Large-area nanopatterning of self-assembled monolayers of alkanethiolates by interferometric lithography.

We demonstrate that interferometric lithography provides a fast, simple approach to the production of patterns in self-assembled monolayers (SAMs) with high resolution over square centimeter areas. As a proof of principle, two-beam interference patterns, formed using light from a frequency-doubled argon ion laser (244 nm), were used to pattern methyl-terminated SAMs on gold, facilitating the introduction of hydroxyl-terminated adsorbates and yielding patterns of surface free energy with a pitch of ca. 200 nm. The photopatterning of SAMs on Pd has been demonstrated for the first time, with interferometric exposure yielding patterns of surface free energy with similar features sizes to those obtained on gold. Gold nanostructures were formed by exposing SAMs to UV interference patterns and then immersing the samples in an ethanolic solution of mercaptoethylamine, which etched the metal substrate in exposed areas while unoxidized thiols acted as a resist and protected the metal from dissolution. Macroscopically extended gold nanowires were fabricated using single exposures and arrays of 66 nm gold dots at 180 nm centers were formed using orthogonal exposures in a fast, simple process. Exposure of oligo(ethylene glycol)-terminated SAMs to UV light caused photodegradation of the protein-resistant tail groups in a substrate-independent process. In contrast to many protein patterning methods, which utilize multiple steps to control surface binding, this single step process introduced aldehyde functional groups to the SAM surface at exposures as low as 0.3 J cm(-2), significantly less than the exposure required for oxidation of the thiol headgroup. Although interferometric methods rely upon a continuous gradient of exposure, it was possible to fabricate well-defined protein nanostructures by the introduction of aldehyde groups and removal of protein resistance in nanoscopic regions. Macroscopically extended, nanostructured assemblies of streptavidin were formed. Retention of functionality in the patterned materials was demonstrated by binding of biotinylated proteins.

Mussel (Mytilus edulis) byssus deposition in response to variations in surface wettability.

Mussels (Mytilus edulis) are economically important in their role as an aquaculture species and also with regard to marine biofouling. They attach tenaciously to a wide variety of submerged surfaces by virtue of collagenous attachment threads termed 'byssi'. The aim of this study was to characterize the spreading of the byssal attachment plaque, which mediates attachment to the surface, on a range of surfaces in response to changes in wettability. To achieve this, well characterized self-assembled monolayers of omega-terminated alkanethiolates on gold were used, allowing correlation of byssal plaque spreading with a single surface characteristic--wettability. The present results were inconsistent with those from previous studies, in that there was a positive correlation between plaque size and surface wettability; a trend which is not explained by conventional wetting theory for a three-phase system. A recent extension to wetting theory with regard to hydrophilic proteins is discussed and the results of settlement assays are used to attempt reconciliation of these results with those of similar previous studies and, also, with recent data presented for the spreading of Ulva linza spore adhesive.

Evidence for vertical phase separation in densely grafted, high-molecular-weight poly(-isopropylacrylamide) brushes in water.

The detailed conformational change of poly(N-isopropylacrylamide) (PNIPAM) brushes at high grafting density in D2O was investigated as a function of temperature using neutron reflection. PNIPAM chains were grafted at high surface density from gold and silicon oxide surfaces by atom transfer radical polymerization. Whereas single layer profiles were observed for temperatures below and above the transition region, bilayer profiles were observed for a narrow range of temperatures near the transition. This nonmonotonic change in the concentration profile with temperature is discussed in the context of theoretical models of vertical phase separation within a brush.

Performance of in-line microfluidic mixers in laminar flow for high-throughput flow cytometry.

We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require the mixing of components that have been introduced into laminarflow. The scheme is based on high-throughput flow cytometry (HyperCyt) where samples from multi-well plates that have been picked up by an autosampler can be separated during delivery by the small air bubbles introduced during the transit of the autosampler probe from well to well. Here, either cell or particle samplesflowing continuously and driven by a syringe are brought together in a Y with reagent samples from wells driven by a peristaltic pump. The mixing is driven by a magnetic microstirrer contained within the sample line. The mixing is assessed using fluorescence of both cell calcium responses and bead-based fluorescence unquenching. In the analysis stream, the particles and reagents are mixed with eithera "wire" or "bar". The bar is more efficient than the wire, and the efficiency of either depends on the spinning action. The high-throughput approach and mixing in HyperCyt integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.

Mixing small volumes for continuous high-throughput flow cytometry: performance of a mixing Y and peristaltic sample delivery.

BACKGROUND: Online mixing for continuous high-throughput flow cytometry has not been previously described. A simple, general high-throughput method for mixing and delivery of submicroliter volumes in laminar flow at low Reynolds numbers would be widely useful. MATERIALS AND METHODS: We describe a micromixing approach that is compatible with commercial autosamplers, flow cytometry, and other detection schemes that require mixing of components that have been introduced into laminar flow. The scheme is based on a previous approach to high-throughput flow cytometry (HyperCyt, Kuckuck et al.: Cytometry 44:83-90, 2001). We showed that samples from multiwell plates that have been picked up by an autosampler can be separated during delivery by the small air bubbles introduced during the transit of the autosampler probe from well to well. Here, a particle sample flowing continuously is brought together in a Y with reagent samples from wells, which have been separated by bubbles. RESULTS: In the effluent stream, the particles and reagents are mixed, most likely as a result of peristaltic action, and reagents from individual wells can be resolved. The sample volumes that can be mixed with this technology are submicroliter in volume, and samples can be mixed at rates up to at least 100/samples per minute. With the current device, carryover between samples can be eliminated if the mixing system is flushed with several volumes of buffer. The anticipated throughput for screening is expected to be at least 20 samples per minute. CONCLUSIONS: The high-throughput approach and peristaltic mixing in HyperCytTM serve to integrate autosamplers with submicroliter detection volumes for analysis in flow cytometry or in microfluidic channels.

Robust and efficient synthetic method for forming DNA microarrays.

The field of DNA microarray technology has necessitated the cooperative efforts of interdisciplinary scientific teams to achieve its primary goal of rapidly measuring global gene expression patterns. A collaborative effort was established to produce a chemically reactive surface on glass slide substrates to which unmodified DNA will covalently bind for improvement of cDNA microarray technology. Using the p-aminophenyl trimethoxysilane (ATMS)/diazotization chemistry that was developed, microarrays were fabricated and analyzed. This immobilization method produced uniform spots containing equivalent or greater amounts of DNA than commercially available immobilization techniques. In addition, hybridization analyses of microarrays made with ATMS/diazotization chemistry showed very sensitive detection of the target sequence, two to three orders of magnitude more sensitive than the commercial chemistries. Repeated stripping and re-hybridization of these slides showed that DNA loss was minimal, allowing multiple rounds of hybridization. Thus, the ATMS/diazotization chemistry facilitated covalent binding of unmodified DNA, and the reusable microarrays that were produced showed enhanced levels of hybridization and very low background fluorescence.

Detection of epitope-tagged proteins in flow cytometry: fluorescence resonance energy transfer-based assays on beads with femtomole resolution.

Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, and M5. We describe novel approaches to the detection of epitope-tagged proteins via fluorescence resonance energy transfer on beads. We have synthesized and characterized biotinylated and fluorescein-labeled FLAG peptides and examined the binding of FLAG peptides to commercial streptavidin beads using flow cytometric analysis. A requirement of assay development is the elucidation of parameters that characterize the binding interactions between component systems. We have thus compiled a set of Kd values determined from a series of equilibrium binding experiments with beads, peptides, and antibodies. We have defined conditions for binding biotinylated and fluoresceinated FLAG peptides to beads. Site occupancies of the peptides were determined to be on the order of several million sites per bead and Kd values in the 0.3-2.0 nM range. The affinity for antibody attachment to peptides was determined to be in the low nanomolar range (less than 10 nM) for measurements on beads and solution. We demonstrate the applicability of this methodology to assay development, by detecting femtomole amounts of N-terminal FLAG-bacteria alkaline phosphatase fusion protein. These characterizations form the basis of generalizable and high throughput assays for proteins with known epitopes, for research, proteomic, or clinical applications.

A surface plasmon resonance array biosensor based on spectroscopic imaging.

We have developed a multi-element transduction system which combines conventional SPR spectroscopy with one-dimensional SPR microscopy to create an effective platform for monitoring binding events on macro- or micro-patterned receptor arrays created on disposable sensor chips. This creates an effective platform for monitoring simultaneous binding events on each of the regions patterned with the receptors. This system has been specifically designed with commercially available components to allow relatively easy duplication. Furthermore, this system can use a proven, simple method to compensate for changes in the bulk index of refraction of the solution containing the analytes due to changes in temperature or solute concentration with simple modifications to the sensor chips alone. Preliminary results demonstrate how this system can be used to monitor several independent biospecific binding events simultaneously.

Technique for detecting changes in fluorescence lifetime by means of optoelectronic circuit auto-oscillation.

We present a new, simple, inexpensive, and highly precise approach to excited-state fluorescence-lifetime-based measurements. The detection system consists of a closed-loop optoelectronic arrangement containing a radio frequency resonance amplifier, a fluorescence excitation light source, a fiber-optic delay line, and a photodetector. The system exhibits auto-oscillations in the form of intensity modulation. The oscillation frequency varies with the modulation phase shift of the fluorescent light. This frequency is used as the detection parameter, which is advantageous because frequency may be measured easily, inexpensively, and with high precision. This technique is well suited for chemical or biosensor applications.

Use of self-assembled monolayers of different wettabilities to study surface selection and primary adhesion processes of green algal (Enteromorpha) zoospores.

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH(3)) or hydroxyl (OH) groups or mixtures of CH(3)- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and "searching" above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to "sense" that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.

Iatrogenic exposure to mercury after hepatitis B vaccination in preterm infants.

Thimerosal, a derivative of mercury, is used as a preservative in hepatitis B vaccines. We measured total mercury levels before and after the administration of this vaccine in 15 preterm and 5 term infants. Comparison of pre- and post-vaccination mercury levels showed a significant increase in both preterm and term infants after vaccination. Additionally, post-vaccination mercury levels were significantly higher in preterm infants as compared with term infants. Because mercury is known to be a potential neurotoxin to infants, further study of its pharmacodynamics is warranted.

Peptides, antibodies, and FRET on beads in flow cytometry: A model system using fluoresceinated and biotinylated beta-endorphin.

BACKGROUND: Particulate surfaces such as beads are routinely used as platforms for molecular assembly for fundamental and practical applications in flow cytometry. Molecular assembly is transduced as the direct analysis of fluorescence, or as a result of fluorescence resonance energy transfer. Binding of fluorescent ligands to beads sometimes alters their emission yield relative to the unbound ligands. Characterizing the physical basis of factors that regulate the fluorescence yield of bound fluorophores (on beads) is a necessary step toward their rational use as mediators of numerous fluorescence based applications. METHODS: We have examined the binding between two biotinylated and fluoresceinated beta-endorphin peptides and commercial streptavidin beads using flow cytometric analysis. We have analyzed the assembly between a specific monoclonal antibody and an endorphin peptide in solution using resonance energy transfer and compared the results on beads in flow cytometry using steady-state and time-resolved fluorescence. RESULTS: We have defined conditions for binding biotinylated and fluoresceinated endorphin peptides to beads. These measurements suggest that the peptide structure can influence both the intensity of fluorescence and the mode of peptide binding on the bead surface. We have defined conditions for binding antibody to the bead using biotinylated protein A. We compared and contrasted the interactions between the fluoresceinated endorphin peptide and the rhodamine- labeled antibody. In solution we measure a K(d) of <38 nM by resonance energy transfer and on beads 22 nM. DISCUSSION: Some issues important to the modular assembly of a fluorescence resonance energy transfer (FRET) based sensing scheme have been resolved. The affinity of peptides used herein is a function of their solubility in water, and the emission intensity of the bound species depends on the separation distance between the fluorescein and the biotin moiety. This is due to the quasi-specific quenching interaction between the fluorescein and a proximal binding pocket of streptavidin. Detection of antibodies in solution and on beads either by FRET or capture of fluorescent ligands by dark antibodies subsequently enables the determination of K(d) values, which indicate agreement between solution and flow cytometric determinations.

Poison center planning for mass gatherings: the Georgia Poison Center experience with the 1996 Centennial Olympic Games.

BACKGROUND: The host city for the Olympic Games needs to prepare for "the world" to visit. This article is the first published report describing the impact of an event such as the Olympics on poison center services. PLANNING AND ACTIONS: We evaluated our operations and identified potential new demands. Operational aspects reviewed included: staffing; communication with foreign language callers; knowledge of international drug products; telephone access; procedures for disaster response and recovery; poison treatment protocols; and handling of hazardous material releases. We collaborated with local, state, and federal planners to delineate the poison center role and to develop protocols for use in a poisoning, including a hazardous material's release at a densely populated site. We enhanced our capability to respond to unusual incidents by forming new alliances. Fortunately, no such events occurred; these plans were therefore not tested. CONCLUSIONS: The Georgia Poison Center developed and implemented new capabilities for the Centennial Olympic Games. Immediate access to poison center resources would have facilitated the care of poisoned patients at multiple hospitals if this had become necessary. Communities preparing for mass gatherings should capitalize on the common interests of poison centers, hazardous materials specialists, and public safety agencies. Preparations should emphasize the most likely events, while recognizing the possibility of the unexpected. Planning an integrated response allows the talents and resources of participating agencies to be maximally utilized.

Surface-grafted, environmentally sensitive polymers for biofilm release.

Controlling bacterial biofouling is desirable for almost every human enterprise in which solid surfaces are introduced into nonsterile aqueous environments. One approach that is used to decrease contamination of manufactured devices by microorganisms is using materials that easily slough off accumulated material (i.e., fouling release surfaces). The compounds currently used for this purpose rely on low surface energy to inhibit strong attachment of organisms. In this study, we examined the possible use of environmentally responsive (or "smart") polymers as a new class of fouling release agents; a surface-grafted thermally responsive polymer, poly(N-isopropylacrylamide) (PNIPAAM), was used as a model compound. PNIPAAM is known to have a lower critical solubility temperature of approximately 32 degrees C (i.e., it is insoluble in water at temperatures above 32 degrees C and is soluble at temperatures below 32 degrees C). Under experimental conditions, >90% of cultured microorganisms (Staphylococcus epidermidis, Halomonas marina) and naturally occurring marine microorganisms that attached to grafted PNIPAAM surfaces during 2-, 18-, 36-, and 72-h incubations were removed when the hydration state of the polymer was changed from a wettability that was favorable for attachment to a wettability that was less favorable. Of particular significance is the observation that an organism known to attach in the greatest numbers to hydrophobic substrata (i.e., H. marina) was removed when transition of PNIPAAM to a more hydrated state occurred, whereas an organism that attaches in the greatest numbers to hydrophilic substrata (i.e., S. epidermidis) was removed when the opposite transition occurred. Neither solvated nor desolvated PNIPAAM exhibited intrinsic fouling release properties, indicating that the phase transition was the important factor in removal of organisms. Based on our observations of the behavior of this model system, we suggest that environmentally responsive polymers represent a new approach for controlling biofouling release.

Computer-controlled laser ablation: a convenient and versatile tool for micropatterning biofunctional synthetic surfaces for applications in biosensing and tissue engineering.

This paper describes laser-based methods for preparing micropatterns of bioactive molecular species in self-assembled monolayers (SAMs) and micropatterns of proteins and other biological molecules immobilized on solid substrates. Applications of these micropatterned surfaces in multianalyte biosensing and tissue engineering are emphasized. The focus of the paper is on the use of a computer-controlled laser ablation system comprising a research-grade inverted optical microscope, a pulsed nitrogen-pumped dye laser emitting at 390 nm, a programmable sample stage, and the computerized control system. The laser system can be implemented in a typical biosensor or tissue culture laboratory to enable the facile and reproducible fabrication of micropatterned surfaces by several methods. Various methods for patterning are discussed with examples given and emphasis placed on (1) laser ablation in the fabrication of photolithography masks, (2) electrochemical patterning of SAMs, and (3) laser desorption of SAMs. The relative merits of each technique are discussed with respect to application in fabrication of active surfaces for biosensing and tissue culture applications.

Assessment of learning by emergency medicine residents and pharmacy students participating in a poison center clerkship.

An AAPCC-designated poison center developed and validated an objective testing instrument to evaluate learning during a poison center clinical rotation for 2nd-year emergency medicine residents and 5th-year pharmacy students. The examination contained multiple-choice, true-false, and fill-in questions pertaining to basic clinical toxicology. A pretest was administered prior to the rotation and a post-test was administered upon completion of the rotation. Overall pre-test mean was 56.2%; physician pre-test mean was 73.8%, and student pre-test mean was 43.9%. Overall post-test mean was 78.7%; physician post-test mean was 85.7%, and student post-test mean was 81%. Pre-test scores ranged from 21 to 86% for the group, and post-test scores ranged from 68 to 96%. The mean difference in pre-test to post-test score was 26.9%. These data suggest that a poison center rotation can result in significant increases in post-test scores in comparison to pre-test scores.

Attachment of bacteria to model solid surfaces: oligo(ethylene glycol) surfaces inhibit bacterial attachment.

Bacterial cell attachment to the surfaces of self-assembled monolayers formed by the adsorption of omega-substituted alkanethiols on transparent gold films has been studied under defined bacterial culture and flow conditions. Phase contrast microscopy was used to quantify the attachment of two organisms, one of medical (Staphylococcus epidermidis) and one of marine (Deleya marina) importance. Self-assembled monolayers terminated with hexa(ethylene glycol), methyl, carboxylic acid and fluorocarbon groups were investigated. Over the range of experimental conditions, self-assembled monolayers formed from HS(CH2)11(OCH2CH2)6OH were found to be uniformly resistant to bacterial attachment, with a 99.7% reduction of attachment for both organisms when compared to the most fouled surface for each organism. On other surfaces, S. epidermidis and D. marina were shown to exhibit very different attachment responses to the wettability of the substratum. While the attachment of S. epidermidis correlated positively with surface hydrophilicity, D. marina showed a preference for hydrophobic surfaces. This study suggests that surfaces incorporating high densities of oligo(ethylene glycol) are good candidates for surfaces that interact minimally with bacteria.

Engineering cell shape and function.

An elastomeric stamp, containing defined features on the micrometer scale, was used to imprint gold surfaces with specific patterns of self-assembled monolayers of alkanethiols and, thereby, to create islands of defined shape and size that support extracellular matrix protein adsorption and cell attachment. Through this technique, it was possible to place cells in predetermined locations and arrays, separated by defined distances, and to dictate their shape. Limiting the degree of cell extension provided control over cell growth and protein secretion. This method is experimentally simple and highly adaptable. It should be useful for applications in biotechnology that require analysis of individual cells cultured at high density or repeated access to cells placed in specified locations.

Imaging of features on surfaces by condensation figures.

Condensation of a vapor to a liquid on a cold surface that is not wet completely by this liquid leads to the formation of an array of droplets. If the surface is heterogeneous in its physical properties (especially its interfacial free energy), the patterns of these arrays reflect this heterogeneity. The distribution of droplets of water (condensation figures or CFs) observed by optical microscopy on a surface can be correlated with the molecular structure of that surface. The substrates used to investigate the formation and morphology of the CFs were patterned, self-assembled monolayers of different alkanethiolates on gold and of alkyl siloxanes on glass. Analysis of CFs is a valuable nondestructive technique for characterizing heterogeneities in surfaces.