Characterization of hydrogen responsive nanoporous palladium films synthesized via a spontaneous galvanic displacement reaction.

A model is presented regarding the mechanistic properties associated with the interaction of hydrogen with nanoporous palladium (np-Pd) films prepared using a spontaneous galvanic displacement reaction (SGDR), which involves PdCl(2) reduction by atomic Ag. Characterization of these films shows both chemical and morphological factors, which influence the performance characteristics of np-Pd microcantilever (MC) nanomechanical sensing devices. Raman spectroscopy, uniquely complemented with MC response profiles, is used to explore the chemical influence of palladium oxide (PdO). These combined techniques support a reaction mechanism that provides for rapid response to H(2) and recovery in the presence of O(2). Post-SGDR processing via reduction of PdCl(2)(s) in a H(2) environment results in a segregated nanoparticle three-dimensional matrix dispersed in a silver layer. The porous nature of the reduced material is shown by high resolution scanning electron microscopy. Extended grain boundaries, typical of these materials, result in a greater surface area conducive to fast sorption/desorption of hydrogen, encouraged by the presence of PdO. X-ray diffraction and inductively coupled plasma-optical emission spectroscopy are employed to study changes in morphology and chemistry occurring in these nanoporous films under different processing conditions. The unique nature of chemical/morphological effects, as demonstrated by the above characterization methods, provides evidence in support of observed nanomechanical response/recovery profiles offering insight for catalysis, H(2) storage and improved sensing applications.

Nanotechnology and chip level systems for pressure driven liquid chromatography and emerging analytical separation techniques: a review.

Pressure driven liquid chromatography (LC) is a powerful and versatile separation technique particularly suitable for differentiating species present in extremely small quantities. This paper briefly reviews main historical trends and focuses on more recently developed technological approaches in miniaturization and on-chip integration of LC columns. The review emphasizes enabling technologies as well as main technological challenges specific to pressure driven separations and highlights emerging concepts that could ultimately overcome fundamental limitations of conventional LC columns.

A comparative study of the enantiomeric separation of labeled amino acids with cyclodextrins and mixed micelles in capillary electrophoresis.

Enantiomeric separations of fluorescently labeled amino acids are studied by capillary electrophoresis (CE) under a novel variety of experimental conditions. Three different labels are evaluated using two different additives: cyclodextrins (beta- and gamma-) and a dual surfactant system of sodium dodecyl sulfate and sodium taurodeoxycholate. Fluorescein-5-isothiocyanate is the best label to use in this cyclodextrin-based system, and dansyl chloride is the best label to use in this dual surfactant system. Possible limitations for separation of the enantiomers using the mixed micelle system include the fact that there is little interaction of the solute with the surfactants, the negative charge of the solute is limiting the separation window of the system, and the amount of the chiral phase available for partitioning is limited. The separations using cyclodextrins as a chiral selector show that the label affects migration order of the enantiomers, and the cyclodextrins are very effective in separating numerous enantiomers. Overall, cyclodextrins are the better buffer additive for CE use, and the dual surfactant systems, including sodium taurodeoxycholate, offer future promise.

Nanofabrication of densely packed metal-polymer arrays for surface-enhanced Raman spectrometry.

A key element to improve the analytical capabilities of surface-enhanced Raman spectroscopy (SERS) resides in the performance characteristics of the SERS-active substrate. Variables such as shape, size, and homogeneous distribution of the metal nanoparticles throughout the substrate surface are important in the design of more analytically sensitive and reliable substrates. Electron-beam lithography (EBL) has emerged as a powerful tool for the systematic fabrication of substrates with periodic nanoscale features. EBL also allows the rational design of nanoscale features that are optimized to the frequency of the Raman laser source. In this work, the efficiency of EBL fabricated substrates are studied by measuring the relative SERS signals of Rhodamine 6G and 1,10-phenanthro-line adsorbed on a series of cubic, elliptical, and hexagonal nanopatterned pillars of ma-N 2403 directly coated by physical vapor deposition with 25 nm films of Ag or Au. The raw analyte SERS signals, and signals normalized to metal nanoparticle surface area or numbers of loci, are used to study the effects of nanoparticle morphology on the performance of a rapidly created, diverse collection of substrates. For the excitation wavelength used, the nanoparticle size, geometry, and orientation of the particle primary axis relative to the excitation polarization vector, and particularly the density of nanoparticles, are shown to strongly influence substrate performance. A correlation between the inverse of the magnitude of the laser backscatter passed by the spectrometer and SERS activities of the various substrate patterns is also noted and provides a simple means to evaluate possible efficient coupling of the excitation radiation to localized surface plasmons for Raman enhancement.

Factors affecting the sorption of model environmental pollutants onto silver polydimethylsiloxane nanocomposite Raman substrates.

The presence of aromatic compounds in water is an important topic in environmental sciences. Silver-polydimethylsiloxane nanocomposites (Ag-PDMS) have recently been demonstrated as promising substrates for the detection of model environmental pollutants via surface-enhanced Raman spectroscopy (SERS). This work discusses how different variables such as pH and matrix composition can affect the sorption and SERS activity of these chemicals. The results show that the conjugate base of weak acids can interact more efficiently with the substrate, leading to an increased signal at higher pH, while amino-aromatic compounds interact more efficiently at a lower pH. The sorption of these chemicals is an essential step in the process and has been attributed to the absorption of the analyte into the PDMS followed by its adsorption to the metallic surface. In addition, the presence of moderate concentrations (1 x 10(-4) M) of a supporting electrolyte such as nitrate or fluoride can improve the sorption of 4-hydroxybenzoic acid to the Ag-PDMS nanoparticles. Other ions such as phosphate and chloride cause rapid oxidation of the substrates even at concentrations as low as 1 x 10(-5) M. The effect of these variables in the analysis of real samples is presented. The potential use of liquid chromatography for isolating the model pollutants from detrimental matrix components in nat- ural waters is also shown.

Use of a sample translation technique to minimize adverse effects of laser irradiation in surface-enhanced Raman spectrometry.

Surface-enhanced Raman spectroscopy (SERS) has proven to be a very powerful tool in the analysis of a wide range of compounds. However, continuous irradiation of the laser beam over the SERS substrate can promote the gross decomposition of the sample analytes and significantly broaden and diminish the intensities of observed spectral bands. In addition, the incident radiation can promote thermal or photolytic fragmentation of analytes, thereby altering the observable bands and possibly leading to a misinterpretation of analytical data. Finally, chemical or morphological changes in the SERS substrate are possible. This work presents the use of a sample translation technique (STT) as a means to minimize these adverse effects. By spinning the sample rapidly, the effective residence time of analytes and substrate within the irradiated zone is dramatically decreased without reduction of spectral acquisition time or the density of analyte in the zone. The technique is studied by acquiring SERS spectra of Naproxen USP, riboflavin, folic acid, Rhodamine 6G, and 4-aminothiophenol using silver islands on glass and silver-poly(dimethylsiloxane) composite substrates under various spinning and stationary conditions. In all cases, spectra show improvements upon spinning at laser powers as low as 4.2 (+/- 0.1) mW. Specific differences in the appearance of the spectra and the potential use of STT for improved SERS qualitative and quantitative determinations are presented.

Enantioselective sensors based on antibody-mediated nanomechanics.

The use of microfabricated cantilevers as bioaffinity sensors was investigated. Since many bioaffinity interactions involve proteins as receptors, we conducted studies of the magnitude, kinetics, and reversibility of surface stresses caused when common proteins interact with microcantilevers (MCs) with nanostructured (roughened) gold surfaces on one side. Exposure of nanostructured, unfunctionalized MCs to the proteins immunoglobulin G and bovine serum albumin (BSA) resulted in reversible large tensile stresses, whereas MCs with smooth gold surfaces on one side produced reversible responses that were considerably smaller and compressive. The response magnitude for nanostructured MCs exposed to BSA is shown to be concentration dependent, and linear calibration over the range of 1-200 mg/L is demonstrated. Stable, reusable protein bioaffinity phases based on unique enantioselective antibodies are created by covalently linking monoclonal antibodies to nanostructured MC surfaces. The direct (label-free) stereoselective detection of trace amounts of an important class of chiral analytes, the alpha-amino acids, was achieved based on immunomechanical responses involving nanoscale bending of the cantilever. The temporal response of the cantilever (delta deflection/delta time) is linearly proportional to the analyte concentration and allows the quantitative determination of enantiomeric purity up to an enantiomeric excess of 99.8%. To our knowledge, this is the first demonstration of chiral discrimination using highly scalable microelectromechanical systems.

Enhancing chemi-mechanical transduction in microcantilever chemical sensing by surface modification.

The use of chemically selective thin-film coatings has been shown to enhance both the chemical selectivity and sensitivity of microcantilever (MC) chemical sensors. As an analyte absorbs into the coating, the coating can swell or contract causing an in-plane stress at the associated MC surface. However, much of the stress upon absorption of an analyte may be lost through slippage of the chemical coatings on the MC surface, or through relaxation of the coating in a manner that minimizes stress to the cantilever. Structural modification of MC chemical sensors can improve the stress transduction between the chemical coating and the MC. Surfaces of silicon MC were modified with focused ion beam milling. Sub-micron channels were milled across the width of the MC. Responses of the nanostructured, coated MCs to 2,3-dihydroxynaphthalene and a series of volatile organic compounds (VOCs) were compared to smooth, coated MCs. The analytical figures of merit for the nanostructured, coated MCs in the sensing of VOCs were found to be better than the unstructured MCs. A comparison is made with a previously reported method of creating disordered nanostructured MC surfaces.

Optimization strategies and modeling of separations of dansyl-amino acids by cyclodextrin-modified capillary electrophoresis.

Presented in this study is an approach to optimize conditions for capillary electrophoresis separations of multianalyte enantiomeric pairs (D- and L-dansyl (Dns)-amino acids) that involves the rational use of combinations of cyclodextrins (CDs) as enantioselective running buffer additives. Migration data is experimentally obtained for a range of concentrations for native CDs used individually and employed to determine inclusion constants for the Dns-amino acids of interest. An expression for the mobility of the amino acids when multiple (two in this work) CDs are present in the running buffer is used to simulate separations for more complex CD systems. A chromatographic response function involving predicted resolution is generated to gauge the quality of these separations. Simplex methods are then employed for the first time to optimize conditions for the separation of amino acid enantiomers. The validity of this approach is demonstrated for separations of five Dns-amino acid enantiomers using gamma- and beta-CDs at various concentrations. Extending the dual-CD approach to other CDs and increasing the number of CDs beyond two should be possible. To this end, preliminary experiments are performed by using several available single-isomer, derivatized CDs (individually) to determine if they have potential for further studies. Although results with these particular derivatized CDs are not encouraging, we did find that molecular mechanics modeling is useful in interpreting those cases in which low inclusion constants possibly contributed to the ineffectiveness of the CDs.

Spatially focused deposition of capillary electrophoresis effluent onto surface-enhanced Raman-active substrates for off-column spectroscopy.

Surface-enhanced Raman scattering (SERS) is employed to obtain distinctive spectra of compounds that are efficiently separated by capillary electrophoresis (CE) and deposited onto planar SERS-active substrates. A simple method is described that explains how to prepare SERS-active substrates by depositing a silver-colloid solution onto frosted-glass microscope slides, using a high-efficiency nebulizer. Scanning electron micrographs reveal a layered coating of fairly uniform-sized, 100-nm silver nanoparticles with interstitial spaces ranging from a few to tens of nanometers. The on-column separation is monitored by laser-induced fluorescence, while electrofilament depositing the CE effluent onto a moving SERS-substrate. Subsequently, the SERS spectra and off-column electropherograms are obtained with a simple confocal Raman spectrometer. The test compounds used to demonstrate this technique include compounds of biological significance: benzyloxyresorufin, riboflavin, and resorufin. CE and Raman conditions are evaluated to determine their affects on the SERS signals. An average off-column efficiency of 100,000 plates/m and a signal reproducibility of 11% relative standard deviation were achieved. Characteristic spectra with major Raman bands exhibiting signal-to-noise ratios of greater than 3 were obtained for a 3.2-nL injection of 10(-6) M (706 fg) resorufin. Forming a self-assembled monolayer (SAM) on the substrate increases the sensitivity of the SERS technique and decreases the on-substrate broadening. Calibration plots for both plain- and SAM-SERS substrates are demonstrated.

Electrofilament deposition and off-column detection of analytes separated by capillary electrophoresis.

Capillary electrophoresis interfaced with electrospray is a convenient technique for continuously transferring column effluent from capillary-to-planar format. Conditions are optimized to produce a narrow (approximately 20 microm) liquid filament (electrofilament), which is capable of depositing spatially focused bands with track widths that are routinely 100 microm. A fiber optic-based, laser-induced fluorescence cell is employed to monitor the separation on-column while the separated bands are deposited onto a moving substrate. The photodetection of deposited bands is accomplished by using either a charge-coupled device camera or a photomultiplier tube. Deterioration of on-column separation performance is observed when the electrofilament voltage is applied. Elevating the inlet of the capillary column, to provide hydrodynamic flow, restores separation performance. Substrate temperature and translational rates are optimized with respect to both off-column separation efficiency and signal intensity. Off-column separation efficiencies of 65 000 plates per meter were achieved. A linear dynamic range of 10(3) and a limit of detection of 10(-8) M were obtained for kiton red deposited onto a reversed phase thin-layer chromatography plate. To demonstrate the applicability of this technique to more complex separation solutions, a dye mixture was successfully separated and deposited with sodium dodecyl sulfate in the running buffer.

Parameters affecting reproducibility in capillary electrophoresis.

It is well known that poor quantitative reproducibility substantially limits the practical implementation of capillary electrophoresis (CE) separations in chemical analysis. The principal sources of variance in observed peak areas are irreproducible flow rate, which influences on-column detector response, and inconsistent injection volume or amount. An overview of studies by researchers to address the reproducibility issue will be presented. In addition, current efforts in our laboratory to assess sources of quantitative variance for separations of dansylated amino acids using an automated CE system are presented and related when appropriate to the body of existing knowledge on this important topic. A comparison of different injection methods (hydrostatic vs. electrokinetic) and approaches (e.g., high vs. low pressure), the effect of random changes in electroosmotic flow (EOF) due to air bubbles in the CE capillary, and choice of certain peak integration parameters in terms of peak area reproducibility are presented. Under optimum conditions relative standard deviation (RSD) values in raw peak area are typically 2.0%. With nonoptimum conditions (e.g., with air bubbles in capillary), RSD values can substantially degrade. However, normalizing with retention times, internal standards, or observed electrophoretic current produces RSD values in a range of 1.4-2.3%.

Modification of micro-cantilever sensors with sol-gels to enhance performance and immobilize chemically selective phases.

A chemical sensor based on the deflection of a surface modified silicon micro-cantilever is presented. A thin film of sol-gel was applied to one side of the micro-cantilever surface using a spin coating procedure. The sensor has been shown to give different responses to vapor phase analytes of varying chemical composition, as well as to varying concentrations of a given analyte. Ethanol, a highly polar molecule, exhibits a strong affinity for the polar sol-gel coating resulting in a large response; pentane, a non-polar hydrocarbon, shows very little response. The sol-gel coating has also been shown to function as a backbone for the immobilization of chemically selective phases on the cantilever surface. Reaction of the sol-gel film with chlorotriethoxysilane and subsequent capping of the remaining reactive surface silanols with hexamethyldisilizane increases the non-polar nature of the film. This results in an increase in the response of the sensor to non-polar analytes. The effects of film thickness and cantilever structure thickness on response were also investigated.

Examination of cyanine intercalation dyes for rapid and sensitive detection of DNA fragments by capillary electrophoresis.

Recently, a series of cyanine dyes has been developed that are reported to offer superior fluorescence upon intercalation relative to ethidium bromide (EB). The dimeric cyanine dyes, such as POPO-3 (Molecular Probes, Eugene, OR, U.S.A.), are reported to offer the highest fluorescence enhancement of the cyanine dyes. Although POPO-3 does offer an improvement in sensitivity, achieving reproducible, high-efficiency results by employing pre-column, on-column, and postcolumn labeling strategies proved to be problematic. Evaluation of the labeling rate of these dimeric dyes indicates that perhaps the most limiting factor to using this dye for rapid CE analysis of DNA fragments is the apparent slow rate at which the dye fully intercalates into the DNA structure. Use of the monomeric analog PO-PRO-3 (Molecular Probes) resulted in much better separations and low limits of defection when employed for on-column intercalation. However, we find that the kinetics are too slow to employ this dye in a sheath flow cell for postcolumn derivatization.

Evaluation of a sheath flow cuvette for postcolumn fluorescence derivatization of DNA fragments separated by capillary electrophoresis.

The investigation and evaluation of the sheath flow cell as a reaction chamber to postcolumn fluorescently derivatize DNA fragments separated by capillary electrophoresis is described herein. Use of the sheath flow cell arrangement facilitates the mixing of the intercalating dye, ethidium bromide (EB), and the effluent from the separation capillary by diffusion without a high degree of band dispersion. Theoretical plate counts of >1 × 10(6) are reported with the postcolumn derivatization technique, and resolution of all of the fragments in a φx-174-HaeIII digest is achieved. Optimization of experimental parameters such as flow rate, position of the detection zone, and EB concentration is examined. A limit of detection in the low nanograms-per-milliliter range with a linear dynamic range over 3 orders of magnitude is reported for a sample of φx-174-HaeIII digest. Evaluation of postcolumn derivatization for the investigation of DNA-protein interactions is demonstrated. The integrity of a DNA-trp-repressor protein interaction is maintained with the postcolumn approach but is compromised when EB is added to the running buffer.

Temporal analysis of DNA restriction digests by capillary electrophoresis.

We demonstrate a facile means for temporal analysis of DNA restriction enzyme digests by capillary electrophoresis-laser-induced fluorescence (CE-LIF) detection. phi X-174 DNA was digested with HaeIII restriction enzyme under conditions that allowed the monitoring of digestion as it proceeded toward completion. Separation by a polymer solution of methylcellulose in a polyacrylamide coated capillary allowed high resolution and a high degree of reproducibility between sequential runs. At pre-selected time intervals an injection of the digest, directly from the reaction mixture, was made. Sensitive detection was achieved by using ethidium bromide as an intercalation dye and allowing intercalation to occur on-column. It is demonstrated that the course of the digestion (i.e., the creation and diminishing of fragment peaks) can be followed using this methodology. Also demonstrated is the ability to use temporal analysis to determine ideal conditions for producing a single cut within a cloning and expression vector (pET3a-PAI-1) which contains 11 potential restriction endonuclease cleavage sites. This initial attempt to follow a restriction digest on-column not only provides meaningful information for the biochemical researcher, but also furthers the use of CE a diagnostic tool for the biochemical laboratory.

Examination of band dispersion during size-selective capillary electrophoresis separations of DNA fragments.

Versatile capillary electrophoresis instrumentation that permits the rapid and precise translation of a laser fluorometric detection zone along the capillary wall has been used to examine the factors which cause band broadening during size-selective separations of DNA fragments. Separations are performed using capillaries containing entangled polymer solutions. The scanning capabilities of this instrumentation facilitates the determination of diffusion coefficients under static conditions without the need to discontinue and reapply an electric field. The ability to rapidly translate the detection zone along the column allows the monitoring of the separation at various points along the capillary which enables the examination of the sources of band dispersion under kinetic conditions. Results from experiments utilizing various concentrations of both high and low molecular mass methyl cellulose polymers as sieving media are presented. It is shown that axial diffusion, even when adjusted for kinetic conditions using the Einstein relationship, does not account for the total observed band variance. Possible explanations for this behavior are presented.

Development of a new capillary electrophoresis-based fibre optic sensor.

A new fluorescence-based fibre optic sensor is described which combines the sensitivity offered by laser-induced fluorescence with the selectivity offered by capillary electrophoresis (CE). A single optical fibre directly probes the terminus of a 5-8 cm separation capillary. The linear geometry associated with this sensor necessitates a 'single reservoir' design, thus presenting major challenges to overcome in comparison to the conventional two-reservoir configuration common to a typical laboratory setup. Some of the challenges confronted by the design features presented in this work include the reduction of gravity-driven hydrostatic flow, the ejection of electrolytic gases evolved at the detection-side electrode and the establishment a suitable compromise between detectability and separation performance. The success of such design features demonstrates the feasibility of a CE-based sensor which offers several amenities particularly useful for in situ sensing. Such attributes include selectivity, diminutive size, flexibility, reusability, high sensitivity, speed, and remote control. Detailed descriptions of sensor fabrication are included, including two variations on a general design concept. In addition, the single-fibre optical detection system is described. Separation characteristics of the new CE-based sensor are presented, highlighted by an observed separation efficiency of up to 8000 theoretical plates (for a 5 cm capillary). The separation of a three-component mixture of the laser dyes, Rhodamine 6G, fluorescein isothyocyanate and sodium fluorescein, is demonstrated.

Chemiluminescence detection in capillary electrophoresis.

Capillary electrophoresis (CE) and related techniques yield highly efficient separations while requiring only minute amounts of sample. Thus, these techniques are amenable to analyses of complex samples in diverse matrices and in situations where sample is extremely limited. The constraints of on-column detection generally result in poor detection limits and have reduced the overall application of CE. One logical approach to increased sensitivity in CE detection has been the development of chemiluminescence (CL)-based detectors. The current state of post-column detector development, CL applications, and limitations of the technique are reported herein.

Capillary Electrokinetic Chromatography Employing p-(Carboxyethyl)calix[n]arenes as Running Buffer Additives.

Calixarenes, a class of macrocyclic phenolic compounds with a basket-like shape, are used as capillary electrophoresis reagents for separations of native and substituted polycyclic aromatic hydrocarbons. The p-(carboxyethyl)calix[n]arenes reported herein are a series of charged, moderately water soluble macrocyclic molecules that can form complexes with neutral molecules. Electrokinetic chromatographic separations are based on the differential distribution of molecules between a running buffer phase, which is transported by electroosmotic flow, and an electrophoretically mediated calixarene. The size of the calixarene influences separation performance, illustrating the importance of cavity size and geometry in the complexation process. p-(Carboxyethyl)calix[7]arene provides the best efficiency (>10(5) plates/m) and selectivity in these studies. The influences of pH, organic solvent, and field strength on elution range, capacity factors, efficiency, and selectivity are also reported. In general, capacity factors are rather low, but the high charge-to-mass ratios of certain calixarenes produce relatively wide elution ranges. Molecular modeling data and solubility data are used to interpret the observed selectivity.