UC San Diego

Nonlinear laser wave-mixing spectroscopy is demonstrated as a sensitive, selective and fast detection method for a range of bioanalytical applications. Laser wave mixing is an ultrasensitive optical absorption-based detection method, and hence, it is effective for label-free native biomolecules. Advantages of this technique over conventional techniques include enhanced sensitivity, shorter analysis times, smaller sample requirements, and higher throughput. Two input laser beams are focused and mixed to create the small wave-mixing probe to detect small sample sizes, yielding excellent mass detection limits and high spatial resolution. In the presence of absorbing analytes, laser-induced thermal gratings diffract incoming photons to produce the wave-mixing signal beams that are coherent laser-like beams, and they can be captured on an inexpensive photodetector with high S/N. In addition, the signal intensity has a quadratic dependence on analyte concentration and a cubic dependence on laser power, making wave mixing an ideal sensor for monitoring small chemical or biological changes using compact low-power lasers. Various peptides or proteins can be detected either in a flowing capillary or in biological specimens fixed to a glass slide. The analytes can be investigated in their native (unlabeled) or labeled with fluorophore or chromophore labels. Laser wave mixing is demonstrated for sensitive detection of heart failure biomarkers pro-atrial natriuretic peptide and brain natriuretic peptide. The peptides are conjugated with a dye that absorbs at the visible wavelength range using compact visible solid-state lasers. Due to the similar chemical and physical properties of the peptides, the wave-mixing detector is interfaced to a custom-built capillary electrophoresis separation system to enhance both chemical selectivity and detection sensitivity. The final two chapters focus on monitoring myoglobin oxygen saturation in various media using laser wave mixing. Myoglobin is a chromophoric protein, and its absorption is highly dependent upon its oxidation states. A reducing agent, such as sodium dithionite, is employed to deoxygenate myoglobin reversibly and irreversibly. The reversible myoglobin deoxygenation is performed to monitor the oxygen transfer from oxyhemoglobin to deoxymyoglobin. The irreversible myoglobin deoxygenation helps in visualizing the spatial distribution of oxygen in the myofibers of mice skeletal muscles.