is applied that forward biases the membrane and permits the analyte to enter the spray channel. Once the injection is complete, the bias potential is switched off, and the spray voltage is increased to generate the ESI of the injected analyte plug. Consecutive injections of a 10 microM bovine insulin solution are reproducible and produce sample plugs with limited band broadening and high quality mass spectra. Peptide signals are observed following transport through the NCAM, even when the peptide is dissolved in solutions containing up to 20% seawater. The multilayer emitter shows great potential for performing multidimensional chemical manipulations on-chip, followed by direct ESI with negligible dead volume for online MS analysis.

Dimethylpolysiloxanes↗
PubMed2004

Microfluidic separation and gateable fraction collection for mass-limited samples.

Integrating multiple analytical processes into microfluidic devices is an important research area required for a variety of microchip-based analyses. A microfluidic system is described that achieves preparative separations by intelligent fraction collection of attomole quantities of sample. The device consists of a main microfluidic channel used to perform electrophoresis, which is interconnected at 90 degrees to two vertically displaced channels via a nanocapillary array membrane. The membrane interconnect contains nanometer-diameter pores that provide fluidic communication between the channels. Sample injection and analyte collection are controlled by application of an electrical bias between the microfluidic channels across the nanocapillary array. After the separation, the automated transfer of the FITC-labeled Arg, Gln, and Gly bands occurs; a fluorescence detector located at the separation/collection channel interconnect is used to generate a triggering signal that initiates suitable voltages to allow near-quantitative transfer of analyte from the separation channel to the second fluidic layer. The ability to achieve such sample manipulations from mass-limited samples enables a variety of postseparation processing events.

Algorithms↗
PubMed2004

Anisotropic in-plane gradients of poly(acrylic acid) formed by electropolymerization with spatiotemporal control of the electrochemical potential.

Laterally varying thickness gradients of poly(acrylic acid) (PAA) were formed by Zn(II)-catalyzed electropolymerization of acrylic acid (AA) in the presence of an in-plane electrochemical potential gradient applied to Au working electrodes. In the static potential gradient (SPG) approach, two ends of a Au working electrode were clamped at distinct potentials for the duration of the electropolymerization process, thereby generating a time-independent in-plane electrochemical potential gradient, V(x). A dynamic potential gradient (DPG) approach was also used, in which the two end potentials were varied in time, while maintaining a constant voltage offset, to generate an in-plane electrochemical potential gradient, V(x,t). Because the kinetics of heterogeneous electron transfer vary with the local overpotential, these two methods produce PAA films with laterally varying thickness gradients, although they exhibit different spatial characteristics. X-ray photoelectron spectroscopy (XPS) and surface plasmon resonance (SPR) imaging were used to characterize the PAA gradients. The in-plane thickness variations of PAA gradients formed by both SPG and DPG approaches agree with predictions of the Butler-Volmer equation at small absolute overpotentials, while at large (negative) overpotentials, mass transport dominates, and the thickness reaches a plateau value independent of local potential. DPG-produced PAA gradients are generally broader than SPG gradients with the same initial potential and comparable effective growth time, indicating that the DPG approach is more suitable for formation of thicker gradients.

Journal Article↗
PubMed2004

Surface adsorption and transfer of organomercaptans to colloidal gold and direct identification by matrix assisted laser desorption/ionization mass spectrometry.

The adsorption characteristics of two different organomercaptan adsorbates, 5-((2-(and-3)-S-(acetylmercapto)succinoyl)amino)fluorescein (SAMSA) and the peptide Cys-Lys-Trp-Ala-Lys-Trp-Ala-Trp (CKWAKWAK), on colloidal Au were studied, and the conjugates produced were characterized by UV-vis spectroscopy, transmission electron microscopy, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Fluorescence difference measurements of free thiols in solution were used to assemble surface adsorption isotherms on Au colloid revealing surface coverages of 1.0 x 10(14) molecules cm(-2) for SAMSA and 3.1 x 10(14) molecules cm(-2) for CKWAKWAK. The free energies of adsorption were calculated to be -48.4 kJ/mol for SAMSA and -49.2 kJ/mol for CKWAKWAK. UV-visible absorption spectroscopy and transmission electron microscopy reveal that the thiol/colloid conjugates flocculate under conditions where the net charge per colloid is small or neutral and that flocculated colloids can be resuspended by a change in pH to more basic for the acidic SAMSA/Au conjugates or to more acidic for the basic CKWAKWAK/Au conjugates. The reversible flocculation allows the conjugates to be readily separated from free adsorbate in solution and thereby prepared for further characterization. CKWAKWAK/colloid conjugates were analyzed by MALDI-MS, and the mass spectra show (M + H)(+), (M + Na)(+), and (M + K)(+) ions attributable to the peptide. The manipulations studied here constitute a powerful complement to microfluidic-based separation and analysis methods. Conjugating mass-limited analytes to Au colloids makes it possible to sequester and transfer small quantities of analytes with high efficiency.

Adsorption↗
PubMed2004

Quantitative submonolayer spatial mapping of Arg-Gly-Asp-containing peptide organomercaptan gradients on gold with matrix-assisted laser desorption/ionization mass spectrometry.

Peptides containing the tripeptide sequence Arg-Gly-Asp (RGD) have the ability to bind to members of the integrin superfamily of cell-surface receptors and direct cellular adhesion and haptotaxis. The goal of this work is the development of a rapid and effective method for the quantitative submonolayer spatial composition mapping of surfaces displaying molecular assemblies of RGD-containing organomercaptan peptides on a Au surface using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-MS). Quantitation of the RGD peptide is achieved by determining the peak intensity of the protonated molecular ion, (M + H)+, relative to the (M + H)+ peak for an internal standard, which is similar chemically but with glutamic acid (E) substituted for aspartic acid (D). Using optimized sample preparation procedures, a bilinear calibration was obtained between the quantitative peak intensity ratio and the mole fraction of the RGD-containing peptide. Quantitative compositions were determined with relative standard deviations of <10%, even in the presence of 10x spot-to-spot variations in the absolute signal intensities, by using this internal standard approach. This MALDI-MS quantitative analysis method was employed to probe variable-width two-component counterpropagating electrochemically generated gradients of the two peptides, prepared by coupling in-plane electrochemical potential gradients with the electrosorption reactions of organothiols to vary the composition laterally. The measured lateral composition profiles match the quasi-linear potential gradient model and yield profiles that overlap to a high degree of fidelity in potential space. Thus, MALDI-MS spatial composition mapping should become a powerful tool for the preparation of designed surfaces facilitating the study of cellular adhesion and motility and cell-cell interactions.

Gold↗
PubMed2003

Nanocapillary array interconnects for gated analyte injections and electrophoretic separations in multilayer microfluidic architectures.

An electrokinetic injection technique is described which uses a nuclear track-etched nanocapillary array to inject sample plugs from one layer of a microfluidic device into another vertically separated layer for electrophoretic separations. Gated injection protocols for analyte separations, reported here, establish nanocapillary array interconnects as a route to multilevel microfluidic analytical designs. The hybrid nanofluidic/microfluidic gated injection protocol allows sample preparation and separation to be implemented in separate horizontal planes, thereby achieving multilayer integration. Repeated injections and separations of FITC-labeled arginine and tryptophan, using 200-nm pore-diameter capillary array injectors in place of traditional cross injectors are used to demonstrate gated injection with a bias configuration that uses relay switching of a single high-voltage source. Injection times as rapid as 0.3 s along with separation reproducibilities as low as 1% for FITC-labeled arginine exemplify the capability for fast, serial separations and analyses. Impedance analysis of the micro-/nanofluidic network is used to gain further insight into the mechanism by which this actively controlled nanofluidic-interconnect injection method works. Gated sample introduction via a nanocapillary array interconnect allows the injection and separation protocols to be optimized independently, thus realizing the versatility needed for real-world implementation of rapid, serial microchip analyses.

Arginine↗