Fabricating microfluidic devices in polymers for bioanalytica applications

The research presented in this document focuses on the fabrication, characterization and application of microfluidic systems fabricated in poly(methyl methacrylate) (PMMA) with the emphasis focused on the fabrication processing steps. Microfluidic devices were produced in PMMA using X-ray lithography. The fabrication methods investigated were sacrificial mask, polyimide membrane mask and embossing techniques. PMMA microfluidic devices fabricated using X-ray lithography were characterized using scanning electron microscopy (SEM) and optical microscopy, while analytical techniques such as electroosmotic flow determination, separations, and fluorescent microscopy were used to characterize fluid transport in these devices. A novel method for the heat annealing of PMMA to PMMA to create a closed system is described. Characterization of this technique was carried out by optical microscopy and scanning electron microscopy. The manufacturing techniques utilized in producing mold inserts for hot embossing and injection molding is discussed as well. Both the mold insert and devices produced from the inserts were characterized using scanning electron microscopy. Devices produced can be used to perform a number of analytical techniques including single molecule detection and fluorescence lifetime monitoring. The primary goal of this research was to develop molding tools consisting of high-aspect-ratio microstructures using robust and reproducible processing steps

Sample extraction and injection with a microscale preconcentrator.

This report details the development of a microfabricated preconcentrator that functions as a fully integrated chemical extractor-injector for a microscale gas chromatograph (GC). The device enables parts-per-billion detection and quantitative analysis of volatile organic compounds (VOCs) in indoor air with size and power advantages over macro-scale systems. The 44 mm{sup 3} preconcentrator extracts VOCs using highly adsorptive, granular forms of graphitized carbon black and carbon molecular sieves. The micron-sized silicon cavities have integrated heating and temperature sensing allowing low power, yet rapid heating to thermally desorb the collected VOCs (GC injection). The keys to device construction are a new adsorbent-solvent filling technique and solvent-tolerant wafer-level silicon-gold eutectic bonding technology. The product is the first granular adsorbent preconcentrator integrated at the wafer level. Other advantages include exhaustive VOC extraction and injection peak widths an order of magnitude narrower than predecessor prototypes. A mass transfer model, the first for any microscale preconcentrator, is developed to describe both adsorption and desorption behaviors. The physically intuitive model uses implicit and explicit finite differences to numerically solve the required partial differential equations. The model is applied to the adsorption and desorption of decane at various concentrations to extract Langmuir adsorption isotherm parameters from effluent curve measurements where properties are unknown a priori

Laboratory technology research: Abstracts of FY 1998 projects

The Laboratory Technology Research (LTR) program supports high-risk, multidisciplinary research partnerships to investigate challenging scientific problems whose solutions have promising commercial potential. These partnerships capitalize on two great strengths of the country: the world-class basic research capability of the DOE Office of Science (SC) national laboratories and the unparalleled entrepreneurial spirit of American industry. Projects supported by the LTR program in FY 1998 explore the applications of basic research advances relevant to DOE`s mission over a full range of scientific disciplines. The program presently emphasizes three critical areas of mission-related research: advanced materials, intelligent processing and manufacturing research, and environmental and biomedical research. Abstracts for 85 projects are contained in this report

Attachment of oligonucleotide probes to polymer biochips and its application for the detection of point mutations

This dissertation is on the fabrication of polymer-based microfluidic arrays for the detection of genetic mutations. Poly(methyl methacrylate) was chosen as one of the polymer substrate materials due to its low background noise, low adsorption of biomolecules, and low assembly temperature. The surface modification of polymer substrates for covalent attachment of oligonucleotide probes, the construction of fluidic channels/arrays, and hybridization kinetics will be covered. As an example of the application, point mutation detection using immobilized arrays constructed in microfluidic devices will be demonstrated. The PMMA surface was derivatized with N-lithioethylenediamine solution to introduce amine groups, which were utilized for the covalent immobilization of terminal amino modified oligonucleotide probes via a homo-bi-functional linker molecule. The coupling bonds formed were stable enough to withstand multiple denaturation/rehybridization cycles. To overcome the drawbacks associated with conventional 2-D flat microarrays, such as long hybridization times and large sample consumption, oligonucleotide arrays were constructed into the microfluidic channels hot embossed into PMMA substrate. With the use of these fluidic channels we observed increased hybridization kinetics as compared to that on the flat arrays. Another benefit is that the channel-attached oligonucleotide probes allow the detection of target concentrations down to pM levels. As such, the specially designed oligonucleotide probes, which have similar melting temperatures, were constructed in microfluidic channels. Low-abundance point mutations in K-ras genes were successfully detected by using a ligase detection reaction (LDR) combined with the microfluidic hybridization. Near-IR laser induced fluorescence technique was used for the detection of surface conducted bioanalytical reactions and high detection sensitivity was obtained. In addition, preliminary work was also conducted on direct photo-patterning of deep ultraviolet (UV) light for immobilizing oligonucleotides on poly(methyl methacrylate) and polycarbonate substrates. Deep UV patterning using a through-hole mask indicated that more oligonucleotide molecules were immobilized on the UV-exposed areas than the non-exposed area in the presence of EDC conjugating reagent. However, the proper dose of UV-exposure and the appropriate EDC concentration need to be optimized in future work to increase the contrast on the immobilization efficiency between the exposed regions and un-exposed regions

Environmental implications of higher order fullerenes and conjugated nanostructures

In quest of harnessing emergent properties and achieving multifunctionality in the materials realm, synthesis and manipulation at the nano-scale has moved its focus from simple passive nanomaterials (NMs) to hierarchical nanostructures. Such nanostructures include higher order fullerenes (HOFs), carbon allotropes composed of more than 60 carbon atoms per fullerene cage, and conjugated nanohybrids (NHs), prepared from materials of multiple chemical origin. The advantages in their electronic, optical, physicochemical, and magnetic properties have inspired their research and use in photovoltaics, nano-electronics, biomedical imaging and drug delivery, catalysis, energy generation and storage, and environmental remediation and sensing. Not only as research grade materials, a global market of bio-imaging and fuel-cell applications have been integrating use of HOFs, and NHs, respectively. Thus it is an exciting time for materials engineering to expand the spectrum of these ‘horizon materials’ by putting together a variety of chemical ‘building blocks’ and build a wide range of multifunctional hierarchical structures. However, such conjugation leading to complex hierarchical structures also introduces unknown environmental risks. The emergent properties of these hierarchical structures necessitate careful assessment of their environmental health and safety. This dissertation is one of the first organized efforts to identify hierarchical nanostructures and assess their environmental implications. This research, through extensive literature review of these novel nanostructures, proposes a working definition of NH from environmental perspective, classifies a wide array of NHs based on chemical origin, and identifies their emerging and altered physicochemical properties with potential to generate unprecedented environmental fate, transport, transformation, and toxicity. Furthermore, this dissertation makes an effort to address three major data gaps: i.e., a) challenges in aqueous solubilization of HOFs, b) possible correlation of carbon numbers on fullerene molecules with their aggregation behavior, and c) influence of hybridization on aggregation kinetics and antimicrobiality of an important electrocatalyst NH (metal-carbon). To address the first data gap, aqueous suspensions of nC₆₀, nC₇₀, nC₇₆, and nC₈₄ were prepared using a calorimetry-based solvent exchange method. Non-aggregating and size-specific aqueous nC₆₀ and nC₇₀ fullerene clusters also were prepared using a non-ionic polymer, pluronic acid (PA). The environmental processes section of this research assessed aggregation kinetics of nHOFs and NHs as well as antimicrobiality of TiO₂ conjugated oxidized multiwalled carbon nanotube (OMWNT-TiO₂) NH. Aqueous solubilization of C₇₀, C₇₆, and C₈₄ was performed being guided by molecular dynamics (MD) simulations. Increased energy demand reflects favorability of HOF-water interaction. The experimental findings suggest that nHOF clusters obtained via solvent-exchange solubilization method remains stabilized by electrostatic repulsion. Similarly, non-ionic triblock co-polymer PA F-127 stabilized aqueous C₆₀ and C₇₀s were prepared. Experimental results suggest that size uniformity of aqueous fullerenes increased with the increase in PA concentration, yielding optimum 58.8±5.6 and 61.8±5.6 nm nC₆₀s and nC₇₀s, respectively (0.10 %w/v PA). Fullerene aqueous suspensions also manifested colloidal stability even in presence of 1 M NaCl or in biological media, i.e., DMEM and RPMI. MD simulations results indicate encapsulation of fullerene clusters by PA molecules and subsequent steric stabilization. The results from this study may facilitate mechanistic environmental and toxicological studies with size-specific fullerenes that do not aggregate in high ionic strength biological media. Aqueous suspensions of nC₆₀ and three nHOFs (i.e., nC₇₀, nC₇₆, and nC₈₄) obtained via solvent-exchange method were systematically studied to determine their aggregation kinetics in a wide range of mono- (NaCl) and divalent (CaCl₂) electrolytes. Experimentally obtained critical coagulation concentration (CCC) values of nC₆₀ and nHOFs displayed a strong negative correlation with the carbon number in fullerenes. The aggregation mechanism was dominated by van der Waals interaction as enumerated via MD simulation and modified Derjaguin-Landau-Verwey-Overbeek (DLVO) model. Natural macromolecules profoundly stabilized all fullerene clusters, even at 100 mM NaCl concentration. The results from this study can be utilized to predict aggregation kinetics of nHOFs other than the ones studied here. To understand the aggregation behavior of carbon-metal NHs, oxidized MWNTs were hybridized sequentially with undoped or Nb-doped TiO₂ and Pt NPs. OMWNT-TiO₂, OMWNT-TiNbO₂, OMWNT-TiO₂, and OMWNT-TiNbO₂-Pt and the component materials were characterized and their aggregation behavior was studied systematically. Experimental findings show that CCC values OMWNT were reduced by TiO₂ attachment; however, Nb-doping and Pt attachment increased their colloidal stability and CCC values. The aggregation mechanism was elucidated by modified DLVO energy calculations using composition-averaged Hamaker constants for NHs. Natural macromolecules stabilized all the NHs and the component materials. Antimicrobiality of OMWNT-TiO₂ NH was studied via in vitro cell viability tests. Opportunistic pathogen Pseudomonas aeruginosa PAO1 strain was exposed to OMWNT, TiO₂, and OMWNT-TiO₂ NH at different concentrations in dark and UV-irradiated conditions. OMWNT-TiO₂ NH showed higher antimicrobial activity compared to the component materials under UV-irradiation. Extracellular reactive oxygen species (ROS) measurement by using fluorescence molecular probes for H₂O₂ identifies UV-induced enhanced ROS generation by the NH as a likely antimicrobial mechanism. The research presented in this dissertation takes the first attempt toward EHS assessment of complex and hierarchical nanostructures. The research findings present new insights into these ‘horizon materials’ and likely will spark interests on this necessary line of research to better understand the environmental fate, transport, and effects of HOFs and NHs. As nanotechnology is advancing from passive singular nanostructures to active and complex nano-systems; such undertakings become imperative to evaluate implications of material complexity at the environmental interface.Civil, Architectural, and Environmental Engineerin

Integrated modular microfluidic system for forensic Alu DNA typing

Driven by the numerous applications of genome-related research, fully integrated microfluidic systems have been developed that have advanced the capabilities of molecular and, in particular, genetic analyses. A brief overview on integrated microfluidic systems for DNA analysis is given in Chapter 1 followed by a report on micro-capillary electrophoresis (µCE) of Alu elements with laser-induced fluorescence (LIF) detection, in which the monomorphic Alu insertions on the X and Y chromosomes were utilized to detect male DNA in large female DNA background (Y: X = 1:19) without cell sorting prior to the determination. The polymorphic Alu loci with known restricted geographical distribution were used for ethnicity determination. A valveless integrated microsystem that consists of three modules is discussed as well: (1) A solid-phase extraction (SPE) module microfabricated on polycarbonate, for DNA extraction from whole cell lysates (extraction bed capacity ~209 ±35.6 ng/cm² of total DNA). (2) A continuous-flow polymerase chain reaction (CFPCR) module fabricated in polycarbonate (Tg ~150 ºC) in which selected gene fragments were amplified using biotin and fluorescently-labeled primers accomplished by continuously shuttling small packets of PCR reagents and template through isothermal zones. (3) µCE module fabricated in poly(methylmethacrylate), which utilized a bioaffinity selection and purification bed (2.9-µL) to preconcentrate and purify the PCR products generated from the CFPCR module prior to µCE. Biotin-labeled CFPCR products were hydrostatically pumped through the streptavidin-modified bed where they were extracted onto the surface of the poly(methylmethacrylate) micropillars (50-µm width; 100-µm height; total surface area of ~117 mm²). This SPE process demonstrated high selectivity for biotinylated amplicons and utilized the strong streptavidin/biotin interaction (Kd =10-15M) to generate high recoveries. The SPE selected CFPCR products were thermally denatured and single stranded DNA released for size-based separations and LIF detection. The multiplexed SPE-CFPCR-µCE yielded detectable fluorescence signal (S/N≥3; LOD ~75 cells) for Alu DNA amplicons for gender and ethnicity determinations with a separation efficiency of ~1.5 x105 plates/m. Compared to traditional cross-T injection procedures typically used for µCE, the affinity preconcentration and injection procedure generated signal enhancements of 17-40 fold, critical for CFPCR thermal cyclers due to Taylor dispersion associated with their operation

Current Air Quality Issues

Air pollution is thus far one of the key environmental issues in urban areas. Comprehensive air quality plans are required to manage air pollution for a particular area. Consequently, air should be continuously sampled, monitored, and modeled to examine different action plans. Reviews and research papers describe air pollution in five main contexts: Monitoring, Modeling, Risk Assessment, Health, and Indoor Air Pollution. The book is recommended to experts interested in health and air pollution issues