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.

Nonlinear laser wave mixing is presented as a highly sensitive absorption-based detection method for biomarkers in a capillary electrophoresis system, and on glass surfaces or microscope slides. It offers significant advantages, including outstanding sensitivity and selectivity levels, high spatial resolution, small sample volume requirement, small probe volume (nL to pL), and portable and compact designs. Different laser wave-mixing detectors can be interfaced to capillary electrophoresis systems for enhanced chemical selectivity and microscope glass slides for inexpensive and fast detection of millimeter-thin samples.Laser wave-mixing detection interfaced with capillary electrophoresis further enhances detection sensitivity by on-line sample concentration methods. Selectivity levels are improved by separating excess labels or dyes from dye-conjugated proteins. Different modes of capillary electrophoresis can be used to separate proteins and biomarkers, including capillary zone electrophoresis, micellar electrokinetic chromatography, and capillary sieving electrophoresis. Since wave mixing is an absorption-based detection method, one can use both fluorophores and chromophores to label proteins and biomarkers. For example, Chromeo P503 and Chromeo P540 are used to label pancreatic cancer biomarker CA 19-9 with the optimal molar ratio of dye to protein. The concentration and mass detection limits for CA 19-9 are determined to be 0.0090 U/mL and 6.8 x 10-10 U, respectively. Colorimetric assays (Bradford assay and BCA assay) are used to quantify CA 19-9, and the corresponding concentration and mass detection limits for CA 19-9 are determined to be 75 pM (picomolar) and 5.6 zeptomole, respectively. This study also demonstrates ultrasensitive detection of biomarkers held between microscope glass slides or air-dried on a microscope slide for convenient and fast detection of biomarkers and viruses. Preliminary concentration and mass detection limits are determined to be 160 U/mL and 680 attomole for CA 19-9; 40 ng/mL and 91 zeptomole for human epidermal growth factor receptor 2; and 125 pg/mL and 1 zeptomole for HIV-1 p24 antigen.

Optical nonlinear degenerate four-wave mixing (D4WM) spectroscopy is a presented as an ultrasensitive method for characterizing ad quantifying low abundance molecules, such as, chemical and biological agents, carcinogenic compounds, and biomarker protein in complex biological/environmental systems. This is achieved through refractive index change within an absorbing 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. The purpose of this study is to demonstrate optical degenerate four-wave mixing (DFWM) as an effective optical tool for standoff/surface detection of explosives, as well an ultrasensitive tool for detection of carcinogenic pollutants and disease markers.

Standoff/surface detection of is of great interest in these times of active terrorism. Optical techniques remain the most promising approach for standoff/surface detection of trace residues on surfaces. Laser-based approaches, such as LIBS (Laser Induced Breakdown Spectroscopy), Raman Spectroscopy or CARS (Coherent Anti-Stokes Raman Spectroscopy), and LIF (Laser Induced Fluorescence) are amongst a variety of optical techniques that have shown great potentials for standoff/surface detection of explosives. However, the determination of analytes depends on the measurement of scattered light and functional groups above background noise which can be difficult. DFWM on the other end is a coherent laser-like signal which can be measured with almost 100% efficiency over background noise and does depend on the measurement of functional groups, giving rise to higher sensitivity and specificity.

D4WM is employed as the main detector coupled to high-throughput (1D or 2D) separation methods for the determination and quantitation of environmental toxicants, colorectal protein marker conjugated to a chromophore, metabolomic studies of mouse heart Secretome, and surface detection of explosives. This study demonstrates the applicability of field-deployable D4WM instrumentation for use at port of entries. The first to be verified by 3rd party scientist from John Hopkins University.

Objectives

Methods

Results

Conclusions