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Degenerate four-wave mixing interfaced with capillary electrophoresis as a bioanalytical method for small molecules, peptides, and proteins
Iwabuchi, Manna Frances
Tauber, Michael J
Tong, Willam G
Smith, Diane K
xxv, 187 pages : illustrations (some color).
Degenerate four-wave mixing (DFWM) is demonstrated as an ultrasensitive detection method for the study of neurodegenerative disease-related biomolecules in a capillary electrophoresis system. Nonlinear absorption-based wave mixing offers important advantages including high spatial resolution, small probe volumes down to picoliter, small sample requirements, effective signal collections, and low background noise levels. The laser wave mixing is easily interfaced with microcapillaries for a continuous-flow mode detection and a capillary electrophoresis-mode detection. The optimal conditions for laser wave mixing and capillary electrophoresis is investigated to perform ultrasensitive detection and molecular weight-based separation for proteins. A cost-effective fluorophore is employed to target analytes, bovine serum albumin (BSA), antibody of HIV-1 capsid protein p24, and monoclonal antibody of breast cancer marker CA15-3. Concentration (and mass) detection limits of 1.4 x 10⁻¹⁰ M (1.0 x 10⁻²⁰ mol), 6.6 x 1010⁻¹⁰ M (5.1 x 10⁻²⁰ mol), and 6.7 x 10⁻¹² M (5.2 x 10⁻²² mol) are determined for BSA, p24 antibody, and CA15-3 antibody. For the first time, [alpha]-synuclein is analyzed using fluorescein isothiocyanate (FITC), QSY35 acetic acid succinimidyl ester, and ChromeoTM P503 by wave mixing-based capillary zone electrophoresis and capillary sieving electrophoresis. Detection limits of 1.4 x 10⁻¹³ M (1.1 x 10⁻²³ mol), 1.4 x 10⁻¹⁰ M (1.1 x 1010⁻²⁰ mol), and 3.8 x 10⁻¹³ M (3.0 x 10⁻²³ mol) are determined for FITC, QSY35, and ChromeoTM P503-conjugated [alpha]-synuclein. Tetramer [alpha]-synuclein is observed to be the most abundant species in the buffer system in which separation is executed. Native label-free adenosine and dopamine, and chromophore-conjugated glutamate and [gamma]-aminobutyric acid are detected at ultrasensitive concentration levels using wave mixing-based micellar electrokinetic chromatography. Detection limits of 1.0x10⁻¹³ M (5.6x10⁻²⁴ mol corresponds to 4 molecules), 1.2x10⁻⁹ M (6.6x10⁻²⁰ mol), 3.7x10⁻¹⁰ M (2.9x10⁻²⁰ mol), and 4.1x10⁻¹¹ M (3.2x10⁻²¹ mol) are determined for native label-free adenosine and dopamine, dabsylated glutamate and [gamma]-aminobutyric acid. [Beta]-Amyloid 1-42 and 1-40 peptides are investigated using wave mixing based capillary zone electrophoresis. Detection limits of 8.2 x 10⁻¹³ M (6.5 x 10⁻²³ mol) and 1.2 x 10⁻¹² M (9.5 x 10⁻²³ mol) are determined for A[Beta]1-42 and A[Beta]1-40.
San Diego State University
Doctor of Philosophy (Ph.D.) University of California, San Diego and San Diego State University, 2015
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