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SDSU Template, Version 11.1 NONLINEAR LASER WAVE-MIXING DETECTION FOR CAPILLARY ELECTROPHORESIS AND MULTI-CHANNEL ARRAYS FOR BIOMEDICAL AND ENVIRONMENTAL APPLICATIONS _______________ A Thesis Presented to the Faculty of San Diego State University _______________ In Partial Fulfillment of the Requirements for the Degree Master of Science in Chemistry _______________ by Eric J. Maxwell Spring 2015 iii Copyright © 2015 by Eric J. Maxwell All Rights Reserved iv DEDICATION To my wife, Selena, for supporting me through all of the long nights and weekends of work that this program required. I cannot wait to start the next chapter in this journey. v ABSTRACT OF THE THESIS Nonlinear Laser Wave-Mixing Detection for Capillary Electrophoresis and Multi-Channel Arrays for Biomedical and Environmental Applications by Eric J. Maxwell Master of Science in Chemistry San Diego State University, 2015 Degenerate Four-Wave Mixing is demonstrated as a highly sensitive nonlinear spectroscopic detection method for biomedical and environmental targets. This is achieved through refractive index change within an absorbing liquid medium, which produces a laser- like signal beam. This signal has high spatial resolution, and may be collected with high efficiency against a nearly 100% dark background. The cubic dependence on laser power and square dependence on analyte concentration allow for high signal intensity in trace analysis applications. In this work, the Degenerate Four-Wave Mixing technique is coupled with capillary electrophoresis, immunoprecipitation or color-forming reactions to provide specificity. The veterinary drugs malachite green and crystal violet are shown to be detectable at concentrations as low as 6.9 x 10-10 M (2.5 x 10-19 mol) and 8.3 x 10-11 M (3.0 x 10-20 mol) respectively (S/N = 2). Capillary electrophoresis is used in conjunction with a 2- laser Degenerate Four-Wave Mixing detector to allow simultaneous identification of both analytes. For another small molecule target, ammonium nitrate, sample preparation with diphenylamine is used to produce a colored compound capable of absorbing 635 nm light. This explosive component is of significant forensics interest due to its use in the manufacture of improvised explosive devices. The limit of detection for ammonium nitrate in 1 mm ID capillary cells is determined to be 1.5 x 10-9 M (5.2 x 10-18 mol). The detection limit in a 1.5 mm thin-film cell is found to be 3.2 x 10-7 M (2.0 x 10-15 mol) for S/N = 2. In addition to small molecule environmental targets, a detector for the cancer biomarker carcinoembryonic antigen is demonstrated with a combination of magnetic immunoprecipitation, multi-channel capillary arrays and Degenerate Four-Wave Mixing. Isolated protein samples are reacted with bicinchoninic acid (BCA) to yield a colored product, and absorbance of a 532nm laser produces a wave-mixing signal. Multiple samples may be processed rapidly though computer-controlled positioning of the multi-channel capillary array. The limit of detection for carcinoembryonic antigen is determined to be 3.3 x 10-12 M (0.59 ng/mL), with a corresponding mass detection limit of 1.2 x 10-21 mol (0.22 fg) for S/N = 2. vi TABLE OF CONTENTS PAGE ABSTRACT ...............................................................................................................................v LIST OF TABLES ................................................................................................................... ix LIST OF FIGURES ...................................................................................................................x ACKNOWLEDGEMENTS ................................................................................................... xiv CHAPTER 1 INTRODUCTION ........................................................................................................1 1.1. Detection of Biomedical and Environmental Targets .......................................1 1.1.1. Motivation ........................................................................................1 1.1.2. Spectroscopic Detection Methods....................................................2 1.1.3. Lasers ...............................................................................................4 1.1.4. Laser Spectroscopic Methods ..........................................................4 1.2. Nonlinear Wave-Mixing ...................................................................................5 1.3. History of Nonlinear Wave Mixing ..................................................................6 1.4. Thesis Outline ...................................................................................................8 2 FOUR-WAVE MIXING SPECTROSCOPY ...............................................................9 2.1. Overview ...........................................................................................................9 2.1.1. Backward-Scattering Degenerate Four-Wave Mixing.....................9 2.1.2. Forward-Scattering Degenerate Four-Wave Mixing .....................10 2.2. Interference Patterns .......................................................................................10 2.3. Laser-Induced Dynamic Gratings ...................................................................12 2.4. Wave-Mixing Signal .......................................................................................14 2.4.1. Signal Intensity ..............................................................................16 vii 2.4.2. Signal Collection ............................................................................17 3 DETECTION OF MALACHITE GREEN AND CRYSTAL VIOLET BY DEGENERATE FOUR-WAVE MIXING AND CAPILLARY ELECTROPHORESIS .................................................................................................19 3.1. Abstract ...........................................................................................................19 3.2. Introduction .....................................................................................................20 3.3. Experimental ...................................................................................................23 3.3.1. Chemicals .......................................................................................23 3.3.2. Degenerate Four-Wave Mixing Apparatus ....................................23 3.3.3. Capillary Electrophoresis System ..................................................25 3.3.4. Capillary Treatment .......................................................................27 3.4. Results and Discussion ...................................................................................29 3.4.1. Continuous Flow Detection ...........................................................29 3.4.2. Background Electrolytes ................................................................30 3.4.3. Field Amplified Sample Stacking ..................................................36 3.4.4. Bare Capillary Separation ..............................................................39 3.4.5. Effects of Capillary Dynamic Coating ...........................................41 3.4.6. Detection Limits and Linearity ......................................................43 3.5. Conclusions .....................................................................................................45 4 AMMONIUM NITRATE EXPLOSIVE SCREENING USING DEGENERATE FOUR-WAVE MIXING SPECTROSCOPY ...................................48 4.1. Abstract ...........................................................................................................48 4.2. Introduction .....................................................................................................48 4.3. Experimental ...................................................................................................50 4.3.1. Chemicals .......................................................................................50 4.3.2. Degenerate Four-Wave Mixing Optical Setup ...............................50 4.3.3. Capillary Sample Cells ..................................................................52 4.3.4. Thin-Film Sample Cells .................................................................52 4.3.5. Nitrate Derivatization.....................................................................55 4.4. Results and Discussion ...................................................................................58 4.4.1. Nitrate Derivatization Kinetics ......................................................58 4.4.2. Detection in Capillary Cells ...........................................................60 4.4.3. Detection in Thin-Film Cells .........................................................66 viii 4.4.4. Linearity of Response ....................................................................69 4.5. Conclusions .....................................................................................................71 5 SENSITIVE DETECTION OF CARCINOEMBRYONIC ANTIGEN BY DEGENERATE FOUR-WAVE MIXING SPECTROSCOPY ...................................74 5.1. Abstract ...........................................................................................................74 5.2. Introduction .....................................................................................................74 5.2.1. Degenerate Four-Wave Mixing .....................................................76 5.2.2. Bradford Protein Assay ..................................................................77 5.2.3. BCA Protein Assay ........................................................................77
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