The research presented in this work explores the application of microfluidics to the field of proteomics through the design of a multi-channel microfluidic platform and the investigation of individual components of the system. The design of this microfluidic device allows the integration of several protein sample preparation steps for automated electrospray ionization mass spectrometric (ESI-MS) analysis, including protein separation, fractionation and collection, preconcentration and cleanup, and protein digestion. In order for the multi-channel system to function properly, I first evaluated each individual component of the device. Several areas were explored: (i) optimization of polymer monolith for solid-phase extraction (SPE) preconcentration; (ii) investigation of cationic coatings for microchannel surface modification to facilitate positive electrospray of peptides and proteins for chip-MS coupling; (iii) combination of the hydrophobic monolith and the PolyE-323 coating in a single channel device for on-chip SPE and on-bed tryptic digestion with on-line coupling to ESI-MS. Multiplexed microfluidic devices for protein analysis, which integrate a series of microfluidic features, were then designed, built and tested. The multiplexed microfluidic architecture employed a separation channel, a fractionator, an array of microchambers to accommodate monolithic polymer for SPE preconcentration, and an elution channel for the detection of eluted sample using fluorescence detector or mass spectrometer. The performance of the multiplexed devices for integration of multiple analytical steps was explored with sequential fractionation, collection, and elution of fluorescent sample, evaluating the ability to trap and release individual fractions without cross-contamination. Thorough analysis of each of the individual components on the multiplexed microfluidic platform provides valuable insights into the design of such systems, which brings us closer to our final goal of a proteomic processing microchip.

The capability to easily and inexpensively fabricate microfluidic devices with negligible dependence on specialized laboratory equipment continues to be one of the primary forces driving the widespread use of plastic-based devices. These devices are typically produced as replicas of a rigid mold or master incorporating a negative image of the desired structures. The negative image is typically constructed from either thick photoresists or etched silicon substrates using conventional photolithographic fabrication processes. While these micromachining techniques are effective in constructing masters with micron-sized features, the need to produce masters rapidly in order to design, fabricate, and test microfluidic devices, is a major challenge in microfluidic technology. In this research, we use inexpensive photosensitized copper clad circuit board substrates to produce master molds using conventional printed circuit technology. The techniques provide the benefits of parallel fabrication associated with photolithography without the need for cleanroom facilities, thereby offering a degree of speed and simplicity that allows microfluidic master molds to be constructed in approximately 30 minutes in any laboratory. These techniques are used to produce a variety of microfluidic channel networks using PDMS (polydimethylsiloxane) and melt-processable plastic materials.

Proteome profiling is the identification and quantitation of all proteins in biological samples. An important application of proteome profiling that has received much attention is clinical proteomics, a field that promises the discovery of biomarkers that will be useful for early diagnosis and prognosis of diseases. While clinical proteomic methods vary widely, a common characteristic is the need for (i) extraction of proteins from complex biological fluids and (ii) extensive biochemical processing (reduction, alkylation and enzymatic digestion) prior to analysis. However, the lack of standardized sample handling and processing in proteomics is a major limitation for the field. The conventional macroscale manual sample handling requires multiple containers and transfers, which often leads to sample loss and contamination. For clinical proteomics to be adopted as a gold standard for clinical measures, the issue of irreproducibility needs to be addressed. A potential solution to this problem is to form integrated systems for sample handling and processing, and in this dissertation, I describe my work towards realizing this goal using digital microfluidics (DMF). DMF is a technique characterized by the manipulation of discrete droplets (100 nL – 10 L) on an array of electrodes by the application of electrical fields. It is well-suited for carrying out rapid, sequential, miniaturized automated biochemical assays. This thesis demonstrates how DMF can be a powerful tool capable of automating several protein handling and processing steps used in proteomics.

A prototype acoustic streaming pump for microfluidic applications was developed. A novel integration scheme was devised based on the acoustic reflector concept. Numerical simulations were conducted to predict the flow patterns around the transducer. Ultrasound transducers using P(VDF-TrFE) as the piezoelectric element were fabricated using lithography-based microfabrication technology. Silicon channels were fabricated using anisotropic etching. A heat-press bonding technique was adopted to bond the transducers with the silicon chips using CYTOP fluoropolymer as the adhesive. The piezoelectric transducers were characterized to have a resonance frequency of 82 MHz. Micro-PIV experiments were performed in the near and far-fields of the ultrasonic transducer/pump. The near field experiments showed complex flow patterns that could enhance mixing. Estimates of the pumping pressure were obtained using transient flow velocities in the far-field. Conservative estimates indicate the total back pressure the micropump can pump against is 39 Pa. Future research directions were suggested.

A prototype acoustic streaming pump for microfluidic applications was developed. A novel integration scheme was devised based on the acoustic reflector concept. Numerical simulations were conducted to predict the flow patterns around the transducer. Ultrasound transducers using P(VDF-TrFE) as the piezoelectric element were fabricated using lithography-based microfabrication technology. Silicon channels were fabricated using anisotropic etching. A heat-press bonding technique was adopted to bond the transducers with the silicon chips using CYTOP fluoropolymer as the adhesive. The piezoelectric transducers were characterized to have a resonance frequency of 82 MHz. Micro-PIV experiments were performed in the near and far-fields of the ultrasonic transducer/pump. The near field experiments showed complex flow patterns that could enhance mixing. Estimates of the pumping pressure were obtained using transient flow velocities in the far-field. Conservative estimates indicate the total back pressure the micropump can pump against is 39 Pa. Future research directions were suggested.

Microfluidic biosensors and bioreactors based on immobilized biomaterials are described in this dissertation. Photocrosslinkable hydrogel or polymeric microbeads were used as a supporting matrix for immobilizing E.coli or enzymes in a microfluidic device. This dissertation covers a microfluidic bioreactor based on hydrogel-entrapped E.coli, a microfluidic biosensor based on an array of hydrogel-entrapped enzymes, and a microfluidic bioreactor based on microbead-immobilized enzymes. Hydrogel micropatches containing E.coli were fabricated within a microfluidic channel by in-situ photopolymerization. The cells were viable in the hydrogel micropatch and their membranes could be porated by lysating agents. Entrapment of viable cells within hydrogels, followed by lysis, could provide a convenient means for preparing biocatalysts without the need for enzyme extraction and purification. Our results suggested that hydrogel-entrapped cells, immobilized within microfluidic channels, can act as sensors for small molecules and as bioreactors for carrying out reactions. A microfluidic biosensor based on an array of hydrogel-entrapped enzymes could be used to simultaneously detect different concentrations of the same analyte or multiple analyte in real time. The concentration of an enzyme inhibitor could be quantified using the same basic approach. Isolations of the microchannels within different microfluidic channels could eliminate the possibility of cross talk between enzymes. Finally, we characterized microfluidic bioreactors packed with microbead-immobilized enzymes that can carry out sequential, two-step enzyme-catalyzed reactions under flow conditions. The overall efficiency of the reactors depended on the spatial relationship of the two enzymes immobilized on the beads. Digital simulations confirmed the experimental results.

Thesis (M.S.)--Rutgers University, 2009. / "Graduate Program in Mechanical and Aerospace Engineering." Includes bibliographical references (p. 78-81).

Thesis (M.S.)--University of Central Florida, 2008. / Adviser: Larry Chew. Includes bibliographical references (p. 146-148).

In this dissertation, three types of paper-based analytical devices for point-of-care biosensing, a potentiometric method for analyzing percent hemoglobin A1c (%HbA1c) and a PDMS-glass microelectrochemical device for highly reproducible amperometric measurement in microdroplet, are described. The first paper-based sensing device is fabricated using the principles of origami (paper folding). The three-dimensional origami paper analytical device (oPAD) is fabricated on a single sheet of flat paper in a single photolithographic step and assembled by simply folding the paper by hand. Following analysis, the device can be unfolded to reveal each layer for optical and fluorescent read-out. The second type of paper-based device has an integral aluminum/air battery as the power source and reports its output using Prussian blue as an electrochromic indicator. The integrated aluminum/air battery powers both the electrochemical sensor and the electrochromic read-out. The applicability of the device to point-of-care sensing is demonstrated by qualitative detection of glucose and H2O2 in artificial urine. The third type of paper-based device (oPAD 2) uses an aptamer to recognize the analyte, adenosine, a glucose oxidase tag to modify the relative concentrations of an electroactive redox couple, and a digital multimeter to transduce the result of the assay. Adenosine is quantitatively determined using this device with a detection limit of 11.8 uM. The method for measuring HbA1c concentration, hemoglobin concentration, and thus %HbA1c in human blood is based on potentiometry. We use Alizarin red s (ARS) as a redox indicator. The potential shift of ARS owing to diol-boronic acid complexation is used to determine the HbA1c, which is a competitor of ARS for the complexation reaction. The concentration of Hb is determined by reacting it with Fe(CN)₆³⁻ and measuring the potential shift arising from the reduction of Fe(CN)₆³⁻ by Hb. The results obtained for %HBA1c in human blood are in good agreement with those determined using a reference method. The method for highly reproducible chronoamperometric analysis of the contents of microdroplets is developed. Aqueous microdroplets (~ 1 nL) and separated by a fluorocarbon solvent are generated within a microfluidic device using a T-shaped junction. Highly reproducible quasi-steady-state currents (relative standard deviations = ~ 2%) are observed when the microdroplets are stretched by a factor of 10 in a narrowed segment of a microchannel, which leads to desirable intradroplet mass transfer characteristics. Importantly, the design of the microelectrochemical device ensures direct contact between intradroplet redox molecules and the electrode surface to study inner-sphere electrocatalytic processes such as the oxygen reduction reaction. Finite-element simulations are presented that are in accord with the experimental findings. / text

The capability to easily and inexpensively fabricate microfluidic devices with negligible dependence on specialized laboratory equipment continues to be one of the primary forces driving the widespread use of plastic-based devices. These devices are typically produced as replicas of a rigid mold or master incorporating a negative image of the desired structures. The negative image is typically constructed from either thick photoresists or etched silicon substrates using conventional photolithographic fabrication processes. While these micromachining techniques are effective in constructing masters with micron-sized features, the need to produce masters rapidly in order to design, fabricate, and test microfluidic devices, is a major challenge in microfluidic technology. In this research, we use inexpensive photosensitized copper clad circuit board substrates to produce master molds using conventional printed circuit technology. The techniques provide the benefits of parallel fabrication associated with photolithography without the need for cleanroom facilities, thereby offering a degree of speed and simplicity that allows microfluidic master molds to be constructed in approximately 30 minutes in any laboratory. These techniques are used to produce a variety of microfluidic channel networks using PDMS (polydimethylsiloxane) and melt-processable plastic materials.