Micropillar Array Based Microchips for Electrospray Ionization Mass Spectrometry, Microreactors, and Liquid Chromatographic Separation

This dissertation deals with the design, fabrication, and applications of microscale electrospray ionization chips for mass spectrometry. The microchip consists of microchannel, which leads to a sharp electrospray tip. Microchannel contain micropillars that facilitate a powerful capillary action in the channels. The capillary action delivers the liquid sample to the electrospray tip, which sprays the liquid sample to gas phase ions that can be analyzed with mass spectrometry. The microchip uses a high voltage, which can be utilized as a valve between the microchip and mass spectrometry. The microchips can be used in various applications, such as for analyses of drugs, proteins, peptides, or metabolites. The microchip works without pumps for liquid transfer, is usable for rapid analyses, and is sensitive. The characteristics of performance of the single microchips are studied and a rotating multitip version of the microchips are designed and fabricated. It is possible to use the microchip also as a microreactor and reaction products can be detected online with mass spectrometry. This property can be utilized for protein identification for example. Proteins can be digested enzymatically on-chip and reaction products, which are in this case peptides, can be detected with mass spectrometry. Because reactions occur faster in a microscale due to shorter diffusion lengths, the amount of protein can be very low, which is a benefit of the method. The microchip is well suited to surface activated reactions because of a high surface-to-volume ratio due to a dense micropillar array. For example, titanium dioxide nanolayer on the micropillar array combined with UV radiation produces photocatalytic reactions which can be used for mimicking drug metabolism biotransformation reactions. Rapid mimicking with the microchip eases the detection of possibly toxic compounds in preclinical research and therefore could speed up the research of new drugs. A micropillar array chip can also be utilized in the fabrication of liquid chromatographic columns. Precisely ordered micropillar arrays offer a very homogenous column, where separation of compounds has been demonstrated by using both laser induced fluorescence and mass spectrometry. Because of small dimensions on the microchip, the integrated microchip based liquid chromatography electrospray microchip is especially well suited to low sample concentrations. Overall, this work demonstrates that the designed and fabricated silicon/glass three dimensionally sharp electrospray tip is unique and facilitates stable ion spray for mass spectrometry.Väitöskirjatyö käsittelee mikrofluidististen massaspektrometriaan liitettävien sähkösumutusmikrosirujen suunnittelua, valmistamista ja sovelluksia. Mikrosirut koostuvat mikropilarikkokanavasta ja terävästä sähkösumutuskärjestä. Hydrofiilisen mikropilarikkokanavan ansiosta mikrosiru täyttyy nesteillä kapillaarivoimien avulla. Kapillaari-ilmiön avulla nestemäinen näyte siirtyy passiivisesti täyttyen näytteen syöttökohdasta terävään sähkösumutuskärkeen, joka sumuttaa näytteen ioneiksi kaasufaasiin. Nämä molekyyli-ionit voidaan massaspektrometrisesti analysoida. Tämä menetelmä mahdollistaa näytteen syötön ilman pumppua tai erillistä jännitettä elektro-osmoottista virtausta varten. Mikrosirut toimivat korkeajännitteellä, jota voidaan hyödyntää myös sähköisenä venttiilinä mikrosirun ja massaspektrometrin välillä. Mikrosiruja voidaan käyttää useissa eri sovelluksissa, kuten esimerkiksi lääkeaineiden, proteiinien, peptidien tai metaboliittien herkissä ja nopeissa analyyseissä. Väitöskirjassa on selvitetty yksittäisten ja monikärkisten mikrosirujen toimintakykyä. Mikrosiruja on mahdollista käyttää myös mikroreaktoreina, joiden reaktiotuotteet voidaan välittömästi analysoida massaspektrometrisesti. Tätä ominaisuutta voidaan hyödyntää esimerkiksi proteiinien tunnistamisessa. Proteiinit voidaan pilkkoa entsymaattisesti mikrosirulla, ja reaktiotuotteet tunnistaa massaspektrometrisesto. Menetelmän etuna voidaan pitää sitä, että analyysissä tarvittavien proteiinien määriä voidaan pienentää, koska diffuusiorajoitteiset reaktiot tapahtuvat nopeammin mikroskaalassa pienempien diffuusiomatkojen ansiosta. Mikrosirut sopivat erityisen hyvin pinta-aktivoituihin reaktioihin, koska tiheällä mikropilarikolla on korkea pinta-ala-tilavuussuhde. Esimerkiksi mikrosiru, johon on valmistettu nanokerros titaanidioksidia mikropilarikon päälle liitettynä UV-säteilytykseen tuottaa fotokatalyyttisiä reaktioita, joita voidaan hyödyntää lääkeainemetabolian ennustamisessa. Nopea mikrosirupohjainen lääkeainemetabolian ennustaminen helpottaa mahdollisten myrkyllisten yhdisteiden tunnistamista jo lääkekehityskaaren prekliinisessä vaiheessa, ja siksi menetelmä voisi nopeuttaa uusien lääkeaineiden tutkimusta. Mikropilarikkosiruja voidaan hyödyntää myös nestekromatografisten kolonnien valmistuksessa. Tarkasti muodostunut mikropilarikko on hyvin homogeeninen kolonni, jossa tapahtuneet kromatografiset erotukset on voitu demonstroida sekä laser indusoidulla fluoresenssilla että massaspektrometrisesti. Pienien dimensioidensa ansiosta nestekromatografinen kolonni ja sähkösumutuskärki integroituna samalle mikrosirulle, toimii erityisen hyvin pienien näytemäärien kanssa

Microfluidic devices interfaced to matrix-assisted laser desorption/ionization mass spectrometry for proteomics

Microfluidic interfaces were developed for off-line matrix-assisted laser desorption/ionization mass spectrometry (MALDI). Microfluidic interfaces allow samples to be manipulated on-chip and deposited onto a MALDI target plate for analysis. For this research, microfluidic culturing devices and automated digestion and deposition microfluidic chip platforms were developed for the identification of proteins. The microfluidic chip components were fabricated on a poly(methyl methacrylate), PMMA, wafer using the hot embossing method and a molding tool with structures prepared via micromilling. One of the most important components of the chip system was a trypsin microreactor. An open channel microreactor was constructed in a 100 µm wide and 100 µm deep channel with a 4 cm effective channel length. This device integrated frequently repeated steps for MALDI-based proteomics such as digestion, mixing with a matrix solution, and depositing onto a MALDI target. The microreactor provided efficient digestion of proteins at a flow rate of 1 µL/min with a residence time of approximately 24 s in the reaction channel. An electrokinetically driven microreactor was also developed using a micropost structured chip for digestion. The micropost chip had a higher digestion efficiency due to the higher surface area-to-volume ratio in the channel. Also, the electrokinetic flow eliminated the need for an external pumping system and gave a flat flow profile in the microchannel. The post microreactor consisted of a 4 cm × 200 µm × 50 µm microfluidic channel with trypsin immobilized on an array of 50 µm in diameter micropost support structures with a 50 µm edge-to-edge inter-post spacing. This micropost reactor was also used for fingerprint analysis of whole bacterial cells. The entire tryptic digestion and deposition procedure for intact bacteria took about 1 min. A contact deposition solid-phase bioreactor coupled with MALDI-TOF MS allowed for low-volume fraction deposition with a smaller spot size and a higher local concentration of the analyte. A bacterial cell-culturing chip was constructed for growing cells on-chip followed by off-line MALDI analysis. Coupling MALDI-TOF MS whole cell analysis with microfluidic culturing resulted in more consistent spectra as well as reduction of the total processing time. The microfluidic cell culturing was performed in a PMMA chip with a polydimethylsiloxane (PDMS) cover to allow gas permeation into the culture channel, which contained a 2.1 μL volume active culture chamber. After incubation of E. coli in a microfluidic culture device at 37 ℃ for 24 h, the cultured cells were analyzed with MALDI MS. Also, a microfluidic cell culture device containing continuous perfusion of culture medium was developed using a polycarbonate membrane. This microfluidic culturing format was improved with a fluidic manifold and thermostatted microheaters. Fingerprint mass spectra distinguishing E. coli strains tested were obtained after a 6 h incubation time, which was shorter compared to the 24 h incubation time using conventional culturing techniques. In addition, an enhanced identification procedure for bacteria was achieved by integrating on-chip digestion of cultured bacteria

Platforms and Protocols for the Multidimensional Microchip Electrophoretic Analysis of Complex Proteomes

The need for rapid, portable and high-throughput systems in proteomics is now prevalent because of demands for generating new protein-based disease biomarkers. However, 2-D protein profile patterns are lending themselves as potential diagnostic tools for biomarker discovery. It is difficult to identify protein biomarkers which are low abundant in the presence of highly abundant proteins, especially in complex biological samples like serum. Protein profiles from 2-D separation of the protein content of cells or body fluids, which are unique to certain physiological or pathological states, are currently available on internet databases. In this work, we demonstrate the ability to separate a complex biological sample using low cost, disposable, polymer-based microchips suitable for a multidimensional techniques that employed sodium dodecyl sulfate micro-capillary gel electrophoresis (SDS µ-CGE) in the 1st dimension and micellar electrokinetic capillary chromatography (MEKC) or microemulsion electrokinetic capillary chromatography (MEEKC) in the 2nd dimension. The peak capacity generated by this microchip technique was about 3-fold greater compared to conventional 2-D separation methods and the complete separation time was 60X faster. To minimize electroosmotic flow effects, we dynamically coated the channels with methylhydroxyethyl cellulose. Proteins were detected by laser-induced fluorescence following their labeling with dyes. To mitigate challenges posed by labeling the proteins, we investigated the use of a label-free technique that relied upon conductivity measurements. Preliminary data are presented on the fabrication of on-chip electrodes using a conductive SU-8 polymer via lithography

Electrokinetic Transport, Trapping, and Sensing in Integrated Micro- and Nanofluidic Devices

Thesis (Ph.D.) - Indiana University, Chemistry, 2009Microfluidics is rapidly becoming a mature field, and improved fabrication methods now routinely produce sub-micrometer features. As device dimensions shrink, physical phenomena that are negligible at larger length scales become more important, and by integrating nanofluidic elements with microchannels, new analytical techniques can be developed based on the unique behavior of matter at the nanoscale. This work addresses the fabrication, operation, and application of in-plane nanochannels and out-of-plane nanopores in lab-on-a-chip devices. In planar nanofluidic devices, we demonstrate a method to produce micro- and nanoscale features simultaneously with a single UV exposure step and evaluate flow control and sample dispensing with nanofluidic cross structures. Modification of the pinched injection method makes it applicable to variable-volume, attoliter-scale injections, including the smallest volume electrokinetically-controlled injections to date. As an alternative approach, track-etch nanopore membranes are explored as out-of-plane nanofluidic components. The random distribution of pores in these membranes is overcome by lithographic and microchannel-based methods to isolate and address specific pores. Microfluidic isolation improves mass transport to the pore(s), provides easy coupling of electrical potentials, and facilitates additional sample processing steps up- and downstream. These integrated microchannel-nanopore devices are used for diffusion-based dispensing, electrokinetic trapping, and resistive pulse sensing. In a high pore density device, diffusion-based dispensing establishes a stable chemical gradient for bacterial chemotaxis assays. For lower pore density devices, the nanopores are the most resistive components in the fluidic circuit, and application of an electric potential produces localized regions of high electric field strength and field gradient. These high field regions are applied to electrokinetic trapping of particles and cells in multiple-pore devices and to single particle detection by resistive pulse sensing in devices with a single isolated pore. To better understand factors influencing ion current in single nanoscale conduits, we systematically examine ion current rectification as a function of pore diameter, ionic strength, and pH to improve understanding of ion current through nanopores and to characterize preferred operating parameters for sensing applications. These results are applied to detection of virus capsids, and future work is proposed to investigate capsid assembly

Solid State Circuits Technologies

The evolution of solid-state circuit technology has a long history within a relatively short period of time. This technology has lead to the modern information society that connects us and tools, a large market, and many types of products and applications. The solid-state circuit technology continuously evolves via breakthroughs and improvements every year. This book is devoted to review and present novel approaches for some of the main issues involved in this exciting and vigorous technology. The book is composed of 22 chapters, written by authors coming from 30 different institutions located in 12 different countries throughout the Americas, Asia and Europe. Thus, reflecting the wide international contribution to the book. The broad range of subjects presented in the book offers a general overview of the main issues in modern solid-state circuit technology. Furthermore, the book offers an in depth analysis on specific subjects for specialists. We believe the book is of great scientific and educational value for many readers. I am profoundly indebted to the support provided by all of those involved in the work. First and foremost I would like to acknowledge and thank the authors who worked hard and generously agreed to share their results and knowledge. Second I would like to express my gratitude to the Intech team that invited me to edit the book and give me their full support and a fruitful experience while working together to combine this book

Fundamental studies of AC/DC electrokinetic phenomena for the realization of microchip capillary electrophoresis for single-cell analysis

The goal of this research was to investigate AC and DC electrokinetic phenomena to better understand their individual and combined effects on particle and fluid motions in microchannels in order to realize microchip capillary electrophoresis for single cell analysis. AC-DC electroosmotic flow interaction was studied by observing the motions of polystyrene microbeads suspended in deionized water in a microchannel as the main AC and DC electrokinetics parameters were varied.Particle-particle interactive dielectrophoretic (DEP) force under electrohydrodynamic flow conditions was studied by performing experiments on a microchannel - microelectrode system containing polystyrene beads and comparing the experimental results with numerical simulation results using the Maxwell stress tensor calculation. Efficient sample injection and separation is another key to successful microchip CE. Accurate numerical studies were performed for understanding 3-D characteristics of the dispersion of sample species that is injected and carried by electroosmotic flow in diverse microchannel geometries. The following three cases were investigated; 1) non-rectangular cross section of microchannels, 2) different zeta potential for the top and bottom microchannel substrates, and 3) development of internal pressure gradient by variation of electric or electrokinetic properties along the channel direction. The results of the numerical study for the aforementioned 3 cases showed that 3-D modeling is crucial for accurate predictions of sample injection and migration in microchip electrophoresis system. Finally, continuous cell lysis in microchip CE devices was investigated experimentally by adopting a combination of electrical and osmotic cell lysis methods. The concept of continuous single cell lysis and CE was proven by analysis of single red blood cells labeled with FITC.Ph.D., MechanicalEngineering and Mechanics -- Drexel University, 201

Rational control of hydrothermal nanowire synthesis and its applications

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Materials Science and Engineering, 2010.Includes bibliographical references (p. 172-182).Hydrothermal nanowire synthesis is a rapidly emerging nanowire discipline that enables low temperature growth and batch process. It has a major impact on the development of novel energy conversion devices, high density electronics, and optical devices. However, detailed growth mechanism is still in early stage of its development. This thesis presents the fundamental understanding of controlled zinc oxide nanowire synthesis in a hydrothermal system based on thermodynamic / kinetic analysis of heterogeneous chemical reactions. Governing parameters of hydrothermal growth were evaluated with experimental growth rates and calculated solubility plots. Supersaturation was shown to be a key parameter for the hydrothermal nanowire synthesis. Morphology control of the nanowire synthesis was tested with various additional cations during synthesis. Changes in morphology and aspect ratio with different cations were explained by electrostatic competing ion model. Based on experimental results and complex ion charge distribution, the growth direction was biased via electrostatic competition from cation-complexes that adsorb to the crystal in a face-specific manner, thereby reducing zinc ion-complex adsorption and suppressing growth along that face. Dynamic control of nanowire synthesis was investigated under microfluidic environment with continuous flow. Microfluidic growth conditions were analyzed with the parametric experiments and finite element modeling. Nanowire growth under complex geometry was also evaluated. This rational control of hydrothermal nanowire synthesis was applied to fabricate high efficiency alternative current electroluminescent devices, in-situ fabricated light emitting diodes, photovoltaic devices, and field emission devices.by Jaebum Joo.Ph.D

Development of Analytical Methodology for Neurochemical Investigations

David J. Fischer Department of Pharmaceutical Chemistry University of Kansas Neurochemical Applications of Microchip Electrophoresis The development of sensitive and selective analytical tools has facilitated the investigation of complex neurological pathways and enhanced our understanding of neurodegenerative diseases. The development of sensitive analytical methodology for the determination of neurotransmitters and proteins related to neurodegenerative disease is described. The goal of the work performed in the first part of this dissertation was to develop analytical methodology for the analysis of catecholamine neurotransmitters (NTs) by microchip electrophoresis with electrochemical (EC) detection. Much of this work focused on the fabrication and characterization of the novel carbon-based electrode material, pyrolyzed photoresist. The fabrication of pyrolyzed photoresist film (PPF) electrodes was optimized for use in microchip electrophoresis and analytical performance was characterized using catecholamine NTs. In addition, an extensive comparison of the analytical performance of several commonly used electrode materials and electrode alignment schemes and the PPF electrode material was performed. Aspects such as sensitivity, limit of detection (LOD), resolution, reproducibility, and ease of fabrication were examined. In addition to the development of EC detection methods for catecholamine NTs, analytical methods for the determination of myc-tagged proteins were developed. The development of an electrophoretic immunoaffinity assay for the detection of a myc-tagged protein expressed in cell culture is described. While this is a general assay that can be applied to a variety of myc-tagged proteins, mutant huntingtin protein (mHtt) was used as a specific example. The development and optimization of capillary and microchip electrophoresis assays were performed for this purpose. In addition, the results obtained using these methods were directly compared to traditional analysis by Western blotting. The long term goal of this project is integrate both of these assays into a single lab-on-a-chip device capable of detecting NT release and mHtt protein in single cells

A Reliable and Simple Method for Fabricating a Poly(Dimethylsiloxane) Electrospray Ionization Chip with a Corner-Integrated Emitter

Monolithically integrated emitters have been increasingly applied to microfluidic devices that are coupled to mass spectrometers (MS) as electrospray ionization sources (ESI). A new method was developed to fabricate a duplicable structure which integrated the emitter into a poly(dimethylsiloxane) chip corner. Two photoresist layers containing a raised base which guaranteed the precise integration of the electrospray tip emitter and ensured that the cutting out of the tip exerted no influence even during repeated prototyping were used to ease the operation of the process. Highly stable ESI-MS performance was obtained and the results were compared with those of a commercial fused-silica capillary source. Furthermore, chip-to-chip and run-to-run results indicated both reliability and reproducibility during repeated fabrication. These results reveal that the proposed chip can provide an ideal ion source for MS across many applications, especially with the perspective to be widely used in portable MS during on-site analysis